To Örebro University

Titanium dioxide nanoparticles (TiO₂NPs) are widely produced engineered nanomaterials with ongoing human exposure through consumer and occupational uses. Conventional in vitro assays often focus on cytotoxicity and may therefore overlook early or sublethal cellular perturbations. Here, we applied Cell Painting-based phenomics to resolve size-dependent sub-lethal phenotypic signatures of TiO2NP exposure in human HepG2 hepatocytes. Two TiO2NPs (<25 nm and <100 nm) were characterized by field emission scanning electron microscopy and evaluated following 24-hour exposure at five concentrations: 6.25, 12.5, 25, 50, and 100 µg/mL. Cell viability was assessed using the alamarBlue assay, and high-dimensional phenotypic profiles were generated using Cell Painting-based phenomics, including automated high-content imaging and CellProfiler-based feature extraction. TiO2NP exposure induced modest reductions in viability at the highest concentration, indicating limited acute cytotoxicity. In contrast, phenomic profiling revealed clear, concentration-dependent phenotypic perturbations for both size fractions, with markedly stronger and more consistent effects for the < 100 nm TiO2NPs. At 100 µg/mL, the < 100 nm TiO2NPs altered 50.9% of the measured phenotypic features, compared with 28.9% for the < 25 nm particles, with prominent contributions from endoplasmic reticulum-, actin/Golgi/plasma membrane-, mitochondria-, and RNA-associated features. Dimensionality reduction and correlation analyses confirmed reproducible, concentration-dependent phenotypic trajectories. Importantly, the TiO2NP-induced phenotypes were distinct from those induced by the reference chemical CA-074Me, which produced broad perturbations and served as a reference chemical to verify assay sensitivity and dynamic range. Overall, Cell Painting phenomics sensitively captures size-dependent, sublethal cellular phenotypes induced by TiO2NPs, supporting its value as a New Approach Methodology for nanosafety assessment beyond conventional viability endpoints.

Environmental pollutants often induce morphological alterations in developing organisms, yet assessments are commonly subjective, limiting reproducibility and sensitivity. We developed and validated a semi-automated brightfield high-content imaging (HCI) pipeline to quantitatively detect morphological changes in zebrafish embryos. Using FishInspector software, we adapted image analysis for microscopy systems without automated embryo positioning, extending applicability across standard laboratory setups.To validate the approach, zebrafish embryos were exposed for 96 hours to two previously characterized pollutant mixtures (PFOS + PCB126; PFOS + B[a]P + arsenate) known to cause developmental effects. The pipeline sensitively quantified phenotypes including reduced swim bladder and shortened body length. These endpoints reflect developmental delay, highlighting the method's ability to capture mechanistically relevant effects. Such changes may reduce physiological performance and behavior, ultimately impacting fish populations.While earlier subjective scoring identified some similar alterations, our findings underscore the advantages of quantitative, semi-automated morphology assessment. The method improves reproducibility, enables standardized comparisons across studies, and increases sensitivity to detecting subtle morphological effects. By integrating brightfield imaging with semi-automated analysis, this approach broadens the toxicological toolbox for developmental hazard assessment and mixture toxicity research.

As the volume of plastic waste from electrical and electronic equipment (WEEE) continues to rise, a significant portion is disposed of in the environment, with only a small fraction being recycled. Both disposal and recycling pose unknown health risks that require immediate attention. Existing knowledge of WEEE plastic toxicity is limited and mostly relies on epidemiological data and association studies, with few insights into the underlying toxicity mechanisms. Therefore, this study aimed to perform comprehensive chemical screening and mechanistic toxicological assessment of WEEE plastic-associated chemicals. Chemical analysis, utilizing suspect screening based on high-resolution mass spectrometry, along with quantitative target chemical analysis, unveiled numerous hazardous compounds including polyaromatic compounds, organophosphate flame retardants, phthalates, benzotriazoles, etc. Toxicity endpoints included perturbation of morphological phenotypes using the Cell Painting approach, inflammatory response, oxidative stress, and endocrine disruption. Results demonstrated that WEEE plastic chemicals altered the phenotypes of the cytoskeleton, endoplasmic reticulum, and mitochondria in a dose-dependent manner. In addition, WEEE chemicals induced inflammatory responses in resting macrophages and altered inflammatory responses in lipopolysaccharide-primed macrophages. Furthermore, WEEE chemicals activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, indicating oxidative stress, and the aryl hydrocarbon receptor (AhR). Endocrine disruption was also observed through the activation of estrogenic receptor-α (ER-α) and the induction of anti-androgenic activity. The findings show that WEEE plastic-associated chemicals exert effects in multiple subcellular sites, via different receptors and mechanisms. Thus, an integrated approach employing both chemical and toxicological methods is essential for comprehensive assessment of the toxicity mechanisms and cumulative chemical burden of WEEE plastic-associated chemicals.

