Caenorhabditis elegans As a Promising Alternative Model for Environmental Chemical Mixture Effect Assessment-A Comparative Study.

A key challenge of mixture toxicity testing is that a multitude of substances with even more combinations need to be tested in a broad dose range. Consequently testing in rodent bioassays, the current gold standard of toxicity testing, is hardly feasible. High-throughput compatible cell culture systems, however, suffer from limitations with respect to toxicokinetics, tissue interactions, and compensatory mechanisms. Therefore, simple organisms like the nematode Caenorhabditis elegans, combining relevant advantages of complex in vivo and fast in vitro assays might prove highly valuable within a testing strategy for mixtures. To investigate the comparability between results obtained with C. elegans and traditional rodent assays, we used five azole fungicides as well investigated model substances. Our findings suggest that azoles act additively in C. elegans which is in line with previous results in rats. Additionally, we show that toxicokinetics are one important factor for the differences in the relative toxicity of the azoles in both species. Importantly, we also demonstrate that in contrast to most rodent in vivo studies, C. elegans assays provide well-defined concentration-response relationships which are a very good basis for the prediction of mixture effects. We conclude that C. elegans may be an appropriate model for mixture toxicity testing at least within a first step to identify and prioritize relevant mixtures for further testing.

Regulatory toxicology in the twenty-first century: challenges, perspectives and possible solutions.

The advent of new testing systems and "omics"-technologies has left regulatory toxicology facing one of the biggest challenges for decades. That is the question whether and how these methods can be used for regulatory purposes. The new methods undoubtedly enable regulators to address important open questions of toxicology such as species-specific toxicity, mixture toxicity, low-dose effects, endocrine effects or nanotoxicology, while promising faster and more efficient toxicity testing with the use of less animals. Consequently, the respective assays, methods and testing strategies are subject of several research programs worldwide. On the other hand, the practical application of such tests for regulatory purposes is a matter of ongoing debate. This document summarizes key aspects of this debate in the light of the European "regulatory status quo", while elucidating new perspectives for regulatory toxicity testing.

Prediction of Acute Hepatotoxicity With Human Pluripotent Stem Cell-Derived Hepatic Organoids.

Recent development of hepatic organoids (HOs) derived from human pluripotent stem cells (hPSCs) provides an alternative in vitro model that can mimic the human liver detoxification pathway for drug safety assessment. By recapitulating the high level of maturity and drug-metabolizing capacity of the liver in a three-dimensional organoid culture, HOs may allow researchers to assess drug toxicity and metabolism more accurately than animal models or hepatocellular carcinoma cells. Although this promising potential has contributed to the development of various protocols, only a few protocols are available to generate functional HOs with guaranteed CYP450 enzymatic activity, the key feature driving toxic responses during drug metabolism. Based on previously published protocols, we describe an optimized culture method that can substantially increase the expression and activity of CYP450s, in particular CYP3A4, CYP2C9, and CYP2C19, in HOs. To generate mass-produced and highly reproducible HOs required as models for toxicity evaluation, we first generated hepatic endodermal organoids (HEOs) from hPSCs capable of in vitro proliferation and cryopreservation. The stepwise protocol includes generating HEOs as well as efficient methods to enhance CYP450 expression and activity in terminally differentiated HOs. Furthermore, we present a simple protocol for the assessment of HO cytotoxicity, one of the hallmarks of drug-induced acute hepatotoxicity. The protocols are relatively straightforward and can be successfully used by laboratories with basic experience in culturing hPSCs. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hepatic endodermal organoids from human pluripotent stem cells Basic Protocol 2: Expansion and cryopreservation of hepatic endodermal organoids Basic Protocol 3: Differentiation of hepatic organoids from hepatic endodermal organoids Basic Protocol 4: Evaluation of hepatotoxicity using hepatic organoids Support Protocol: Human pluripotent stem cell culture.

hiPSC-derived macrophages improve drug sensitivity and selectivity in a macrophage-incorporating organoid culture model.

Accurate simulation of different cell type interactions is crucial for physiological and precisein vitrodrug testing. Human tissue-resident macrophages are critical for modulating disease conditions and drug-induced injuries in various tissues; however, their limited availability has hindered their use inin vitromodeling. Therefore, this study aimed to create macrophage-containing organoid co-culture models by directly incorporating human-induced pluripotent stem cell (hiPSC)-derived pre-macrophages into organoid and scaffold cell models. The fully differentiated cells in these organoids exhibited functional characteristics of tissue-resident macrophages with enriched pan-macrophage markers and the potential for M1/M2 subtype specialization upon cytokine stimulation. In a hepatic organoid model, the integrated macrophages replicated typical intrinsic properties, including cytokine release, polarization, and phagocytosis, and the co-culture model was more responsive to drug-induced liver injury than a macrophage-free model. Furthermore, alveolar organoid models containing these hiPSC-derived macrophages also showed increased drug and chemical sensitivity to pulmonary toxicants. Moreover, 3D adipocyte scaffold models incorporating macrophages effectively simulated in vivo insulin resistance observed in adipose tissue and showed improved insulin sensitivity on exposure to anti-diabetic drugs. Overall, the findings demonstrated that incorporating hiPSC-derived macrophages into organoid culture models resulted in more physiological and sensitivein vitrodrug evaluation and screening systems.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

The 4th dimension of in vitro systems - Time to level up.

