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.

Effective early warning systems for aquatic contamination require monitoring strategies capable of detecting subtle, long-term shifts in mixture-driven biological activity. Suspended particulate matter (SPM) serves as a carrier and reservoir for complex contaminant mixtures, facilitating their transport and persistence in aquatic systems, yet systematic toxicological time series for archived SPM remain scarce. Regulatory monitoring predominantly targets Priority Substances and River Basin Specific Pollutants, leaving the temporal trends of particle-associated mixture toxicity largely unresolved. Leveraging 18 years (2005–2022) of cryogenically archived annual SPM composites from the Rhine River, we conducted a spatiotemporal effect-based assessment integrating receptor-mediated effects, oxidative stress analysis and untargeted Cell Painting phenomics. This integrated toolbox enabled evaluation of pathway-specific responses and multi-compartment cellular perturbations associated with particle-bound contaminant mixtures. Polar SPM-associated chemicals elicited oxidative stress response and caused endocrine disruption through estrogen receptor α (ERα) activation and androgen receptor inhibition (anti-AR). Trend analysis showed spatiotemporal variation along the river, with statistically increasing trends of oxidative stress and anti-AR activity over time at Koblenz, driven by polar chemicals. Both polar and non-polar SPM extracts activated the aryl hydrocarbon receptor (AhR), indicating presence of compounds capable of triggering xenobiotic response pathways. Several subcellular compartments were affected, with mitochondrial features being among the most affected. These findings demonstrate that SPM-associated chemicals elicit diverse toxicological effects by acting on several receptors and impacting diverse cellular structures. Combining targeted and phenomics-based effect approaches provided comprehensive mechanistic insights and valuable information to support the early warning systems for chemical contamination in aquatic environments.

Metal additive manufacturing (AM) relies on alloy feedstock powders that may come into contact with the workers' skin during handling, yet skin-relevant data on metal release and biological reactivity remain limited. Here, we assessed the cutaneous bioactivity of the fine particle fraction of four gas-atomized Fe-based AM powders (316L stainless steel, Fe-powder A, and tooling steels B and C). Powders were sieved to <10 mu m and characterized by scanning electron microscopy and X-ray photoelectron spectroscopy before and after incubation in artificial sweat (ASW). Metal biodissolution was quantified in ASW and keratinocyte culture medium using atomic absorption spectrophotometry. Cellular responses were evaluated in HaCaT keratinocytes using Cell Painting-based phenomics and multiplex cytokine/chemokine profiling and in an ex vivo full-thickness human skin explant model, including superficial barrier disruption, IL-8/CXCL8 quantification, and histological assessment. ASW exposure induced marked shifts in the outermost surface composition across powders, indicating sweat-driven surface transformation. Biodissolution was low and medium-dependent, with Fe dominating the release in ASW, and with an overall metal release remaining limited in cell culture medium. In HaCaT cells, MCP-1/CCL2, IL-6, and IL-8/CXCL8 were quantifiable but showed no significant changes following powder exposure. Cell Painting revealed subtle, shared phenotypic signatures, primarily involving mitochondrial-associated features, without evidence of broad cellular stress. In the ex vivo skin model, AM powders did not increase IL-8/CXCL8 secretion, the particles remained localized to the skin surface without detectable penetration, and coexposure with Staphylococcus epidermidis did not enhance bacterial colonization or induce inflammation. To the best of our knowledge, this is the first study that applies a human skin explant model to evaluate dermal responses to metal AM powders. Overall, the tested AM powders showed low short-term cutaneous reactivity under skin-relevant conditions, providing human-relevant evidence to inform occupational risk assessment in AM environments.

Nanoplastics have recently been detected in human liver tissue, raising concerns about their potential impact on liver function. However, early hepatocyte responses associated with nanoplastics exposure remain poorly understood. Here, we combined high-throughput Cell Painting-based phenomics, untargeted metabolomics, and Seahorse mitochondrial functional assay to investigate the effects of 100 nm polystyrene nanoplastics on human HepaRG hepatocytes, a surrogate for primary human hepatocytes. At the tested concentrations (6.25-100 µg/mL), exposure did not induce overt cytotoxicity, enabling assessment of early sublethal cellular responses. Phenomics revealed widespread subcellular perturbations, with 16.4% of the measured phenotypic features significantly altered. Mitochondria-associated features represented the dominant altered phenotypic signature, showing pronounced changes in granularity, texture, and radial distribution, alongside alterations in endoplasmic reticulum- and cytoskeleton-associated features. Untargeted metabolomics of intracellular metabolites and the extracellular secretome revealed metabolic alterations, characterized by changes consistent with altered β-oxidation, lipid handling, membrane stress, and central carbon metabolism, including changes in the tricarboxylic acid (TCA) cycle and amino acid catabolism. Pathway analysis identified the TCA cycle as one of the most significantly affected pathways (FDR = 0.028). Integrated phenomic-metabolomic analysis revealed strong correlations between mitochondrial phenotypic features and metabolites involved in lipid and energy metabolism, indicating a coordinated structural-metabolic response to polystyrene nanoplastics exposure. Functional assessment using Seahorse assay showed reduced basal and maximal respiration and decreased ATP-linked O2 consumption. Together, these findings provide evidence that 100 nm polystyrene nanoplastics elicit early mitochondria-associated phenotypic, metabolic, and functional responses prior to overt cytotoxicity under the tested conditions. They also highlight the value of phenomic-metabolomic-functional integration for profiling sublethal nanotoxicological responses and guiding future targeted mechanistic studies.

Formation of the NLRP3 inflammasome occurs in response to a wide range of triggers and leads to pyroptosis and release of IL-1 beta and IL-18. This has widely been considered an "all or nothing" response leading to similar cellular outcomes each time, once the inflammasome has formed, a view that has been challenged over the years. This assumption has largely been based on endpoint measurements at the population level, producing metrics that fail to adequately capture dynamic responses. Herein, we utilize live-cell imaging combined with an algorithmic approach to track individual ASC-GFP specks over time, formed in response to the triggers ATP, monosodium urate and nigericin. Using this approach, we report trigger-dependent differences in speck lifetime. The use of the proposed algorithm requires a relatively small dataset, while still providing insights into NLRP3 inflammasome dynamics downstream of inflammasome activation at the single-cell level.

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.

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.

Tire wear particles (TWP) stand out as a major contributor to microplastic pollution, yet their environmental impact remains inadequately understood. This study delves into the cocktail effects of TWP leachates, employing molecular, cellular, and organismal assessments on diverse biological models. Extracted in artificial seawater and analyzed for metals and organic compounds, TWP leachates revealed the presence of polyaromatic hydrocarbons and 4-tert-octylphenol. Exposure to TWP leachates (1.5 to 1000 mg peq L-1) inhibited algae growth and induced zebrafish embryotoxicity, pigment alterations, and behavioral changes. Cell painting uncovered pro-apoptotic changes, while mechanism-specific gene-reporter assays highlighted endocrine-disrupting potential, particularly antiandrogenic effects. Although heavy metals like zinc have been suggested as major players in TWP leachate toxicity, this study emphasizes water-leachable organic compounds as the primary causative agents of observed acute toxicity. The findings underscore the need to reduce TWP pollution in aquatic systems and enhance regulations governing highly toxic tire additives.

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.