High-throughput Toxicity Research Articles

Predictive value of the ToxCast/Tox21 high throughput toxicity screening data for approximating in vivo ecotoxicity endpoints and ecotoxicological risk in eco- surveillance applications

Ecotoxicology has long relied on assessing the hazard potential of chemicals through traditional in vivo testing methods to understand the possible risk exposure could pose to ecological taxa. In the past decade, the development of non-animal new approach methods (NAMs) for assessing chemical hazard and risk has quickly grown. These methods are often cheaper and faster than traditional toxicity testing, and thus are amenable to high-throughput toxicity testing (HTT), resulting in large datasets. The ToxCast/Tox21 HTT programs have produced in vitro data for thousands of chemicals covering a large space of biological activity. The relevance of these data to in vivo mammalian toxicity has been much explored. Interest has also grown in using these data to evaluate the risk of environmental exposures to taxa of ecological importance such as fish, aquatic invertebrates, etc.; particularly for the purpose of estimating the risk of exposure from real-world complex mixtures. Understanding the relationship and relative sensitivity of NAMs versus standardized ecotoxicological whole organism models is a key component of performing reliable read-across from mammalian in vitro data to ecotoxicological in vivo data. In this work, we explore the relationship between in vivo ecotoxicity data from several publicly available databases and the ToxCast/Tox21 data. We also performed several case studies in which we compare how using different ecotoxicity datasets, whether traditional or ToxCast-based, affects risk conclusions based on exposure to complex mixtures derived from existing large-scale chemical monitoring data. Generally, predictive value of ToxCast data for traditional in vivo endpoints (EPs) was poor (r ≤ 0.3). Risk conclusions, including identification of different chemical risk drivers and prioritized monitoring sites, were different when using HTT data vs. traditional in vivo data.

Development of a Lymnaea stagnalis embryo bioassay for chemicals hazard assessment

The validation of high-throughput toxicity tests with invertebrate species is a key priority to improve hazard assessment of new chemicals and increase the available test guidelines with organisms from a representative set of taxa. This work aimed to contribute to the validation of an embryo test with the freshwater gastropod Lymnaea stagnalis, which has been identified by Organization for Economic Co-operation and Development (OECD) as a potential invertebrate test model, and provide the basis for such an endeavor. Recently, a L. stagnalis reproductive test was standardized by the OECD. However, to encompass the entire life cycle, it is crucial to addresses embryogenic development — a phase highly susceptible to various anthropogenic chemicals, which is covered in the proposed methodology. The approach used in the present study is in line with the OECD guidelines and other published studies, namely the Detailed Review Paper (DRP) on Mollusks life-cycle toxicity testing.Here, the assay quality criteria such as basal mortality and abnormality rates, development, growth and hatching rates, the appropriated testing media, and the optimal assay duration were investigated. Cadmium was chosen as the positive test substance, due to the available data and the verified model sensitivity to this compound, namely in the OECD reproductive test validation process.The obtained data demonstrate that L. stagnalis embryogenesis using the developed methodology is highly sensitive to cadmium. High concentration-response correlation was observed using this reference compound, the EC10 and EC50 for growth are 13.57 and 21.84 μg/L, respectively, after 168 h of exposure. The development EC's 10 and 50 were 15.75 and 38.66 μg/L, respectively, after 240 h. This demonstrates the model sensitivity to this compound when compared with other embryo test models, as well as the model sensitivity during the embryogenesis, if compared with the adult stage. Further, given the determined sensitivity parameters, and incubation times, the test can be performed at 240 h as over 95 % of the control embryos were hatched and no further significant changes in the exposure groups were determined.Overall, the findings of the present study demonstrate that the embryo test with L. stagnalis has potential to high-throughput testing and the model has a high sensitivity to cadmium during this life cycle period. The background data provide by this study will be essential to foster the future standardization of this assay.

