Toxicological Sciences最新文献

The EPA continues to evaluate strategies to implement new approach methods (NAMs) for screening chemicals that disrupt the thyroid endocrine system. Validation of NAMs is a critical milestone toward establishing confidence in data sources that could be used in a regulatory decision-making context. The objective of this study was to conduct an interlaboratory validation of the human thyroid microtissue assay to evaluate its relevance and reliability. In coordination with the U.S. validation authority, NICEATM, and collaboration with industry partners (LifeNet Health, Bayer Crop Science, Corteva Agrisciences), the study aims were to 1) define the study design and establish standard operating procedures, 2) conduct test method transfer, training, and within-laboratory model performance evaluation, 3) perform interlaboratory reference chemical testing and assay performance evaluation. Progress was independently monitored by a validation management team comprised of an international group of experts in thyroid physiology, in vitro test methods, and regulatory toxicology. Results indicated the thyroid microtissue model could be reliably transferred to new laboratories with reproducible effects on thyroid hormone synthesis. Interlaboratory testing of four blinded reference chemicals (three true positive, one true negative) across three independent human donors revealed consistent bioactivity across the reference set and performance metrics (dynamic range, precision, screening quality) that met acceptance criteria and shed insight into areas for improvement. In the context of a NAM-based testing strategy, a validated human thyroid microtissue assay enables direct measurement of thyroid hormone synthesis perturbations, reducing reliance on animal testing and addressing a critical mode-of-action that is of regulatory concern.

Chronic liver disease (CLD) is a global health concern that progresses to liver cirrhosis and cancer. This progression can be partly replicated in rodents through experimental administration of thioacetamide (TAA). Hepatic iron accumulation is a relatively common finding in CLD patients, as excess intracellular iron promotes the progression of CLD by generating reactive oxygen species. We previously reported that dietary iron overload abrogates TAA-induced liver cirrhosis in rats, raising a possibility that hepatic iron accumulation exerts a cytoprotective function. Here we investigated the role of Kelch-like ECH-associated protein 1-NF-E2-related factor 2 (Keap1-Nrf2) system in the protective effects of iron overload in TAA-induced chronic liver injury. The suppression of TAA-induced liver cirrhosis by dietary iron overload, demonstrated in wild-type rats, was cancelled in Nrf2 knockout (KO) rats, suggesting that Nrf2 contributes to the protective effect. In wild-type rats treated with both TAA and iron, major Nrf2-target gene products, NAD(P)H quinone dehydrogenase 1 and placental glutathione S-transferase (GSTP), were specifically overexpressed in hepatocytes around the fibrotic lesions. This overexpression was accompanied by iron accumulation and expression of cytochrome P450 2E1, which converts TAA into its toxic metabolites. In addition, wild-type rats treated with TAA alone developed multiple GSTP-positive preneoplastic foci, characterized by strong activation of Nrf2, partially involved by p62-dependent selective autophagy. GSTP expression was absent in hepatocytes of Nrf2 KO rats. These results suggest that Nrf2 protects liver cirrhosis and promotes formation of preneoplastic hepatocellular nodules during TAA-induced chronic liver injury depending on the hepatic iron condition.

Next Generation Risk Assessment (NGRA) aims to improve safety testing of pharmaceuticals, agrochemicals, and industrial chemicals. NGRA employs new approach methodologies, such as novel in vitro assays coupled with exposure modeling, to minimize the use of animal models, which can fail to predict specific biological effects in humans. The strategy of the 'Omics for Assessing Signatures for Integrated Safety (OASIS) Consortium combines multi-omics technologies (including transcriptomics, proteomics, and Cell Painting [high-content imaging]) and multiple cell model systems (ranging from simple cell cultures to complex organotypic models). By integrating these approaches with internal exposure estimates, the consortium aims to improve the translation between in vitro and in vivo test systems, ultimately enhancing the relevance of safety assessment to human biology. OASIS's integrated approach aims to better translate the biological effects across different chemical and biological spaces, starting with the liver as a use case. By using compounds with well-characterized in vivo and in vitro nonclinical safety and toxicology data related to adverse organ-specific effects in rats and humans, OASIS aims to create novel integrated methods that improve safety assessment while reducing animal use. Ideally, these efforts will contribute to regulatory science across sectors and support the adoption of more predictive, efficient, and cost-effective toxicological models.