Various in vitro model systems have been established over the last decades to understand physiological processes, the causalities of diseases and the response of humans to environmental and industrial chemicals or therapeutic drugs. Common to all is a limited biological significance due to the impairment of functionality, for instance by the lack of physiological 3D tissue architecture or the loss of fundamental regulatory mechanisms including the circadian rhythm. The circadian rhythm is an adaption of living organisms to rhythmic environmental changes of the day-night cycle and coordinates behavior as well as various crucial physiological processes in a 24-hour pattern. Here, we discuss the impact of integrating circadian regulation in experimental approaches and toxicological assessments to improve the biological relevance of the obtained results. In particular, it is known for some time that an ongoing disruption of the circadian rhythmicity is associated with an increased risk for cardiovascular disease, metabolic dysfunction or cancer. In the context of health recovery, the importance of circadian control mechanism is recognized by chronopharmacological concepts to increase the efficiency of pharmacological treatment strategies. Despite the undeniable circadian dependency and the biological relevance of manifold cellular and molecular processes, the impact of circadian regulation is hardly considered in a wide range of biomedical and toxicological research areas. Reactivating the circadian regulation holds the promise to enhance the biological relevance and reliability of in vitro approaches. In the context of human health protection the implementation of a circadian regulation will subsequently generate advanced physiologically relevant in vitro approaches and allows an improved toxicological assessment of health risks. In addition, the establishment of circadian disruption as a novel toxicological endpoint will provide a better understanding of toxicological mode of actions of environmental and industrial chemicals or drugs and enlarge the knowledge of disease development.

Quantitative high-throughput phenotypic screening for environmental estrogens using the E-Morph Screening Assay in combination with in silico predictions.

BACKGROUND: Exposure to environmental chemicals that interfere with normal estrogen function can lead to adverse health effects, including cancer. High-throughput screening (HTS) approaches facilitate the efficient identification and characterization of such substances. OBJECTIVES: We recently described the development of the E-Morph Assay, which measures changes at adherens junctions as a clinically-relevant phenotypic readout for estrogen receptor (ER) alpha signaling activity. Here, we describe its further development and application for automated robotic HTS. METHODS: Using the advanced E-Morph Screening Assay, we screened a substance library comprising 430 toxicologically-relevant industrial chemicals, biocides, and plant protection products to identify novel substances with estrogenic activities. Based on the primary screening data and the publicly available ToxCast dataset, we performed an insilico similarity search to identify further substances with potential estrogenic activity for follow-up hit expansion screening, and built seven insilico ER models using the conformal prediction (CP) framework to evaluate the HTS results. RESULTS: The primary and hit confirmation screens identified 27 'known' estrogenic substances with potencies correlating very well with the published ToxCast ER Agonist Score (r=+0.95). We additionally detected potential 'novel' estrogenic activities for 10 primary hit substances and for another nine out of 20 structurally similar substances from insilico predictions and follow-up hit expansion screening. The concordance of the E-Morph Screening Assay with the ToxCast ER reference data and the generated CP ER models was 71% and 73%, respectively, with a high predictivity for ER active substances of up to 87%, which is particularly important for regulatory purposes. DISCUSSION: These data provide a proof-of-concept for the combination of in vitro HTS approaches with insilico methods (similarity search, CP models) for efficient analysis of large substance libraries in order to prioritize substances with potential estrogenic activity for subsequent testing against higher tier human endpoints.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

Developmental toxicity testing in the 21st century: the sword of Damocles shattered by embryonic stem cell assays?

Modern society faces an inherent dilemma. In our globalized society, we are spoilt for choice by an ever-increasing number of products, many of which are made of new materials and compound mixtures. At the same time, as consumers we got accustomed to the idea of a life minimized for risk, including our own exposure to chemicals from the environment or to compounds present in and released from everyday products. Chemical safety testing bridges these obviously diverging interests, and the corresponding legislation has hence been tremendously extended (e.g., introduction of the European legislation REACH in 2007). However, the underlying regulatory toxicology still relies mainly on animal testing, which is relatively slow, expensive, and ethically arguable. Meanwhile, recent years have seen a surge in efforts to develop alternative testing systems and strategies. Expectations are particularly high for the applicability of stem cells as test systems especially for developmental toxicity testing in vitro. For the first time in history, test systems can be based on differentiating cells and tissue progenitors in culture, thus bringing the 'vision of toxicity testing in the 21st century' a step closer.