Bringing CE-MS into the regulatory toxicology toolbox: Application to neuroinflammation screening

Despite its suitability to analyze polar metabolites using minute amounts of sample and its large peak capacity, CE-MS has traditionally been considered to lack the robustness required by untargeted metabolomics, especially in regulatory environments. This belief comes from the difficulty to adequately identify metabolites based on their migration times due to the variability of such parameter. In the present work, we demonstrate how this limitation can be circumvented by using standardized CE-MS conditions and automatically converting CE-MS files into electrophoretic mobility (μeff) scale. This strategy allows to conveniently exploit the advantages of CE-MS for low-volume samples generated during the toxicological risk assessment of potential neuroinflammatory substances, performed via the evaluation of astrocyte reaction. Human astrocyte cells were exposed to tumor necrosis factor alpha (TNFα) as a model compound and to digoxin at different concentrations as a tested chemical. The induced metabolic profiles were then characterized by means of CE-MS metabolomics and the success of the annotation step was evaluated and compared when one or two reference compounds were used as markers for the conversion into the effective electrophoretic mobility scale. The use of two markers resulted in a more reliable metabolite identification across all the conditions. As a result, a total of 68 anionic and cationic metabolites were annotated in both CE polarities. Unsupervised and supervised multivariate analysis enabled the comparison of the metabolomic profiles induced by each compound, highlighting common and differential metabolites, suggesting a similar but specific mechanism of activation for digoxin with regard to TNFα. CE-MS-based metabolomics is an advantageous tool for the analysis of minute amounts of samples delivered by new approach methodologies (NAMs) in chemical risk assessment, allowing high throughput toxicity screening.

Amniogenesis in Human Amniotic Sac Embryoids after Exposures to Organophosphate Flame Retardants.

Amniogenesis is a key event in biochemical pregnancy, and its failure may result in human embryonic death. However, whether and how environmental chemicals affect amniogenesis remain largely unknown. The objective of the present study was to screen chemicals that may disrupt amniogenesis in an amniotic sac embryoid model and to investigate the potential mechanism of amniogenesis failure, with a focus on organophosphate flame retardants (OPFRs). This study developed a high-throughput toxicity screening assay based on transcriptional activity of octamer-binding transcription factor 4 (Oct4). For the two positive OPFR hits with the strongest inhibitory activity, we used time-lapse and phase-contrast imaging to assess their effects on amniogenesis. Associated pathways were explored by RNA-sequencing and western blotting, and potential binding target protein was identified through a competitive binding experiment. Eight positive hits exhibiting Oct4 expression were identified, with 2-ethylhexyl-diphenyl phosphate (EHDPP) and isodecyl diphenyl phosphate (IDDPP) showing the strongest inhibitory activity. EHDPP and IDDPP were found to disrupt the rosette-like structure of the amniotic sac or inhibit its development. Functional markers of squamous amniotic ectoderm and inner cell mass were also found disrupted in the EHDPP- and IDDPP-exposed embryoids. Mechanistically, embryoids exposed to each chemical exhibited abnormal accumulation of phosphorylated nonmuscle myosin (p-MLC-II) and were able to bind to integrin (). The amniotic sac embryoid models suggested that OPFRs disrupted amniogenesis likely by inhibiting the pathway, thus providing direct in vitro evidence associating OPFRs with biochemical miscarriage. https://doi.org/10.1289/EHP11958.

Harnessing Human Pluripotent Stem Cell-Derived Pancreatic In Vitro Models for High-Throughput Toxicity Testing and Diabetes Drug Discovery.

The long-standing goals in diabetes research are to improve β-cell survival, functionality and increase β-cell mass. Current strategies to manage diabetes progression are still not ideal for sustained maintenance of normoglycemia, thereby increasing demand for the development of novel drugs. Available pancreatic cell lines, cadaveric islets, and their culture methods and formats, either 2D or 3D, allow for multiple avenues of experimental design to address diverse aims in the research setting. More specifically, these pancreatic cells have been employed in toxicity testing, diabetes drug screens, and with careful curation, can be optimized for use in efficient high-throughput screenings (HTS). This has since spearheaded the understanding of disease progression and related mechanisms, as well as the discovery of potential drug candidates which could be the cornerstone for diabetes treatment. This book chapter will touch on the pros and cons of the most widely used pancreatic cells, including the more recent human pluripotent stem cell-derived pancreatic cells, and HTS strategies (cell models, design, readouts) that can be used for the purpose of toxicity testing and diabetes drug discovery.

