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.

The aim of the study was to determine nano/sub-micron particle and dust exposure levels throughout the whole workflow at a Swedish metal additive manufacturing (AM) facility, focusing on the laser powder bed fusion (L-PBF) method. By evaluating particle levels and composition across different AM processes using both stationary and personal sampling, the study sought to improve exposure assessment and inform protective measures in the metal AM workplaces. Measurements were conducted during five measurement weeks, as five working days Monday-Friday, between October 2020 and October 2023. Personal particle measurements in the breathing zone were performed on Mondays and Fridays for 1 to 3 workers per day. Stationary particle and dust sampling were performed continuously at three locations each week to capture task-specific and temporal variation in emissions. Nano/sub-micron particle concentrations ranged from 0 to 3.3 million particles/cm3, with the highest peaks recorded in the post-processing area. Elevated levels were also detected, near the depowdering machine, by the bandsaw, and in the lunchroom, while levels near the printers were low (<10,000 particles/cm3). Personal exposure peaks occurred during printer cleaning, feedstock powder filling, dust removal with compressed air, post-processing, and packing. In contrast to the increased nano/sub-micron particle levels observed, respirable, and inhalable dust levels were very low. The study highlights the need to monitor particle exposure during both manufacturing and post-processing. Health risks associated with airborne particles are influenced by both exposure levels and the toxicological properties of materials. To ensure a safe and sustainable future for metal AM, comprehensive exposure assessment, risk evaluation, and the implementation of protective measures remain essential.

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.

This study applied a virtual effect-directed analysis (vEDA) approach, integrating effect-based analysis and chemical screening, to identify bioactive compounds in rubber infill from artificial turf. Bioreporter assays targeting diverse toxicological endpoints were selected to detect a wide range of potential endocrine-disrupting and genotoxic compounds. Of 21 samples, all except one showed aryl-hydrocarbon receptor (AhR) activity (14-31,400 ng benzo[a]pyrene equivalents/g), four induced p53 activity (0.04-0.86 µg actinomycin D equivalents/g) and two showed estrogen receptor α (ERα) activity (530 and 1020 pg estradiol equivalents/g). Chemical analysis quantified up to 87 polycyclic aromatic compounds (PAC) and gas chromatography high-resolution mass spectrometry-based suspect screening yielded 281 tentative identifications. Annotation with bioassay activity data from databases and predictive models revealed 29 AhR-, 32 ERα- and 18 p53-active compounds. Univariate analysis was used to prioritize compounds for further chemical and toxicological confirmation. Eighteen AhR agonists were confirmed, contributing 0-98% to the observed AhR activity in the samples. Phenylamine additives, detected at high concentrations, exhibited low AhR activating potency and contributed < 1%. In contrast, methylated chrysene isomers elicited relatively high potencies and contributed substantially (≤65%) to the observed AhR activity. N-Isopropyl-N'-phenyl-p-phenylenediamine (IPPD) was confirmed as p53 active and explained ∼50% of the observed activity in the most p53-active sample. Styrene-butadiene rubber (SBR) showed higher AhR- and p53 activities and concentrations of quantified compounds than the alternative materials. The study highlights differences in chemical hazards among rubber infill materials and demonstrates the utility of vEDA as an early-warning tool for identifying compounds of concern.

The rapid rise of 3D printing, both in industrial and home settings, presents emerging health and environmental risks. While 3D printing enhances sustainability by reducing waste and optimizing resource use, its impact on human health remains poorly understood. The use of metals and polymers linked to health risks, coupled with the release of inhalable particles and volatile organic compounds, raises concerns about respiratory and systemic effects. The absence of clear guidelines creates high public demand for information and limits safe implementation, particularly in schools and homes where millions of 3D printers are expected by 2030. Additionally, improper disposal of 3D printing polymer materials may exacerbate plastic pollution. This article proposes the perspective of a structured risk assessment framework set on particle emissions from industrial 3D printing. It will offer a practical tool to bridge current knowledge gaps and to inform safe practice and policy development, because immediate action is necessary to balance innovation with safety.

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.

The environmental fragmentation of plastics generates a mixture of plastic particles of various sizes, which frequently co-occur with other mobile and persistent environmental pollutants. Despite the prevalence of such scenarios, the interaction between micro- and nanoplastics (MNPs) and their combined effects with environmental pollutants, such as highly toxic hexavalent chromium (Cr(VI)), remain almost entirely unexplored in mammalian species. This study demonstrated that nanoplastic and microplastic particles co-aggregate and together influence Cr bioaccumulation patterns and related physiological alterations in rats. Following a four-week repeated intragastric exposure of Wistar rats to MNPs and Cr(VI), either alone or in combination, MNPs significantly enhanced Cr bioaccumulation in the liver, heart, brain, and skin. Under co-exposure conditions, Cr(VI) was the primary driver of cellular effects observed in the blood, including shifts in immune cell subpopulations (e.g., neutrophils, lymphocytes) and alterations in red blood cell indices, while serum biochemistry reflected limited physiological stress. MNPs per se decreased creatine kinase activity and increased cholesterol levels. In summary, polystyrene MNPs increase Cr(VI) distribution and bioavailability, but co-exposure does not uniformly exacerbate toxicity. Instead, their interaction may selectively alter physiological responses, emphasizing the need for a deeper understanding of their combined effects and potential health risks.