Alternatives to animal testing: current status and future perspectives.

On the occasion of the 20th anniversary of the Center for Alternative Methods to Animal Experiments (ZEBET), an international symposium was held at the German Federal Institute for Risk Assessment (BfR) in Berlin. At the same time, this symposium was meant to celebrate the 50th anniversary of the publication of the book "The Principles of Humane Experimental Technique" by Russell and Burch in 1959 in which the 3Rs principle (that is, Replacement, Reduction, and Refinement) has been coined and introduced to foster the development of alternative methods to animal testing. Another topic addressed by the symposium was the new vision on "Toxicology in the twenty-first Century", as proposed by the US-National Research Council, which aims at using human cells and tissues for toxicity testing in vitro rather than live animals. An overview of the achievements and current tasks, as well as a vision of the future to be addressed by ZEBET@BfR in the years to come is outlined in the present paper.

The E-Morph Assay: Identification and characterization of environmental chemicals with estrogenic activity based on quantitative changes in cell-cell contact organization of breast cancer cells.

Adverse health effects that are caused by endocrine disrupting chemicals (EDCs) in the environment, food or consumer products are of high public concern. The identification and characterization of EDCs including substances with estrogenic activity still necessitates the use of animal testing as most of the approved alternative test methods only address single mechanistic events of endocrine activity. Therefore, novel human-relevant in vitro assays covering more complex functional endpoints of adversity, including hormone-related tumor formation and progression, are needed. This study describes the development and evaluation of a novel high-throughput screening-compatible assay called "E-Morph Assay". This image-based phenotypic screening assay facilitates robust predictions of the estrogenic potential of environmental chemicals using quantitative changes in the cell-cell contact morphology of human breast cancer cells as a novel functional endpoint. Based on a classification model, which was developed using six reference substances with known estrogenic activity, the E-Morph Assay correctly classified an additional set of 11 reference chemicals commonly used in OECD Test Guidelines and the U.S. EPA ToxCast program. For each of the tested substances, a relative ER bioactivity score was derived that allowed their grouping into four main categories of estrogenic activity, i.e. 'strong' (>0.9; four substances, i.e. natural hormones or pharmaceutical products), 'moderate' (0.9-0.6; six substances, i.e. phytoestrogens and Bisphenol AF), 'weak' (<0.6; three substances, i.e Bisphenol S, B, and A), and 'negative' (0.0; four substances). The E-Morph Assay considerably expands the portfolio of test methods providing the possibility to characterize the influence of environmental chemicals on estrogen-dependent tumor progression.

A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction.

Growth and offspring count are two commonly determined toxicological endpoints for chemical- or gene-induced developmental and reproductive effects in Caenorhabditis elegans. Here, we present a protocol for a 96 h, medium-throughput assay, assessing both endpoints quantitatively within an automated framework using open-source software. The assay utilizes whole 96-well fluorescence images taken with a high-content screening system. Alternatively, conventional fluorescence images can also be utilized with only a few adjustments. For complete details on the use and execution of this protocol, please refer to Wittkowski et al. (2019).

Restoring circadian synchrony in vitro facilitates physiological responses to environmental chemicals.

BACKGROUND: The growing requirement of hazard and risk assessment of environmental chemicals and the efforts to minimize animal testing, increases the demand for innovative and predictive in vitro test systems in toxicology, reflecting the physiological conditions of human nature. Here, an elemental factor regulating a variety of physiological processes is the day-night rhythm. This circadian rhythm, describing a biological oscillation with a 24-h period is hardly acknowledged in toxicology and test method development. Whilst, in animals or humans the entire organism exhibits a rigorous cellular circadian synchrony, in conventional in vitro systems each cell follows its own rhythm, due to the absence of appropriate synchronizing signals. OBJECTIVE: Here we investigated whether circadian synchronization of human cells in an in vitro system improves the cellular response and, thus, increases the sensitivity of the test system. Since the circadian regulation of metabolism is particularly well understood, and dioxin and dioxin-like compounds are of major concern for environmental health we focused on the ubiquitous drug metabolizing detoxification system mediated by the aryl hydrocarbon receptor (AHR). METHODS: To this end, we applied various prototypical AHR activators onto different human cell lines under non-synchronized or circadian synchronized conditions and determined the dose response on representative endogenous target genes. RESULTS: Remarkably, the cellular response dynamic upon chemical treatment was substantially enhanced in circadian synchronized cells and followed a rhythmic expression pattern. This broader dynamic range was associated with a strikingly higher induction of AHR target genes and the corresponding enzymatic activity, thereby rather mimicking the in vivo situation. CONCLUSION: Our findings indicate that a synchronized circadian rhythm in a cell culture based test system can improve the physiological relevance of an appropriate in vitro method by reflecting the biological in vivo situation more closely. Accordingly, it is a promising tool to facilitate the wide acceptance of in vitro methods in the field of regulatory toxicology and to further optimize the toxicological assessment of environmental chemicals.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

Estrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.