Accurate assessment of cardiotoxicity using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes is critical for ensuring drug safety during preclinical development. However, existing in vitro methodologies predominantly focus on QT interval prolongation and arrhythmia risk, often lacking the capacity to capture the complex interplay among multiple ion channels or to detect early manifestations of chronic cardiotoxicity-both of which are essential for evaluating long-term cardiac safety. Moreover, reliable prediction of pharmacological mechanisms of action remains a significant challenge. In this study, we employed field potential imaging utilizing an ultra-high-density complementary metal-oxide-semiconductor microelectrode array (MEA) comprising 236,880 electrodes distributed across a 5.9 × 5.5 mm active area. With 91.9% surface coverage by 11 μm electrodes spaced at 0.25 μm, the platform achieves near single-cell resolution across the entire cardiomyocyte monolayer. This system enabled the extraction of high-resolution electrophysiological endpoints, including the number and spatial variability of excitation origins, conduction velocity, and propagation area-thereby extending the analytical capabilities beyond those of conventional MEAs. Pharmacological testing revealed compound-specific alterations: Isoproterenol increased excitation origins, mexiletine reduced conduction velocity, and E-4031 diminished propagation area. Although these agents are well characterized, their effects were visualized with unprecedented spatiotemporal resolution, reflecting their underlying mechanisms of action. Multivariate analysis incorporating both conventional and novel endpoints enabled accurate classification of mechanisms under acute conditions. Furthermore, chronic cardiotoxicity induced by low-dose doxorubicin (0.03 μM) was sensitively detected within 24 h-earlier and at lower concentrations than previously reported-based on significant reductions in conduction velocity and propagation area. Collectively, these findings establish a high-resolution, mechanism-aware framework for in vitro cardiotoxicity profiling, offering improved predictive accuracy by capturing multi-ion channel interactions, spatial conduction abnormalities, and early signs of chronic dysfunction.

The large number and diversity of chemicals currently in use present significant challenges in assessing their human and environmental health risks due to a paucity of toxicological data. To address this shortage, high-throughput screening technologies are used to rapidly evaluate the toxicity of these chemicals. Suitable chemical libraries are crucial to evaluate the performance of these technologies and generate the cognate toxicity data. Unlike traditional chemical libraries designed for specific disease targets or receptor interactions, the PrecisionTox collection prioritizes diversity in targets and mechanisms of toxicity to ensure broad applicability in toxicity predictions to test the concept of phylotoxicology. Phylotoxicology proposes that mechanisms of toxicity are evolutionarily conserved among distantly related species. Furthermore, the application of phylotoxicology can contribute to the reduction of mammalian species in toxicity testing. Here, an approach for generating a chemical library based on chemical properties-physicochemical, biomolecular, and toxicological-as well as practical considerations, including compound availability, cost, purity, and shipping regulations, is reported. From an initial pool of over 1,500 nominees, a set of 200 chemicals was selected based on multiple criteria, including organ toxicity, environmental exposure, structure, modes of action, and toxicological relevance. Additionally, information on baseline toxicity, Absorption, Distribution, Metabolism, and Excretion properties and utility for in vitro testing was collected. This work underscores the necessity of thoughtful chemical selection to refine toxicological models, improve hazard identification, and support regulatory efforts to protect human and environmental health.

Nickel-compound engineered nanomaterials (Ni-X NP) have diverse applications, yet their continued use raises concerns for potential health impacts upon exposure. This study investigated 3 structurally distinct Ni-X-NP-pure NiO (NCZ), NiO@Ni(OH)2 (SIG), and Ni@NiO@Ni(OH)2 (AA)-to determine how core composition and surface functionalization contribute to bioactivity. Each Ni-X NP was modified with surface moieties (-OH, -COOH, and -CH3) to assess the efficacy of surface modifications in reducing bioactivity. Ni-X NP were thoroughly characterized for structure, surface chemistry, and Ni2+ ion release in simulated lysosomal fluid. Red blood cells (RBCs) were used to evaluate the hemolytic capabilities of the nanoparticles, and primary murine alveolar macrophages (AM), and murine ex vivo alveolar macrophages (mexAM) were used to assess uptake, cytotoxicity, IL-1β release, and lysosomal membrane permeability (LMP). Results showed that NiO@Ni(OH)2 nanoparticles induced the greatest hemolysis in RBC, elicited the greatest IL-1β response in AM and mexAM, and produced the most LMP in mexAM. The Ni@NiO@Ni(OH)2 nanoparticle released the most Ni2+ and caused profound reductions in AM cell viability but failed to cause RBC hemolysis or LMP. Pure NiO nanoparticles exhibited minimal bioactivity and low Ni2+ release. Surface modification with (-COOH) or (-CH3) effectively reduced bioactivity in LMP-mediated inflammation but had minimal effect on Ni2+-driven toxicity. This study reveals that Ni-X NP bioactivity depends on both core composition and surface chemistry, and that surface functionalization reduces inflammation only when lysosomal damage is the primary driver. These findings underscore the need for careful design and evaluation of engineered nanomaterials.