Background: Poly- and perfluoroalkyl substances (PFAS) are persistent pollutants affecting wildlife and biodiversity. Perfluorooctane sulfonic acid (PFOS) and one of its short-chain substitutes, perfluorobutane sulfonic acid (PFBS), are widely found in environmental components, especially in water. PFOS has been highlighted as causing deleterious effects on various organisms while PFBS adversity is suspected but requires further investigation. In this study, zebrafish embryos were exposed from 2 h post-fertilization to 28 days post-fertilization to two different concentrations (0.2 mu g/L and 2 mu g/L) of PFOS or PFBS. We then investigated the impacts of these early exposures later in life on adult fish fitness, growth, morphology, behaviour, and liver lipidomic profiles.

Results: PFOS exposure significantly reduced egg production, and both PFOS and PFBS altered growth patterns, organ development, and anxiety-like behaviour. Lipidomic analyses revealed persistent shifts in liver lipid composition that correspond to these phenotypic changes.

Conclusions: Taken together, our findings indicate that early-life exposure to low levels of PFOS and PFBS leads to long-term, sex-specific impairments in zebrafish physiology and behaviour, with disruptions in lipid metabolism emerging as a potential underlying mechanism.

Existing research has proven difficult to understand the interplay between upstream signalinge vents during NLRP3 inflammasome activation. Additionally, events downstream of inflammasome complex formation such as cytokine release and pyroptosis can exhibit variation, further complicating matters. Cell Painting has emerged as a prominent tool for unbiased evaluation of the effect of perturbations on cell morphological phenotypes. Using this technique, phenotypic fingerprints can be generated that reveal connections between phenotypes and possible modes of action. To the best of our knowledge, this was the first study that utilized Cell Painting on human THP-1 macrophages to generate phenotypic fingerprints in response to different endogenous and exogenous NLRP3 inflammasome triggers, and to identify phenotypic features specific to NLRP3 inflammasome complex formation. Our results demonstrated that not only can Cell Painting generate morphological fingerprints that are NLRP3 trigger-specific, but it can identify cellular fingerprints associated with NLRP3 inflammasome activation.

Per- and polyfluoroalkyl substances and nanoplastics frequently co-occur in environmental matrices, yet the effects of co-exposure on cellular responses upon ingestion are poorly understood. Here, we exposed human intestinal Caco-2 cells to perfluorooctanesulfonic acid, nanoplastics, and their combination. Cell painting-based phenomics was used to map phenotypic alterations across subcellular structures, and untargeted metabolomics using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was employed to assess metabolic changes. Results show that perfluorooctanesulfonic acid predominantly affected the actin cytoskeleton, Golgi apparatus, and plasma membrane, while nanoplastics primarily targeted mitochondria. Combined exposure disrupted the endoplasmic reticulum, RNA, and mitochondria. Perfluorooctanesulfonic acid reduced levels of carnitines, free fatty acids, nucleotides, and sugars, whereas nanoplastics inhibited ceramides, triglycerides, sphingomyelins, and additional free fatty acids. Combined exposure produced a metabolic profile resembling that of nanoplastics, with specific differences attributed to perfluorooctanesulfonic acid. Overall, nanoplastics appear as the main drivers of the co-exposure effects.

INTRODUCTION: Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that interferes with multiple biological pathways, with lipid metabolism being particularly vulnerable. Early-life exposure may disrupt hepatic function during development, but the underlying mechanisms are not fully understood.

OBJECTIVES: This study investigated how in ovo exposure to PFOA affects hepatic metabolism in the developing chicken embryo, with a focus on identifying altered metabolic pathways and potential mediators of toxicity.

METHODS: Fertilized chicken eggs (Gallus gallus domesticus) were exposed in ovo to six concentrations of PFOA (0-5 µg/g egg). Embryonic liver tissues were analysed by comprehensive metabolomic profiling using two complementary ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) platforms.

RESULTS: We identified 499 metabolites, including lipids, bile acids, carboxylic acids, amino acids, and phenolic compounds. PFOA exposure caused dose-dependent disturbances in lipid, bile acid, and amino acid metabolism. Notably, multiple secondary bile acids were detected and found to be strongly affected by PFOA, suggesting a central role of bile acid modulation in mediating its effects.

CONCLUSIONS: In ovo exposure to PFOA disrupts hepatic metabolism in developing chicken embryos, particularly through alterations in bile acid, lipid, and amino acid pathways. These metabolic changes may impair energy production, endocrine regulation, and organ development, with possible long-term health consequences.

Climate change is driving extreme weather patterns, leading to prolonged droughts and more frequent intense precipitation events. These environmental changes will impact aquatic systems by altering essential water quality parameters such as temperature, redox potential, pH, suspended solids and organic matter, which influence pollutant solubility and determine ecosystem health as well as drinking water production. In this context, sediments play a crucial role as they represent both a sink and source of pollutants. Therefore, sediment toxicity testing is essential for accurate environmental risk assessments. However, there remains a gap regarding comprehensive sediment testing ap-proaches that integrate multiple biomarker responses.