Targeted Per- and Polyfluoroalkyl substances (PFAS) assessments for high throughput screening: Analytical and testing considerations to inform a PFAS stock quality evaluation framework

Per- and polyfluoroalkyl substances (PFAS) represent a large chemical class lacking hazard, toxicokinetic, and exposure information. To accelerate PFAS hazard evaluation, new approach methodologies (NAMs) comprised of in vitro high-throughput toxicity screening, toxicokinetic data, and computational modeling are being employed in read across strategies to evaluate the larger PFAS landscape. A critical consideration to ensure robust evaluations is a parallel assessment of the quality of the screening stock solutions, where dimethyl sulfoxide (DMSO) is often the diluent of choice. Challenged by the lack of commercially available reference standards for many of the selected PFAS and reliance on mass spectrometry approaches for such an evaluation, we developed a high-throughput framework to evaluate the quality of screening stocks for 205 PFAS selected for these NAM efforts. Using mass spectrometry coupled with either liquid or gas chromatography, a quality scoring system was developed that incorporated observations during mass spectral examination to provide a simple pass or fail notation. Informational flags were used to further describe findings regarding parent analyte presence through accurate mass identification, evidence of contaminants and/or degradation, or further describe characteristics such as isomer presence. Across the PFAS-DMSO stocks tested, 148 unique PFAS received passing quality scores to allow for further in vitro testing whereas 57 received a failing score primarily due to detection issues or confounding effects of DMSO. Principle component analysis indicated vapor pressure and Henry's Law Constant as top indicators for a failed quality score for those analyzed by gas chromatography. Three PFAS in the hexafluoropropylene oxide family failed due to degradation in DMSO. As the PFAS evaluated spanned over 20 different structural categories, additional commentary describes analytical observations across specific groups related to PFAS stock composition, detection, stability, and methodologic considerations that will be useful for informing future analytical assessment and downstream HTS efforts. The high-throughput stock quality scoring workflow presented holds value as a tool to evaluate chemical presence and quality efficiently and for informing data inclusion in PFAS or other NAM screening efforts.

New Worm on the Block: Planarians in (Neuro)Toxicology.

Traditional mammalian testing is too time- and cost-intensive to keep up with the large number of environmental chemicals needing assessment. This has led to a dearth of information about the potential adverse effects of these chemicals, especially on the developing brain. Thus, there is an urgent need for rapid and cost-effective neurotoxicity and developmental neurotoxicity testing. Because of the complexity of the brain, metabolically competent organismal models are necessary to understand the effects of chemicals on nervous system development and function on a systems level. In this overview, we showcase asexual freshwater planarians as an alternative invertebrate ("non-animal") organismal model for neurotoxicology research. Planarians have long been used to study the effects of chemicals on regeneration and behavior. But they have only recently moved back into the spotlight because modern molecular and computational approaches now enable quantitative high-content and high-throughput toxicity studies. Here, we present a short history of the use of planarians in toxicology research, highlight current techniques to measure toxicity qualitatively and quantitatively in planarians, and discuss how to further promote this non-animal organismal system into mainstream toxicology research. The articles in this collection will help work towards this goal by providing detailed protocols that can be adopted by the community to standardize planarian toxicity testing. © 2022 Wiley Periodicals LLC.

Towards reproducible structure-based chemical categories for PFAS to inform and evaluate toxicity and toxicokinetic testing

Per- and Polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals that are in widespread use and present concerns for persistence, bioaccumulation and toxicity. Whilst a handful of PFAS have been characterised for their hazard profiles, the vast majority of PFAS have not been studied. The US Environmental Protection Agency (EPA) undertook a research project to screen ∼150 PFAS through an array of different in vitro high throughput toxicity and toxicokinetic tests in order to inform chemical category and read-across approaches. A previous publication described the rationale behind the selection of an initial set of 75 PFAS, whereas herein, we describe how various category approaches were applied and extended to inform the selection of a second set of 75 PFAS from our library of approximately 430 commercially procured PFAS. In particular, we focus on the challenges in grouping PFAS for prospective analysis and how we have sought to develop and apply objective structure-based categories to profile the testing library and other PFAS inventories. We additionally illustrate how these categories can be enriched with other information to facilitate read-across inferences once experimental data become available. The availability of flexible, objective, reproducible and chemically intuitive categories to explore PFAS constitutes an important step forward in prioritising PFAS for further testing and assessment.