Standard in vitro genotoxicity assays often suffer from low specificity, leading to irrelevant positive findings that require costly in vivo follow-up studies. The TGx-DDI (Toxicogenomic DNA Damage-Inducing) transcriptomic biomarker was developed to address this limitation by identifying DNA damage-inducing compounds through gene expression profiling in human TK6 lymphoblastoid cells. To qualify TGx-DDI as a reliable, reproducible biomarker for augmenting genotoxicity hazard assessment, a multi-site ring-trial was conducted across four laboratories using 14 blinded test compounds and standardized protocols. TK6 cells were exposed to three concentrations of each compound, followed by RNA extraction and digital nucleic acid counting using the NanoString nCounter platform. A three-pronged bioinformatics approach-Nearest Shrunken Centroid Probability Analysis, Principal Component Analysis, and Hierarchical Clustering-was used to assign DDI or non-DDI classifications. TGx-DDI demonstrated 100% sensitivity, 86% specificity, and 91% accuracy in distinguishing DDI from non-DDI compounds under validated test conditions. High interlaboratory concordance was observed (agreement coefficients ≥0.61), and transcriptomic data showed strong cross-site correlation (Pearson r > 0.84). The biomarker reproducibly classified test agents even when conducted across study sites. These results demonstrate that TGx-DDI is a robust and reproducible transcriptomic biomarker that enhances the specificity of genotoxicity testing by distinguishing biologically relevant DNA damage responses. Its integration into genotoxicity testing strategies can support regulatory decision-making, reduce unnecessary animal use, and improve the assessment of human health risks.

Environmental exposure to industrial chemicals, endocrine disruptors, and pharmaceuticals has been increasingly linked to the global decline in male reproductive health. To address the urgent need for efficient and mechanistically informed toxicity screening, we developed a high-throughput screening, high-content analysis (HCA) platform using a 3D in vitro mini-testis model. This system was used to evaluate 87 structurally diverse compounds from the National Toxicology Program chemical library. The model incorporates murine-derived spermatogonia, Sertoli, and Leydig cells embedded in an extracellular matrix, providing a physiologically relevant environment for mechanistic toxicology. Each compound was tested across 10 phenotypic endpoints, including nuclear morphology, cytoskeletal integrity (F-actin), DNA damage (γH2AX), and cell viability by using high-content imaging. Quantitative Points of Departure (PODs) were calculated and integrated into a High-Content Assay Index. Toxicological Priority Index (ToxPi) scores, derived from the PODs, enabled compound ranking and clustering. Compared with existing in vivo reproductive toxicity data, the 3D model demonstrated 91.5% sensitivity, 93.8% specificity, and 93.6% concordance (n = 64 compounds). Notably, 22 compounds lacking reproductive toxicity data were identified as potentially reproductive toxicants. Mechanistic analyses revealed that nuclear morphology, F-actin intensity, and γH2AX were the most sensitive indicators of reproductive toxicity. Cluster and category-level analysis showed that flame retardants and pesticides ranked highest in toxicity. The integration of multi-parametric data via ToxPi facilitated high-resolution chemical prioritization. Given current ethical and technical challenges in sourcing human testicular tissue or differentiating stem cells into testicular cell types, murine cells provide a reproducible and practical alternative for complex multicellular testis modeling. Our results demonstrate that the HCA-integrated 3D mini-testis model offers a robust, scalable, and mechanistically insightful platform for male reproductive toxicity screening, supporting its adoption as New Approach Methodologies aligned with regulatory and ethical testing goals.

Historic animal-based toxicity testing methods cannot keep pace with the need for prioritizing new and existing chemicals for comprehensive risk assessment. New approach methodologies such as high-throughput in vitro transcriptomics screening have emerged to address this challenge. However, most in vitro methods were developed using mammalian cell lines, including human, and may not adequately represent environmental species, potentially limiting the utility of this methodology for supporting environmental risk assessment. The objective of this study was to evaluate whether zebrafish cell lines can generate biologically meaningful chemical effects data in a high-throughput transcriptomics pipeline that is protective of toxicologically relevant aquatic apical endpoints. Forty-two test chemicals were screened in 2 commercially available zebrafish cell lines (ZFL liver and ZEM2S embryonic fibroblast) using the TempO-Seq zS1500+ platform. Transcriptomic points-of-departure (tPODs) were derived using 2 methods: Gene-level analysis (tPODgenes) with BMDExpress software and biological pathway-altering concentrations (BPACs/tPODsignatures) from signature-based dose-response analysis. When converted to predicted external water concentrations using quantitative in vitro-in vivo extrapolation models, tPODs were generally protective of aquatic in vivo endpoints from the ECOTOX Knowledgebase. Differential gene expression and biological pathway analysis revealed potential cell-type-specific effects for several chemicals, highlighting the value of using multiple cell types for capturing tissue-specific responses. Lastly, the biological pathway information was used to extrapolate the chemical effects data across species through an integration of protein-protein interaction network analysis and the Sequence Alignment to Predict Across Species Susceptibility tool, which has significant implications for improving the ecological relevance of these methods.