The SEASON project uses an interdisciplinary approach combining strategies of environmental toxicology, analytical chemistry, andhydro geochemistry. The aim is to develop conceptual models for evaluating, understanding and predicting the impact of climate change effects on the fate, bioavailability, and toxicity of pollutants in the aquatic environment. This project focuses on risks associated with sediments contaminated by organic and inorganic pollutants, specifically metals and PFAS (per- and polyfluoroalkyl substances). By studying factors such as temperature, pH, microbial communities, and sediment-water interactions, the project seeks to understand how different climate change aspects affect pollutant behavior in aquatic ecosystems.

The project consists of four sub-projects. Three investigate different chemical groups and mixtures under varying water conditions, using zebrafish (Danio rerio) as the main model organism. These studies will include in vitro and in vivo assays, microcosm experiments, microbiome studies and chemical analyses. The fourth subproject will integrate the results to develop a predictive model for sediment risk assessment.

Sediment contact assays will be performed to evaluate the effects of contaminated samples on zebrafish embryos by measuring teratogenicity, developmental toxicity, behavioral changes, and gene expression. In microcosm studies, we will vary pH and mimic increased precipitation events to assess pollutant toxicity in adult zebrafish including sex-related toxicity differences, reproduction, and behavior. Effect-directed analysis (EDA) will be used to identify key toxicants in the samples. Microbiome analysis using metagenomic sequencing will focus on how contaminated sediments alter bacterial communities, which in turn can affect pollutant distribution and bioavailability. Chemical analyses will quantify PFAS, metals, and their speciation throughout the study. Ultimately, the project will integrate these data to develop models that increase our understanding of the impact of climate change on sediment contamination and aquatic ecosystem health.

This study presents a comprehensive chemical and toxicological screening of chemicals extracted from WEEE (waste from electrical and electronic equipment) plastics. Chemical identification was conducted through suspect and target screening methods, revealing a diverse array of hazardous compounds including polycyclic aromatic compounds (PACs), organophosphate flame retardants (OPFRs), phthalates, benzotriazoles, and others. Toxicological endpoints included cell morphological phenotypes, inflammatory response, aryl hydrocarbon receptor (AhR) activation, activation of estrogenic receptor, and anti-androgenic activity. Results demonstrated that WEEE plastic chemicals significantly altered cell morphological phenotypes, particularly affecting the cytoskeleton, endoplasmic reticulum (ER), and mitochondrial measures. Moreover, WEEE chemicals induced inflammatory responses in resting human macrophages and altered ongoing inflammatory responses in lipopolysaccharide (LPS)-primed macrophages. Furthermore, WEEE chemicals exhibited potent AhR agonistic activity, activated estrogen receptor-α (ERα), and inhibited androgen receptor (AR) activation. The findings suggest that WEEE plastic chemicals exert their effects through multiple modes of action, targeting various subcellular sites. Thus, a combined approach utilizing non-target and target screening tools is essential for comprehensively assessing the toxic effects and health hazards associated with WEEE plastic chemicals.

Due to their exceptional properties and cost effectiveness, polyamides or nylons have emerged as widely used materials, revolutionizing diverse industries, including industrial 3D printing or additive manufacturing (AM). Powder-based AM technologies employ tonnes of polyamide microplastics to produce complex components every year. However, the lack of comprehensive toxicity assessment of particulate polyamides and polyamide-associated chemicals, especially in the light of the global microplastics crisis, calls for urgent action. This study investigated the physicochemical properties of polyamide-12 microplastics used in AM, and assessed a number of toxicity endpoints focusing on inflammation, immunometabolism, genotoxicity, aryl hydrocarbon receptor (AhR) activation, endocrine disruption, and cell morphology. Specifically, microplastics examination by means of field emission scanning electron microscopy revealed that work flow reuse of material created a fraction of smaller particles with an average size of 1-5 µm, a size range readily available for uptake by human cells. Moreover, chemical analysis by means of gas chromatography high-resolution mass spectrometry detected several polyamide-associated chemicals including starting material, plasticizer, thermal stabilizer/antioxidant, and migrating slip additive. Even if polyamide particles and chemicals did not induce an acute inflammatory response, repeated and prolonged exposure of human primary macrophages disclosed a steady increase in the levels of proinflammatory chemokine Interleukin-8 (IL-8/CXCL-8). Moreover, targeted metabolomics disclosed that polyamide particles modulated the kynurenine pathway and some of its key metabolites. The p53-responsive luciferase reporter gene assay showed that particles per se were able to activate p53, being indicative of a genotoxic stress. Polyamide-associated chemicals triggered moderate activation of AhR and elicited anti-androgenic activity. Finally, a high-throughput and non-targeted morphological profiling by Cell Painting assay outlined major sites of bioactivity of polyamide-associated chemicals and indicated putative mechanisms of toxicity in the cells. These findings reveal that the increasing use of polyamide microplastics may pose a potential health risk for the exposed individuals, and it merits more attention.