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- Analysis Of The Cytotoxicity And Genotoxicity of Digested Titanium Dioxide Nanomaterials (TiO2) In Intestinal CellsPublication . Louro, Henriqueta; Vieira, Adriana; Gramacho, Ana Catarina; Rolo, Dora; Vital, Nádia; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana Francisca; Silva, Maria JoãoTitanium dioxide nanomaterials (TiO2) have been frequently applied as food additives, in pharmaceuticals and in personal care products, such as toothpastes. Despite some regulators like EFSA concluded that the absorption of orally administered TiO2 is low, and that the use of TiO2 as a food additive does not raise a genotoxic concern, the presence of TiO2 in human organs was recently reported. This exposure may lead to adverse outcomes and has been poorly investigated. Furthermore, many of the biological effects of TiO2 described in the literature often overlook adequate physicochemical properties and their modification due to NMs interaction with the surrounding physiological matrices happening, e.g, during digestion. This work aimed to investigate in intestinal cells, the cyto- and genotoxic effects of TiO2 after the simulation of the human digestive process using the standardized INFOGEST in vitro digestion method, to better understand their potential negative impacts on the gastrointestinal tract. The TiO2 were characterized before and after digestion using DLS, zeta potential and TEM-EDS. The digestion product was used for cytotoxicity (MTT) and genotoxicity (comet, micronucleus) assays in two types of intestinal cells (Caco-2 and mucus secreting HT29-MTX cells). The results of the cytotoxicity and genotoxicity assays are discussed in view of the TiO2 secondary characteristics, to further understand the potential adverse intestinal outcomes in light of the transformation they suffer during digestion.
- Differentially Expressed miRNAs and Functional Pathways Changes in Human Alveolar Epithelial Cells After 1 and 7 Days Exposure to MWCNT-7 or AsbestosPublication . Ventura, Célia; Vieira, Luís; Silva, Catarina; Silva, Maria JoãoEnvironmental and occupational exposure to multi-walled carbon nanotubes (MWCNT), an engineered nanomaterial widely used in industrial and biomedical applications, has been associated with pulmonary adverse effects upon inhalation. Particularly, MWCNT-7 (Mitsui-7) has been classified as possibly carcinogenic to humans, and many toxicological studies have compared it with asbestos due to their similarity in physical characteristics and biopersistence, and the well-known pulmonary adverse effects of asbestos. Several pulmonary diseases have been associated with miRNA expression changes, thus, in this study, we compare the differentially expressed miRNAs (DEmiRNAs) and enriched KEGG pathways after 1 and 7 days exposure to MWCNT-7 or crocidolite, in order to understand the mechanisms of biological response to these materials over time and their possible similarity. For this purpose, miRNAs were sequenced in exposed and non-exposed A549 cells at both time points by next-generation sequencing, and the enriched cellular pathways identified as previously described with minor modifications1. A higher number of DEmiRNAs was observed at 24 h compared to 7 days (17 vs. 3 and 21 vs. 10 for MWCNT-7 and crocidolite, respectively). Accordingly, the number of enriched pathways was also higher at 24 h (23 vs. 30 at 24 h and 7 vs. 5 at 7 days for MWCNT-7 and crocidolite, respectively). At 7 days, most altered pathways were related to cancer, highlighting the carcinogenic potential of MWCNT-7 and crocidolite. The reduction of the number of affected functional pathways likely reflects a greatest impact of these materials shortly after exposure and/or that the differences in DEmiRNAs between non-exposed and exposed cells are less significant after long-term culture. 1Ventura et al, Toxicology Letters 328: 7-18
