Percorrer por autor "Gramacho, Ana C."
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- Ingested titanium dioxide nanomaterials: new approach to investigate intestinal genotoxicity and key cellular/molecular effectsPublication . Ventura, Célia; Rolo, Dora; Gramacho, Ana C.; Vieira, Adriana; Roque, Rossana; Valente, Ana; Vital, Nádia; Pinto, Fátima; Alvito, Paula; Assunção, Ricardo; Martins, Carla; Bettencourt, Ana; Gonçalves, Lídia; Pereira, Joana; Matos, Paulo; Jordan, Peter; Vieira, Luís; Silva, Catarina; Silva, Maria Joao; Louro, HenriquetaOral exposure to titanium dioxide nanomaterials (TiO2NMs) is due to their presence in food, food contact materials, medicines and cosmetics. The gastrointestinal tract(GIT) represents primary site of contact, that may result in systemic exposure, if biological barriers are surpassed. The INGESTnano project aimed to investigate nano-bio interactions at the cellular/molecular levels within the context of the intestinal tract and digestion processes, for understanding potential effects on human health. A group of three TiO₂NMs(NM-102, NM-103, NM-105) was selected as case study using a new approach methodology(NAM), incorporating the in vitro human digestion simulation prior to biological assays in Caco-2 and HT29-MTX-E12 intestinal cells. The endpoints included cyto- and genotoxicity, cell uptake, intestinal permeability, GIT transport and epigenomic modifications. The results showed a more pronounced cytotoxicity in HT29-MTX-E12 cells for digested NM-105, as compared to undigested, concomitantly with subtle changes in hydrodynamic-size. DNA-damage induction was more relevant for NM-105, and the micronucleus assay showed chromosomal damage in HT29-MTX-E12 cells for all TiO2NMs, especially after in vitro digestion.All NMs, digested or not, were internalized by intestinal cells, but did not affect transepithelial resistance, nor the epithelial markers in polarized enterocytes. NM-102 was retained in lysosomes, while NM-103 and NM-105 showed transcytosis, a potential gateway for systemic distribution. Using Reduced Representation Bisulfite Sequencing, several differentially methylated genes were identified for the TiO₂NMs, either digested or not. Pathway and Gene Ontology analyses showed that each TiO2NMs has a different functional impact on intestinal cells, probably linked to specific physicochemical properties, and digestion seems to reduce this impact. A trend towards CpG hypermethylation was observed upon NM-105 exposure, unlike for the other TiO2NMs. This integrated approach enabled the identification of key events and molecular pathways elicited by TiO2NMs, highlighting the importance of considering the digestion on the induction of adverse outcomes.
- Nano-bio interactions of titanium dioxide nanomaterials in the intestinal moiety after simulated digestion in vitroPublication . Louro, Henriqueta; Rolo, Dora; Gramacho, Ana C.; Pereira, Joana; Matos, Paulo; Jordan, Peter; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana F.; Silva, Maria JoãoThe exponential development of nanomaterials (NMs) contrasts with insufficient risk assessment for human health and the environment, leading to concerns for public health. Their potential to improve many products and processes, namely in agriculture, food and feed industry, has led to increased use of NMs, such as titanium dioxide nanomaterials (TiO2). In fact, oral exposure to TiO2 may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of food/water contaminated due to environmental accumulation. Therefore, the gastrointestinal tract (GIT) appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. Since NMs primary physicochemical properties may define nano-bio interactions1, NMs with the same chemistry but with different shape, diameter, length, surface charge or functionalization may lead to different toxicities. Conversely, since the GIT is chemically and physically complex, ingested NMs will pass through different environments prior to their intestinal uptake, affecting their physicochemical properties, so that these secondary physicochemical properties should be considered while evaluating their safety in the food chain. The aim of this work was to investigate the nano-bio interactions of three TiO2 (NM-102, NM-103, NM-105, from Joint Research Centre, Ispra) in the context of intestinal tract and digestion processes. As alternative to animal testing, an in vitro harmonized digestion method2 was used for simulating the human digestion of NMs and their characteristics were studied in acellular and intestinal (Caco-2) cells context. The NMs were characterized using dynamic light scattering (DLS, for size distribution), electrophoretic light scattering (zeta potential), transmission electron microscopy (TEM) for morphological characterization and X-ray energy dispersive spectrometry (EDS) for elemental chemical analysis. TiO2 samples were labelled directly with a fluorescent probe and used for uptake studies with confocal microscopy (CM). In parallel, a co-culture model of Caco-2 with mucous-secreting HT29-MTX was initiated and exposed to TiO2 to ascertain epithelial barrier translocation. The results did not show major differences in the NMs’ pH, osmolality, charge signal or mean size, except for NM-103. TEM and TEM –EDS analysis showed that it is possible to identify primary particles after digestion process and preliminary CM results suggest that some of the tested TiO2 nanomaterials can be uptake by the cells, suggestive of potential subcellular effects that warrant further investigation. References [1] H. Louro, A. Saruga, J. Santos, M. Pinhão, M.J. Silva, Toxicol. In Vitro. 56 (2019)172–183. [2] A. Brodkorb, L.Egger, M. Alminger, P. Alvito et al. Nat Protoc. 2019 Apr;14(4):991-1014.