ABSTRACT Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants with documented toxic effects, yet their multi- and transgenerational impacts on neurodevelopment and underlying mechanisms remain poorly understood. Here, we present a comprehensive study delineating the effects of developmental exposure to environmentally relevant concentrations of PFOS and PFBS on behavior, transcriptome, and genome-wide DNA methylation patterns in the directly exposed generation (F0) and their unexposed offspring (F1 and F2) in zebrafish. Both PFOS and PFBS altered larval behavior, linked to transcriptomic and DNA methylation changes in neuro-related pathways, even in the unexposed offspring. Importantly, specific DNA methylation changes in F0 were associated with behavioral outcomes in F2 animals, suggesting that these alterations could underlie transgenerational effects. Pathways associated with differentially methylated genes were prominently enriched for response to light and circadian regulation. Our findings demonstrate that developmental exposure to PFAS causes transgenerational behavioral effects in zebrafish and suggest that epigenetic changes induced by direct exposure may serve as markers for predicting outcomes in subsequent, unexposed generations. TEASER PFAS induce circadian-related epigenetic changes in zebrafish associated with behavioral impacts in unexposed offspring.

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.

Methylated and oxygenated derivatives of polycyclic aromatic compounds (PACs) are frequently detected in contaminated environments but remain insufficiently considered in environmental risk assessment, despite increasing evidence that structural modification can substantially alter their toxicity. In the present study we comparatively investigated the embryotoxicity, teratogenicity, behavioral effects, transcriptional responses, and analytical verification of exposure concentrations of five environmentally relevant substituted PACs using zebrafish embryos (Danio rerio). Embryos were exposed to 7,12-dimethylbenz[a]anthracene (DMBaA), 7-methylbenz[a]anthracene (MBaA), 7-methylbenzo[a]pyrene (MBaP), 9-methylacridine (MA), and 9(10H)-acridone (ACR) under semi-static conditions following the Fish Embryo Toxicity test. Behavioral alterations were assessed using the larval photomotor response assay, gene expression was analyzed by qPCR, and exposure concentrations were analytically verified by GC-MS throughout the experiment. Chemical analyses showed declining aqueous exposure concentrations over time for all compounds except ACR. Among the tested PACs, MA and DMBaA caused the highest acute embryotoxicity, with LC50 values of 2.34 and 9.03 µmol/L, respectively. DMBaA induced pronounced developmental abnormalities, including reduced tail length, lordosis, pericardial edema, and cardiovascular abnormalities, whereas MA caused a significantly reduced locomotor activity during the dark phase of the larval photomotor response assay. All compounds induced genes associated with the aryl hydrocarbon receptor (AhR) pathway, with DMBaA causing the strongest induction of ahrrb, cyp1a, and cyp1b1. These results show that methylated and oxygenated PACs differ in their toxic potential and molecular response profiles. While DMBaA primarily induced developmental toxicity accompanied by strong AhR-pathway activation, MA caused pronounced embryotoxic and behavioral effects despite comparatively weak molecular responses. Together, these findings demonstrate that substituted PACs can substantially contribute to environmental hazard and should be considered alongside priority PAHs in environmental monitoring and risk assessment.

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 h 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.

Methylated and oxygenated derivatives of polycyclic aromatic compounds (PACs) are frequently detected in contaminated environments but remain insufficiently considered in environmental risk assessment. Additionally, structural modifications of PACs can substantially alter their toxicity. The present study investigated the embryotoxicity, teratogenicity, behavioral effects, and molecular responses of five PACs in zebrafish embryos (Danio rerio). Embryos were exposed to 7,12-dimethylbenz[a]anthracene (DMBaA), 7-methylbenz[a]anthracene (MBaA), 7-methylbenzo[a]pyrene (MBaP), 9-methylacridine (MA), and 9(10H)-acridone (ACR) using the Fish Embryo Toxicity test under semi-static conditions. Behavioral alterations were assessed using the larval photomotor response assay, gene expression was analyzed by qPCR, and exposure concentrations were verified by GC-MS throughout the experiment. Chemical analyses revealed declining exposure concentrations over time for all compounds except ACR. Among the tested PACs, MA and DMBaA caused acute embryotoxicity with LC50 values of 2.34 and 9.03 µmol/L, respectively. DMBaA induced pronounced teratogenic effects, including reduced tail length, lordosis, pericardial edema, and cardiovascular abnormalities, whereas MA significantly reduced locomotor activity during the dark phase of the larval photomotor response assay. All compounds induced genes associated with the aryl hydrocarbon receptor (AhR) pathway, with DMBaA causing the strongest induction of ahr2, ahrrb, cyp1a, and cyp1b1. These results show that methylated and oxygenated PACs differ in their toxic potential and mode of action. While DMBaA primarily induced developmental toxicity accompanied by strong AhR-pathway activation, MA caused pronounced embryotoxic and behavioral effects despite comparatively weak molecular responses. Together, these findings demonstrate that substituted PACs can contribute substantially to environmental hazard and should be considered alongside priority PAHs in environmental monitoring and risk assessment.