- The use of the in vitro micronucleus assay on the genotoxicity assessment of cellulose nanofibrils in mammalian cell linesPublication . Ventura, Célia; Teixeira, Sara; Marques, Catarina; Vilar, Madalena; Pinto, Fátima; Lourenço, A.F.; Sousa Mendes, A.; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoWith the expansion of innovative cellulose nanofibrils (CNF) applications, either for industrial (e.g., paper industry) or biomedical purposes (e.g., tissue regeneration and dentistry), human exposure has been increasing, raising concerns about their potential health outcomes . Being persistent high aspect-ratio nano-objects, CNFs effects may resemble those of some multi-walled carbon nanotubes, e.g., the MWCNT-7, which has been reported to induce chromosome instability, cytoskeleton alterations and dysfunction of several cancer-related cell signaling pathways . The in vitro cytokinesis-block micronucleus (CBMN) assay is a sensitive and reliable assay for analysis of chromosome damage in mammalian cells, with the potential of providing mechanistic information and predicting carcinogenic effects. In this work we evaluated the use of the CBMN assay in different mammalian cell systems to characterize the genotoxicity of a CNF produced from an industrial bleached Eucalyptus globulus kraft pulp through TEMPO-mediated oxidation followed by a high-pressure homogenization process . The CNF sample was fully characterized in order to assess the most relevant physicochemical properties. It presented a wide distribution of diameters, with the mode in the 20–25 nm range and a length of several micrometers. Preliminary doserange finding through assessment of the cytotoxic effects (MTT and clonogenic assays) in cell lines representative of the respiratory tract (A549, A549 co-cultured with macrophage-like THP-1 cells and V79 cells) and bone (MG63 osteoblasts) showed that the CNF was not cytotoxic, regardless of the exposure period. The results of the CBMN assay (following OECD guideline 487) showed that exposure to low CNF concentrations induced a significant increase of micronuclei in co-cultured alveolar (A549) cells and in osteoblasts, while no significant effect was observed in A549 cells in monoculture. These results support the suitability of the in vitro micronucleus assay to assess the genotoxicity of nanofibers like CNF, highlighting the relevance of the cell system selected. This and other factors related to the experimental design are discussed, pointing to the need of nano-specific guidelines for this assay application in nanogenotoxicology. References Ventura et al. 2020. Cellulose, DOI 10.1007/s10570-020-03176-9 Ventura et al. 2020. Nanotoxicology, 14:479 Ventura et al.2020. Toxicol Lett, 328: 7 Fenech, M. 2007. Nature Protocols 2: 1084. Lourenço et al. 2017. Cellulose 24: 349.
- Validation of Caco-2/HT29-MTX model to assess the potential risk of ingested titanium dioxide nanoparticlesPublication . Rolo, Dora; Pereira, Joana F.S.; Matos, Paulo; Vieira, Adriana; Vital, Nádia; Jordan, Peter; Silva, Maria João; Louro, HenriquetaThe increased use of titanium dioxide nanoparticles (TiO2NPs) as a food additive demands a deep assessment of their potential risk for human health, including their abilities to cross biological barriers. In vitro models of the intestinal barrier are being increasingly used to evaluate NPs exposure risk. Most of these studies have focused on standard monoculture models of Caco-2 monolayers. However, they exhibit several limitations such as the lack of mucus layer and a low paracellular permeability. We aim to study TiO2NPs with an in vitro model of intestinal barrier using co-culture of two types of cells: absorptive Caco-2 and mucus-secreting HT29-MTX. This co-culture confers more physiological intestinal epithelium-like properties to the model, such as mucus secretion and tight junction formation, allowing a more adequate investigation of the cellular effects elicited by NPs. Due to the multiple variables and parameters playing a part when the model's complexity is increased, we characterized the robustness of this model by evaluating cell differentiation by confocal microscopy and Western blot while monitoring epithelial barrier formation, through measurement of both transepithelial resistance (TEER)and paracellular permeability (Lucifer yellow). An optimized model of the intestinal barrier will be used to better understand the uptake, adhesion and localization of TiO2NPs, both directly and after the simulation of the human digestive process using the harmonized in vitro digestion protocol. Preliminary data shows that these complex models can add valuable information to study the potential negative impacts and genotoxicity of TiO2NPs on human health.