Percorrer por autor "Roque, Rossana"
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- Cellular, Molecular and Genotoxic Effects of Digested Titanium Dioxide NanomaterialsPublication . Rolo, Dora; Pereira, Joana F.S; Vieira, Adriana; Roque, Rossana; Gramacho, Ana Catarina; Vital, Nádia; Matos, Paulo; Gonçalves, Lídia; Bettencourt, Ana F.; Silva, Mafalda A.; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Jordan, Peter; Silva, Maria João; Louro, HenriquetaHuman exposure to titanium dioxide nanomaterials(TiO2NMs) occurs particularly by ingestion, due to food/food contact materials and consumer products. However, the possibility of adverse effects in gastrointestinal tract is unclear. Aiming to study the impact of digestion on the NMs’ properties and their cellular/molecular effects, two human intestinal cell lines were used, Caco-2 and HT29-MTX-E12. After exposure to TiO2NMs(NM-102, NM-103, NM-105), undigested or subjected to standardized static in vitro digestion method (mimicking human digestion), the cells were analyzed for toxicity, genotoxicity, reactive oxygen species, NM uptake and intestinal translocation. We showed that in vitro digestion of TiO2NMs may increase their toxicity and DNA-damaging effect, depending on the NM, more relevant for the rutile/anatase NM-105, possibly due to its smaller hydrodynamic size in the cellular medium. Effects on chromosomal integrity were seen in HT29-MTX-E12 cells, for all tested TiO2NMs, especially after digestion. Internalization into early endosomes was confirmed for NM-103 and NM-105, before and after digestion, in monolayers of both cell lines, and at the apical membrane of polarized Caco-2 cells. The internalized NMs accumulated in late endosomes/multivesicular bodies, partially transversing the basolateral membrane of polarized Caco-2 cells without changing transepithelial electrical resistance or epithelial marker abundance. These results suggest that part of the TiO2NMs can be transcytosed through colonic epithelia without disrupting intestinal barrier integrity. Overall, the biological outcomes from TiO2NMs interaction with intestinal cells were more pronounced after digestion, highlighting its relevance in the hazard assessment of ingested NMs.
- Ingested nanomaterials: impact of digestion process in the physicochemical characteristics and biological consequences in intestinal cellsPublication . Vieira, Adriana; Vital, Nádia; Roque, Rossana; Gramacho, Ana Catarina; Rolo, Dora; Gonçalves, Lídia D.; Bettencourt, Ana; Martins, Carla; Assunção, Assunção; Alvito, Paula; Silva, Maria João; Louro, HenriquetaNanomaterials(NMs) provide a basis for key enabling technologies, in view of their potential to improve many products and processes, namely in food and feed industry. That is the case of titanium dioxide NMs(TiO2 NMs), presenting beneficial properties for a broad range of innovative applications such as food additives, toothpaste, pharmaceuticals, food products, etc., that may drive ingestion. The oral exposure can occur directly, by consumption of products/pharmaceuticals or foods containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or environmental sources. As such, the gastrointestinal tract is the first site of contact of the ingested NMs, allowing a systemic exposure if the intestinal barriers is surpassed. This work aimed to investigate how the digestion process affects the physicochemical properties of three different TiO2 NMs(NM-102, NM-103 and NM-105) and their toxic effects on intestinal cells. After undergoing digestion through the standardized static INFOGEST 2.0 in vitro digestion method, the cytotoxicity of the TiO2 NMs was determined in Caco-2 and HT29-MTX-E12 intestinal cells, using the MTT assay. Furthermore, the cytokinesis-blocked micronucleus assay was used to investigate their genotoxicity in both cell lines in order to predict their carcinogenic potential. The results showed that, for one TiO2 NM(NM-105), the digestion caused changes in the hydrodynamic size of the NM and a more pronounced toxicity in HT29-MTX-E12 intestinal cells, as compared to the undigested one. The micronucleus assay suggests effects on the chromosomal integrity in the HT29-MTXE12 cells, for all the tested TiO2 NM especially after the in vitro digestion. Overall, we conclude that including the digestion prior to the in vitro bioassays for the safety evaluation of ingested NMs, allows integrating the physiological modifications that the NMs suffer in the organism, contributing to an improved hazard assessment of ingested NMs.
- 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.
- Investigation of the genotoxicity of digested titanium dioxide nanomaterials in human intestinal cellsPublication . Vieira, Adriana; Vital, Nádia; Rolo, Dora; Roque, Rossana; Gonçalves, Lídia M.; Bettencourt, Ana; Silva, Maria João; Louro, HenriquetaThe widespread use of titanium dioxide nanomaterials (TiO2 NMs) in food and consumer products such as toothpaste or food contact materials, suggests the relevance of human oral exposure to these nanomaterials (NMs) and raises the possibility of adverse effects in the gastrointestinal tract (GIT). We previously showed that the in vitro digestion of TiO2 NMs may increase their toxicity in intestinal cells. In this work, we analyzed the genotoxicity and the intracellular reactive oxygen species induction by physiologically relevant concentrations of three different TiO2 NMs (NM-102, NM-103 and NM-105) in Caco-2 and HT29-MTX-E12 intestinal cells, while considering the potential influence of the digestion process in the NMs' physiochemical characteristics. The results evidenced a DNA-damaging effect dependent on the NM, more relevant for the rutile/anatase NM-105, possibly due to its lower hydrodynamic size in the cells medium. In addition, the results of the micronucleus assay suggest effects on chromosomal integrity, an indicator of cancer risk, in the HT29-MTX-E12 cells, for all the tested TiO2 NMs, especially after the in vitro digestion. This work supports the evidence for concerns on the use of TiO2 NMs as a food additive, recently reported by EFSA, and for their use in applications in consumer products that may drive human exposure through ingestion.