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.

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 assay, 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 signaling events 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 also identify cellular fingerprints associated with NLRP3 inflammasome activation.

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 µg/L and 2 µg/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. 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. 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.

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. 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. 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. 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. 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.

Background Mucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917. Results The HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) – rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/10 8 cfu. Conclusions The CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

PFAS are ubiquitous industrial chemicals with known adverse health effects, particularly on the liver. The liver, being a vital metabolic organ, is susceptible to PFAS-induced metabolic dysregulation, leading to conditions such as hepatotoxicity and metabolic disturbances. In this study, we investigated the phenotypic and metabolic responses of PFAS exposure using two hepatocyte models, HepG2 (male cell line) and HepaRG (female cell line), aiming to define phenotypic alterations, and metabolic disturbances at the metabolite and pathway levels. The PFAS mixture composition was selected based on epidemiological data, covering a broad concentration spectrum observed in diverse human populations. Phenotypic profiling by Cell Painting assay disclosed predominant effects of PFAS exposure on mitochondrial structure and function in both cell models as well as effects on F-actin, Golgi apparatus, and plasma membrane-associated measures. We employed comprehensive metabolic characterization using liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS). We observed dose-dependent changes in the metabolic profiles, particularly in lipid, steroid, amino acid and sugar and carbohydrate metabolism in both cells as well as in cell media, with HepaRG cell line showing a stronger metabolic response. In cells, most of the bile acids, acylcarnitines and free fatty acids showed downregulation, while medium-chain fatty acids and carnosine were upregulated, while the cell media showed different response especially in relation to the bile acids in HepaRG cell media. Importantly, we observed also nonmonotonic response for several phenotypic features and metabolites. On the pathway level, PFAS exposure was also associated with pathways indicating oxidative stress and inflammatory responses. Taken together, our findings on PFAS-induced phenotypic and metabolic disruptions in hepatocytes shed light on potential mechanisms contributing to the broader comprehension of PFAS-related health risks.

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Background Diabetes is a chronic disease with several long-term complications. Several glucose-lowering drugs are used to treat type 2 diabetes mellitus (T2DM), e.g., glimepiride and liraglutide, in which both having different modes of action. Circulating microRNAs (miRNAs) are suggested as potential biomarkers that are associated with the disease development and the effects of the treatment. In the current study we evaluated the effect of glimepiride, liraglutide on the expression of the circulating miRNAs. Methods The present study is a post hoc trial from a previously randomized control trial comparing liraglutide versus glimepiride both in combination with metformin in subjects with T2DM, and subclinical heart failure. miRNAs were determined in the subjects’ serum samples with next generation sequencing. Expression patterns of the circulating miRNAs were analyzed using bioinformatic univariate and multivariate analyses (clinical trial registration: NCT01425580). Results Univariate analyses show that treatment with glimepiride altered expression of three miRNAs in patient serum, miR-206, miR-182-5p, and miR-766-3p. Both miR-182-5p and miR-766-3p were also picked up among the top contributing miRNAs with penalized regularised logistic regressions (Lasso). The highest-ranked miRNAs with respect to Lasso coefficients were miR-3960, miR-31-5p, miR-3613-3p, and miR-378a-3p. Liraglutide treatment did not significantly influence levels of circulating miRNAs. Conclusion Present study indicates that glucose-lowering drugs differently affect the expression of circulating miRNAs in serum in individuals with T2DM. More studies are required to investigate possible mechanisms by which glimepiride is affecting the expression of circulating miRNAs.

Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure.

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.