Browsing by Author "Pereira, Joana F.S."
Now showing 1 - 8 of 8
Results Per Page
Sort Options
- Assessing the impact of TiO2 nanomaterials on intestinal cells: New evidence for epithelial translocation and potential pro-inflammatory effectsPublication . Rolo, Dora; Pereira, Joana F.S.; Gonçalves, Lídia; Bettencourt, Ana; Jordan, Peter; Silva, Maria João; Matos, Paulo; Louro, HenriquetaUnderstanding the potential impact of nanomaterials (NMs) on human health requires further investigation into the organ-specific nano-bio interplay at the cellular and molecular levels. We showed increased chromosomal damage in intestinal cells exposed to some of in vitro digested Titanium dioxide (TiO2) NMs. The present study aimed to explore possible mechanisms linked to the uptake, epithelial barrier integrity, cellular trafficking, as well as activation of pro-inflammatory pathways, after exposure to three TiO2-NMs (NM-102, NM-103, and NM-105). Using confocal microscopy, we show that all NMs, digested or not, were able to enter different types of intestinal cells. At the physiologically relevant concentration of 14 μg/mL, the digested TiO2-NMs did not compromise the transepithelial resistance, nor the levels of epithelial markers E-cadherin and Zonula occludens protein 1 (ZO-1), of polarized enterocyte monolayers. Nonetheless, all NMs were internalized by intestinal cells and, while NM-102 was retained in lysosomes, NM-103 and NM-105 were able to transverse the epithelial barrier through transcytosis. Moreover, 24 h exposure of 14 and 1.4 μg/mL digested NM-105, promoted interleukin IL-1β expression in activated M1 macrophages, indicating a potential pro-inflammatory action in the gut. Taken together, our findings shed light on the cell-specific nano-bio interplay of TiO2-NMs in the context of the intestinal tract and highlight transcytosis as a potential gateway for their systemic distribution. The potential proinflammatory action of digested NM-105 emphasizes the importance of pursuing research into the potential impact of NMs on human health and contribute to the weight of evidence to limit their use in food.
- Cellular effects of Titanium Dioxide Nanoparticles in the intestinePublication . Rolo, Dora; Pereira, Joana F.S.; Matos, Paulo; Gonçalves, Lídia; Bettencourt, Ana Francisca; Jordan, Peter; Silva, Maria João; Louro, HenriquetaIntroduction: The increased use of titanium dioxide nanoparticles (TiO2-NPs) as a food additive demands a thorough assessment of their potential risk for human health. Via oral exposure they may lead to adverse local or systemic outcomes, and few studies have focused on the cellular internalization mechanisms (endocytosis) of TiO2-NPs. The objective was to analyze the mechanisms by which TiO2-NPs (NM-102, NM-103 and NM-105, JRC repository) translocate by the intestinal epithelium layer, using monolayers of human intestinal cell lines (Caco-2 and HT29-MTX), as well as polarized Caco-2 cells, and co-cultures of both cells. Results: We evaluated cell differentiation by transepithelial resistance measurements and the translocation of TiO2-NPs tagged with alizarin through the intestinal barrier by confocal microscopy and we confirmed the internalization of the TiO2-NPs in both cell line models. Co-localization studies suggested that the smallest TiO2-NPs were internalized into EEA1-positive early-endosomes and accumulate in late endosomes (Rab7), with only a small fraction following the degradative pathway to the lysosome (LAMP1). This suggested that at least part of the TiO2-NPs could be redirected to the secretory pathway. Consistently, we detected fluorescence passing from the apical (AP) to the basolateral (BL) chamber, depending on the characteristics of cell model and TiO2-NPs tested. Conclusions: Small TiO2-NPs were endocytosed by Caco-2 cells, with an increase in particle diameter suggesting intracellular aggregation, whereas larger agglomerates deposited mainly extracellularly. Following endocytosis, TiO2 NPs were trafficked through different intracellular compartments including early and late endosomes/endo-lysosomes, with part being subjected to AP to BL transport.
