Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL^® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro models.

Nano-sized titanium dioxide particles (TiO₂ NPs) are a high-production volume nanomaterial widely used in the paints, cosmetics, food and photovoltaics industry. However, the potential carcinogenic effects of TiO₂ NPs in the lung are still unclear despite the vast number of in vitro and in vivo studies investigating TiO₂ NPs. Here, we systematically reviewed the existing in vitro and in vivo mechanistic evidence of TiO₂ NP lung carcinogenicity using the ten key characteristics of carcinogens for identifying and classifying carcinogens. A total of 346 studies qualified for the quality and reliability assessment, of which 206 were considered good quality. Using a weight-of-evidence approach, these studies provided mainly moderate to high confidence for the biological endpoints regarding genotoxicity, oxidative stress and chronic inflammation. A limited number of studies investigated other endpoints important to carcinogenesis, relating to proliferation and transformation, epigenetic alterations and receptor-mediated effects. In summary, TiO₂ NPs might possess the ability to induce chronic inflammation and oxidative stress, but it was challenging to compare the findings in the studies due to the wide variety of TiO₂ NPs differing in their physicochemical characteristics, formulation, exposure scenarios/test systems, and experimental protocols. Given the limited number of high-quality and high-reliability studies identified within this review, there is a lack of good enough mechanistic evidence for TiO₂ NP lung carcinogenicity. Future toxicology/carcinogenicity research must consider including positive controls, endotoxin testing (where necessary), statistical power analysis, and relevant biological endpoints, to improve the study quality and provide reliable data for evaluating TiO₂ NP-induced lung carcinogenicity.

This study represents an attempt toward the standardization of pulmonary NAMs and the development of a novel approach for toxicity testing of nanomaterials. Laboratory comparisons are challenging yet essential for identifying existing limitations and proposing potential solutions. Lung cells cultivated and exposed at the air-liquid interface (ALI) more accurately represent the physiology of human lungs and pulmonary exposure scenarios than submerged cell and exposure models. A triculture cell model system was used, consisting of human A549 lung epithelial cells and differentiated THP-1 macrophages on the apical side, with EA.hy926 endothelial cells on the basolateral side. The cells were exposed to silver nanoparticles NM-300K for 24 h. The model used here showed to be applicable for assessing the hazards of nanomaterials and chemicals, albeit with some limitations. Cellular viability was measured using the alamarBlue assay, DNA damage was assessed with the enzyme-modified comet assay, and the expression of 40 genes related to cell viability, inflammation, and DNA damage response was evaluated through RT² gene expression profiling. Despite harmonized protocols used in the two independent laboratories, however, some methodological challenges could affect the results, including sensitivity and reproducibility of the model.

Air pollution is an environmental factor associated with an increased risk of neurodegenerative diseases, such as Alzheimer's and Parkinson's, characterized by decreased cognitive abilities and memory. The limited models of sporadic Alzheimer's disease fail to replicate all pathological hallmarks of the disease, making it challenging to uncover potential environmental causes. Environmentally driven models of Alzheimer's disease are thus timely and necessary. We used live-cell confocal fluorescent imaging combined with high-resolution stimulated emission depletion (STED) microscopy to follow the response of retinoic acid-differentiated human neuroblastoma SH-SY5Y cells to nanomaterial exposure. Here, we report that exposure of the cells to some particulate matter constituents reproduces a neurodegenerative phenotype, including extracellular amyloid beta-containing plaques and decreased neurite length. Consistent with the existing in vivo research, we observed detrimental effects, specifically a substantial reduction in neurite length and formation of amyloid beta plaques, after exposure to iron oxide and diesel exhaust particles. Conversely, after exposure to engineered cerium oxide nanoparticles, the lengths of neurites were maintained, and almost no extracellular amyloid beta plaques were formed. Although the exact mechanism behind this effect remains to be explained, the retinoic acid differentiated SH-SY5Y cell in vitro model could serve as an alternative, environmentally driven model of neurodegenerative diseases, including Alzheimer's disease.

Organ-specific multicellular in vitro models are used to mimic the lung-blood-brain axis, and to assess the nanomaterials (NMs) safety in humans. We employed a triculture lung model, a whole-blood model, an astrocytes-neurons coculture to examine health outcomes by three cerium dioxide (CeO₂) NMs and silver (Ag) nanowires. Endpoints included cytotoxicity, gene expression, genotoxicity, inflammatory markers at the air-liquid interface (ALI), complement activation, and secondary toxicity in astrocytes-neurons coculture. Post-exposure, CeO₂-3.5 nm high-dose decreased cell viability, no DNA damage was detected. At epithelial-macrophages interface, CeO₂-50 nm upregulated surfactant protein A (SPA), cell surface death receptor (FAS), and heme oxygenase-1 (HMOX1), whereas CeO₂-3.5 nm downregulated SPA. Ag-nanowires upregulated HMOX1, macrophage inflammatory protein-1β (MIP-1β), granulocyte colony-stimulator factor (G-CSF), chemokine C-X-C-motif ligand 1 (CXCL1). At endothelial side, CeO₂-50 nm and - 3.5 nm, and Ag-nanowires upregulated HMOX1. In whole-blood model, CeO₂-3.5 nm high-dose reduced terminal complement complex (TCC) proteins, while CeO₂-50 nm and Ag-nanowires increased them. Nanomaterials activated CD11b⁺ on granulocytes and monocytes. Ag-nanowires conditioned-medium (CM) on astrocytes-neurons coculture, decreased cell viability. CeO₂-50 nm CM upregulated IL1β, NFκB, and HMOX1. Overall, CeO₂-3.5 nm exhibits lung toxicity; CeO₂-50 nm CM triggers inflammatory response and Ag-nanowires CM may induce cytotoxicity in brain cells.