Omics-based high throughput toxicity screening and risk assessment of pollutants

<p indent="0mm">Screening priority pollutants with high toxicity and risk potential from a large number of environmental chemical pollutants is the prerequisite for prevention and control of environmental risks. After entering the living system, toxic pollutants can induce adverse outcomes through a series of disturbed molecular targets and biological pathways. Existing high-throughput toxicity screening methods rely on few and isolated cellular or molecular endpoints, making it difficult to capture the key biological processes that cause toxicity and to determine the direct relationship between chemical pollutants and disease. Meanwhile, the awareness is increasing that we are exposed to a true cocktail of chemicals, among which only a fraction has been identified through analytical methods. Integrated bioanalytical approaches, such as effect-directed analysis and toxicity identification evaluation (TIE), have been used to identify risk drivers in complex mixture pollutants. However, it is still difficult to elucidate the molecular toxicological drivers of chemical stress in the environment. Toxicogenomics take advantage of genome-wide coverage, high throughput, and big data capacity that are associated with omics technologies to achieve technology innovation in screening and risk assessment of chemical pollutants. We reviewed the technical advantages and the recent progress of toxicogenomic methodology in high-throughput toxicological screening and risk assessment of chemical pollutants from four aspects with specific research cases. To identify the molecular events initiated by toxic pollutants, toxicogenomics could uncover a large number of molecular events and pathways potentially disturbed at the early development stage of toxicity. Dose-dependent transcriptomes for high-throughput chemical testing have been developed to quantitatively assess responses induced by chemical pollutants at the molecular level, which improve the sensitivity of identifying bioactivity by 1–3 orders of magnitude. To uncover the biological pathways of adverse health outcome by chemical pollutants, systemic toxicological assessment by omics strategies provide mechanistic information for chemical pollution screening and health risk assessment at multiple biological levels. Through biological pathway enrichment analysis, such as the gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway, the molecular mechanistic information of toxicity could be captured. Moreover, multi-omics integration allows for multi-scale, comprehensive biological network analysis, which provide a systematic understanding of toxicity pathways or adverse outcome pathways (AOPs) to guide ecological and human health risk assessments. For human diseases diagnostics, functional genomics provide a new tool to explore mechanisms of individual disease risk and susceptibility under toxic pollutants exposure. Variations of individual susceptibility to toxicant-induced disease are largely due to genetic susceptibility across different populations. The direct relationship among pollutants, genes and adverse outcomes could be established by combining functional genomics and molecular epidemiological approaches. Finally, for risk management of complex mixture pollutants, it is important to assess the overall toxicity effects of the pool of pollutants and identify the key toxic pollutant. The combination of high-throughput bioassays and chemical analysis can be used to identify the highly bioactive fraction and to screen the key chemical pollutant that pose ecological and health risks. Recently, progresses have been made in integrating transcriptomic techniques with molecular toxicity identification and evaluation (mTIE) to assess the overall effects of mixtures and distinguish the key pollutants in inducing the corresponding biological responses. Particularly, dose-response transcriptome could be used to quantitatively evaluate the overall toxic effects and to guide the key toxicant identification by high mechanistic resolution. In summary, the development of omics-based high-throughput toxicity screening technologies will strongly support accurate environmental risk assessment of conventional and new pollutants in the future.

Optimization of Three-Dimensional Culture Conditions of HepG2 Cells with Response Surface Methodology Based on the VitroGel System

ObjectiveThis study optimizes three-dimensional (3D) culture conditions of HepG2 using response surface methodology (RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues. MethodHepG2 cell was 3D cultured on the VitroGel system. Cell viability was detected using Cell Counting Kit-8 (CCK-8) assay of HepG2 lived cell numbers. The proliferation of HepG2 cell and clustering performance was measured via fluorescence staining test. Albumin concentration in the culture medium supernatant as an index of HepG2 cell biological function was measured with ELISA kit. Independent factor tests were conducted with three key factors: inoculated cell concentration, cultured time, and dilution degree of the hydrogel. The preliminary results of independent factor tests were used to determine the levels of factors for RSM. ResultThe selected optimal culture conditions are as follows: concentration of inoculated cells was 4.44 × 105/mL, culture time was 4.86 days, and hydrogel dilution degree was 1:2.23. The result shows that under optimal conditions, the predicted optical density (OD) value of cell viability was 3.10 and measured 2.978 with a relative error of 3.94%. ConclusionThis study serves as a reference for the 3D HepG2 culture and constructs liver tissues in vitro. Additionally, it provides the foundation for repeated dose high-throughput toxicity studies and other scientific research work.

