Till Örebro universitet

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.

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.

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.

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/108 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.

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.

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.