We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreased Chordin production. Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations. First, dorsalization by lithium chloride treatment was completely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely blocked the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation.

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research.

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

Transgenic Mouse Models Transferred into the Test Tube: New Perspectives for Developmental Toxicity Testing In Vitro?

Despite our increasing understanding of molecular mechanisms controlling embryogenesis, the identification and characterization of teratogenic substances still heavily relies on animal testing. Embryonic development depends on cell-autonomous and non-autonomous processes including spatiotemporally regulated extracellular signaling activities. These have been elucidated in transgenic mouse models harboring easily detectable reporter genes under the control of evolutionarily conserved signaling cascades. We propose combining these transgenic mouse models and cells derived thereof with existing alternative toxicological testing strategies. This would enable the plausibility of in vitro data to be verified in light of in vivo data and, ultimately, facilitate regulatory acceptance of in vitro test methods.

Editor's Highlight: Identification and Characterization of Teratogenic Chemicals Using Embryonic Stem Cells Isolated From a Wnt/ß-Catenin-Reporter Transgenic Mouse Line.

Embryonic stem cells (ESCs) are commonly used for the analysis of gene function in embryonic development and provide valuable models for human diseases. In recent years, ESCs have also become an attractive tool for toxicological testing, in particular for the identification of teratogenic compounds. We have recently described a Bmp-reporter ESC line as a new tool to identify teratogenic compounds and to characterize the molecular mechanisms mediating embryonic toxicity. Here we describe the use of a Wnt/ß-Catenin-reporter ESC line isolated from a previously described mouse line that carries the LacZ reporter gene under the control of a ß-Catenin responsive promoter. The reporter ESC line stably differentiates into cardiomyocytes within 12 days. The reporter was endogenously induced between day 3-5 of differentiation reminiscent of its expression in vivo, in which strong LacZ activity is detected around gastrulation. Subsequently its expression becomes restricted to mesodermal cells and cells undergoing an epithelial to mesenchymal transition. The Wnt/ß-Catenin-dependent expression of the reporter protein allowed quantification of dose- and time-dependent effects of teratogenic chemicals. In particular, valproic acid reduced reporter activity on day 7 whereas retinoic acid induced reporter activity on day 5 at concentrations comparable to the ones inhibiting the formation of functional cardiomyocytes, the classical read-out of the embryonic stem cell test (EST). In addition, we were also able to show distinct effects of teratogenic chemicals on the Wnt/ß-Catenin-reporter compared with the previously described Bmp-reporter ESCs. Thus, different reporter cell lines provide complementary tools for the identification and analysis of potentially teratogenic compounds.

Defining the optimal animal model for translational research using gene set enrichment analysis.

The mouse is the main model organism used to study the functions of human genes because most biological processes in the mouse are highly conserved in humans. Recent reports that compared identical transcriptomic datasets of human inflammatory diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. To reduce susceptibility to biased interpretation, all genes of interest for the biological question under investigation should be considered. Thus, standardized approaches for systematic data analysis are needed. We analyzed the same datasets using gene set enrichment analysis focusing on pathways assigned to inflammatory processes in either humans or mice. The analyses revealed a moderate overlap between all human and mouse datasets, with average positive and negative predictive values of 48 and 57% significant correlations. Subgroups of the septic mouse models (i.e., Staphylococcus aureus injection) correlated very well with most human studies. These findings support the applicability of targeted strategies to identify the optimal animal model and protocol to improve the success of translational research.

Identification of a second Xenopus twisted gastrulation gene.

Twisted Gastrulation (Tsg) is a secreted molecule which regulates BMP signalling in the extracellular space as part of an evolutionarily conserved network of interacting proteins. In Xenopus, maternal xTsg mRNA can be found throughout the early embryo. After gastrulation, xTsg is expressed as part of the BMP4 synexpression group until late tadpole stages. Here we report the identification of a second Xenopus Tsg gene (xTsg-2). Xenopus Tsg-2 is highly homologousto xTsg. In particular, amino acid residues which have been shown to be required for the binding of xTsg to BMP and to Chordin are conserved. The expression of Xenopus Tsg-2 mRNA was restricted to late stages of embryonic development; it was detected at tadpole stages in lateral plate mesoderm, neural crest, branchial arches and head mesenchyme. In microinjection experiments, the activity of xTsg-2 mRNA was similar to that of xTsg. We conclude that two Tsg genes act in distinct temporal and spatial territories in the course of Xenopus embryonic development.