A high-throughput microplate toxicity screening platform based on Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), an established model organism, has been widely used in environmental toxicology research. However, most of the current toxicity testing methods based on worms are time-consuming. In this study we aimed to develop an automated and highly-integrated platform for high-throughput and in situ toxicity testing. Considering the superiority of C. elegans as a neurotoxicological model, this platform mainly evaluates general toxicology and neurotoxicology endpoints, which are usually induced by metals and pesticides, the major environmental contaminants. Microplates were used as a worm culturing system, which have good compatibility with any commercial microplate applicable instruments. We developed a microfluidic-based module for worm dispensing, and an image acquisition/analysis module for monitoring worms and detecting toxicity endpoints in bright filed. These were collectively incorporated with a commercial pipetting workstation for automated food/drug delivery and a high-content analysis system for fluorescence detection. The integrated platform achieved an efficient on-demand worm dispensing, long-term maintenance, regular monitoring and imaging, survival assay and behavioral analyses, and visualized gene reporter assay. Moreover, “Lab on Web” was achieved by connecting the platform to the web for remote operation, worm monitoring, and phenotype calculation. To demonstrate the ability of the platform for automated toxicity testing assays; worms were treated with cadmium and longevity, neurotoxicity, developmental toxicity and gst-4 expression were evaluated. We determined its feasibility and proposed the potential application in high-throughput toxicity screening for environmental risk assessment in the nearest future.

High throughput embryonic zebrafish test with automated dechorionation to evaluate nanomaterial toxicity

Engineered nanomaterials pose occupational health and environmental concerns as they possess unique physical and chemical properties that can contribute to toxicity. High throughput toxicity screening methods are needed to address the increasing number of nanomaterials in production. Here we used a zebrafish photomotor response (PMR) test to evaluate a set of fifteen nanomaterials with military relevance. Automated dechorionation of zebrafish embryos was used to enhance nanomaterials bioavailability. Optimal PMR activity in zebrafish embryos was found at 30–31 hours post-fertilization (hpf). Behavioral and toxicological responses were measured at 30 and 120 hpf; behavioral responses were found for thirteen of the fifteen nanomaterials and acute toxicity (LC50) levels for nine of the fifteen nanomaterials below the maximum test concentration of 500 μg/ml. Physico-chemical characterization of the nanomaterials detected endotoxin and bacterial contamination in two of the tested samples, which may have contributed to observed toxicity and reinforces the need for physical and chemical characterization of nanomaterials use in toxicity testing. The zebrafish PMR test, together with automated dechorionation, provides an initial rapid assessment of the behavioral effects and toxicity of engineered nanomaterials that can be followed up by physico-chemical characterization if toxicity is detected, reducing the amount of time and monetary constraints of physico-chemical testing.

In vitro airway models from mice, rhesus macaques, and humans maintain species differences in xenobiotic metabolism and cellular responses to naphthalene.

The translational value of high-throughput toxicity testing will depend on pharmacokinetic validation. Yet, popular in vitro airway epithelia models were optimized for structure and mucociliary function without considering the bioactivation or detoxification capabilities of lung-specific enzymes. This study evaluated xenobiotic metabolism maintenance within differentiated air-liquid interface (ALI) airway epithelial cell cultures (human bronchial; human, rhesus, and mouse tracheal), isolated airway epithelial cells (human, rhesus, and mouse tracheal; rhesus bronchial), and ex vivo microdissected airways (rhesus and mouse) by measuring gene expression, glutathione content, and naphthalene metabolism. Glutathione levels and detoxification gene transcripts were measured after 1-h exposure to 80 µM naphthalene (a bioactivated toxicant) or reactive naphthoquinone metabolites. Glutathione and glutathione-related enzyme transcript levels were maintained in ALI cultures from all species relative to source tissues, while cytochrome P450 monooxygenase gene expression declined. Notable species differences among the models included a 40-fold lower total glutathione content for mouse ALI trachea cells relative to human and rhesus; a higher rate of naphthalene metabolism in mouse ALI cultures for naphthalene-glutathione formation (100-fold over rhesus) and naphthalene-dihydrodiol production (10-fold over human); and opposite effects of 1,2-naphthoquinone exposure in some models-glutathione was depleted in rhesus tissue but rose in mouse ALI samples. The responses of an immortalized bronchial cell line to naphthalene and naphthoquinones were inconsistent with those of human ALI cultures. These findings of preserved species differences and the altered balance of phase I and phase II xenobiotic metabolism among the characterized in vitro models should be considered for future pulmonary toxicity testing.

Scalable expansion of iPSC and their derivatives across multiple lineages

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for “ready-to-use” cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools.

A High-Throughput Toxicity Screen of 42 Per- and Polyfluoroalkyl Substances (PFAS) and Functional Assessment of Migration and Gene Expression in Human Placental Trophoblast Cells.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants that have been associated with adverse pregnancy outcomes in women and experimental research models. Adverse developmental and reproductive outcomes have been investigated for relatively few PFAS, and such studies are not scalable to address the thousands of unique chemical structures. As the placenta has been reported as a PFAS target tissue, the human placental trophoblast JEG-3 cell line was employed in a high-throughput toxicity screen (HTTS) to evaluate the effects of 42 unique PFAS on viability, proliferation, and mitochondrial membrane potential (MMP). HTTS concentration-response curve fitting determined EC50 values for 79% of tested compounds for at least one of the three endpoints. Trophoblast migratory potential was evaluated for a subset of six prioritized PFAS using a scratch wound assay. Migration, measured as the percent of wound closure after 72 h, was most severely inhibited by exposure to 100 µM perfluorooctanoic acid (PFOA; 72% closure), perfluorooctanesulfonic acid (PFOS; 57% closure), or ammonium perfluoro-2-methyl-3-oxahexanoate (GenX; 79% closure). PFOA and GenX were subsequently evaluated for disrupted expression of 46 genes reported to be vital to trophoblast health. Disrupted regulation of oxidative stress was suggested by altered expression of GPEX1 (300 µM GenX and 3 µM GenX), GPER1 (300 µM GenX), and SOD1 and altered cellular response to xenobiotic stress was indicated by upregulation of the placental efflux transporter, ABCG2 (300 µM GenX, 3 µM GenX, and 100 µM PFOA). These findings suggest the placenta is potentially a direct target of PFAS exposure and indicate that trophoblast cell gene expression and function are disrupted at PFAS levels well below the calculated cytotoxicity threshold (EC50). Future work is needed to determine the mechanism(s) of action of PFAS towards placental trophoblasts.

Population-based high-throughput toxicity screen of human iPSC-derived cardiomyocytes and neurons.

In this study, we establish a population-based human induced pluripotent stem cell (hiPSC) drug screening platform for toxicity assessment. After recruiting 1,000 healthy donors and screening for high-frequency human leukocyte antigen (HLA) haplotypes, we identify 13 HLA-homozygous "super donors" to represent the population. These "super donors" are also expected to represent at least 477,611,135 of the global population. By differentiating these representative hiPSCs into cardiomyocytes and neurons we show their utility in a high-throughput toxicity screen. To validate hit compounds, we demonstrate dose-dependent toxicity of the hit compounds and assess functional modulation. We also show reproducible invivo drug toxicity results using mouse models with select hit compounds. This study shows the feasibility of using a population-based hiPSC drug screening platform to assess cytotoxicity, which can be used as an innovative tool to study inter-population differences in drug toxicity and adverse drug reactions in drug discovery applications.

Risk-Based Prioritization of Organic Chemicals and Locations of Ecological Concern in Sediment From Great Lakes Tributaries.

With improved analytical techniques, environmental monitoring studies are increasingly able to report the occurrence of tens or hundreds of chemicals per site, making it difficult to identify the most relevant chemicals from a biological standpoint. For the present study, organic chemical occurrence was examined, individually and as mixtures, in the context of potential biological effects. Sediment was collected at 71 Great Lakes (USA/Canada) tributary sites and analyzed for 87 chemicals. Multiple risk‐based lines of evidence were used to prioritize chemicals and locations, including comparing sediment concentrations and estimated porewater concentrations with established whole‐organism benchmarks (i.e., sediment and water quality criteria and screening values) and with high‐throughput toxicity screening data from the US Environmental Protection Agency's ToxCast database, estimating additive effects of chemical mixtures on common ToxCast endpoints, and estimating toxic equivalencies for mixtures of alkylphenols and polycyclic aromatic hydrocarbons (PAHs). This multiple‐lines‐of‐evidence approach enabled the screening of more chemicals, mitigated the uncertainties of individual approaches, and strengthened common conclusions. Collectively, at least one benchmark/screening value was exceeded for 54 of the 87 chemicals, with exceedances observed at all 71 of the monitoring sites. Chemicals with the greatest potential for biological effects, both individually and as mixture components, were bisphenol A, 4‐nonylphenol, indole, carbazole, and several PAHs. Potential adverse outcomes based on ToxCast gene targets and putative adverse outcome pathways relevant to individual chemicals and chemical mixtures included tumors, skewed sex ratios, reproductive dysfunction, hepatic steatosis, and early mortality, among others. The results provide a screening‐level prioritization of chemicals with the greatest potential for adverse biological effects and an indication of sites where they are most likely to occur. Environ Toxicol Chem 2022;41:1016–1041. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A High-Throughput Toxicity Screen of 42 Per- and Polyfluoroalkyl Substances (PFAS) and Functional Assessment of Migration and Gene Expression in Human Placental Trophoblast Cells

A High-Throughput Toxicity Screen of 42 Per- and Polyfluoroalkyl Substances (PFAS) and Functional Assessment of Migration and Gene Expression in Human Placental Trophoblast Cells

Accounting for Artifacts in High-Throughput Toxicity Assays.

Compound activity identification is the primary goal in high throughput screening (HTS) assays. However, assay artifacts including both systematic (e.g., compound autofluorescence) and nonsystematic (e.g., noise) complicate activity interpretation. In addition, other than the traditional potency parameter, half-maximal effect concentration [EC50], additional activity parameters (e.g., point-of-departure [POD] and weighted area-under-the-curve [wAUC]) could be derived from HTS data for activity profiling. A data analysis pipeline has been developed to handle the artifacts, and to provide compound activity characterization with either binary or continuous metrics. This chapter outlines the steps in the pipeline using Tox21 estrogen receptor (ER) β-lactamase assays, including the formats to identify either agonists or antagonists, as well as the counterscreen assays for identifying artifacts as examples. The steps can be applied to other lower throughput assays with concentration-response data.

Exploring Potential Carcinogenic Activity of Per- and Polyfluorinated Alkyl Substances Utilizing High-Throughput Toxicity Screening Data.

Per- and polyfluorinated alkyl substances (PFAS) are ubiquitous, persistent, and toxic chemicals that pose public health risks. Recent carcinogenicity concerns have arisen based on epidemiological studies, animal tumor findings, and mechanistic data. Thousands of PFAS exist; however, current understanding of their toxicity is informed by studies of a select few, namely, perfluorooctanoic acid and perfluorooctanesulfonic acid. Hence, the computational, high-throughput screening tool, the US EPA CompTox Chemical Dashboard's ToxCast, was utilized to explore the carcinogenicity potential of PFAS. Twenty-three major PFAS that had sufficient in vitro ToxCast data and covered a range of structural subclasses were analyzed with the visual analytics software ToxPi, yielding a qualitative and quantitative assessment of PFAS activity in realms closely linked with carcinogenicity. A comprehensive literature search was also conducted to check the consistency of analyses with other mechanistic data streams. The PFAS were found to induce a vast range of biological perturbations, in line with several of the International Agency for Research on Cancer-defined key carcinogen characteristics. Patterns observed varied by length of fluorine-bonded chains and/or functional group within and between each key characteristic, suggesting some structure-based variability in activity. In general, the major conclusions drawn from the analysis, that is, the most notable activities being modulation of receptor-mediated effects and induction of oxidative stress, were supported by literature findings. The study helps enhance understanding of the mechanistic pathways that underlie the potential carcinogenicity of various PFAS and hence could assist in hazard identification and risk assessment for this emerging and relevant class of environmental toxicants.