- Cytotoxicity and genotoxicity of MWCNT-7 and crocidolite: assessment in alveolar epithelial cells versus their coculture with monocyte-derived macrophagesPublication . Ventura, Célia; Pereira, Joana F.S.; Matos, Paulo; Marques, Bárbara; Jordan, Peter; Sousa-Uva, António; Silva, Maria JoãoIn the past years, several in vitro studies have addressed the pulmonary toxicity of multi-walled carbon nanotubes (MWCNT) and compared it with that caused by asbestos fibers, but their conclusions have been somewhat inconsistent and difficult to extrapolate to in vivo. Since cell coculture models were proposed to better represent the in vivo conditions than conventional monocultures, this work intended to compare the cytotoxicity and genotoxicity of MWCNT-7 (Mitsui-7) and crocidolite using A549 cells grown in a conventional monoculture or in coculture with THP-1 macrophages. Although a decrease in A549 viability was noted following exposure to a concentration range of MWCNT-7 and crocidolite, no viability change occurred in similarly exposed cocultures. Early events indicating epithelial to mesenchymal transition (EMT) were observed which could explain apoptosis resistance. The comet assay results were similar between the two models, being positive and negative for crocidolite and MWCNT-7, respectively. An increase in the micronucleus frequency was detected in the cocultured A549-treated cells with both materials, but not in the monoculture. On the other hand, exposure of A549 monocultures to MWCNT-7 induced a highly significant increase in nucleoplasmic bridges in which those were found embedded. Our overall results demonstrate that (i) both materials are cytotoxic and genotoxic, (ii) the presence of THP-1 macrophages upholds the viability of A549 cells and increases the aneugenic/clastogenic effects of both materials probably through EMT, and (iii) MWCNT-7 induces the formation of nucleoplasmic bridges in A549 cells.
- A inflamação intestinal como promotor da tumorigénese: pistas sobre a base molecular envolvidaPublication . Pereira, Joana F.S.; Bessa, Cláudia; Matos, Paulo; Jordan, PeterA inflamação intestinal é uma doença crónica debilitante de origem multifatorial que afeta cerca de 25 000 portugueses. É também uma condição promotora para a tumorigénese no cólon. Um microambiente inflamatório fornece sinais de sobrevivência para as células cancerígenas, as quais respondem com mudanças no seu programa genético. RAC1B é um exemplo de um biomarcador que foi identificado num subconjunto de tumores colorretais, e cuja expressão está aumentada em amostras de doentes com doença inflamatória intestinal. Para esclarecer os mecanismos moleculares envolvidos, a expressão da proteína RAC1B foi analisada num modelo celular composto por uma camada epitelial de células colorretais Caco-2 cocultivadas simultaneamente com fibroblastos ou macrófagos. A mera adição da citocina CXCL2/MIP2α purificada, ou uma cocultura com macrófagos do tipo M1, responsáveis pela síntese de MIP2α in vivo, induziram um aumento significativo nos níveis da proteína RAC1B nas células colorretais. Este aumento foi ainda potenciado pela adição de fibroblastos derivados de carcinoma (CAFs) à cocultura com M1. Estes resultados permitiram identificar, molecularmente, como os sinais pró-inflamatórios do microambiente induzem alterações no programa genético das células tumorais colorretais, abrindo caminho para uma melhor compreensão do papel da inflamação na promoção de tumores.
- Investigation of potential respiratory adverse effects of micro/nanofibrillated cellulose and cellulose nanocrystals using human lung cell lines.Publication . Pinto, Fátima; Ventura, Célia; Cadete, João; Lourenço, Ana Filipa; Pedrosa, Jorge F.S.; Vital, Nádia; Pereira, Joana F.S.; Matos, Paulo; Gonçalves, Lídia; Bettencourt, Ana; Silva, Catarina C.; Fernandes, Susete N.; Godinho, Maria Helena; Vieira, Luís; Jordan, Peter; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoMicro/nanofibrillated (CMF/CNF) and nanocrystalline (CNC) celluloses are innovative materials with enormous potential for industrial and biomedical applications. Their expanding production/application urges the investigation of their safety for human health. This study aimed at investigating the potential respiratory outcomes of two CMF/CNF and one CNC produced from bleached Eucalyptus globulus kraft pulp using human alveolar epithelial (A549) cells grown in monoculture or co-cultured with THP-1 monocyte-derived macrophages, by assessing their cellular uptake, cytotoxic, immunotoxic, genotoxic, and epigenetic effects. The nanocelluloses were characterized for their physicochemical properties: CMF displays a low percentage of nanofibrils while CNF comprises 100% fibrils with a diameter (D) circa 11 nm; CNC consists of nanorods with D of 4-5 nm and aspect ratio around 42. TEM analysis evidenced that CMF and CNF were internalised into A549 cells whereas CNC were not. Neither cytotoxicity (colorimetric and clonogenic assays) nor ROS induction was observed for any of the nanocelluloses. CMF caused chromosomal alterations (in vitro micronucleus assay) in A549 cells while negative results were obtained in co-culture and for the other micro/nanocelluloses in mono- or co-culture. Results in progress of DNA damage and gene mutation analyses will complement mutagenesis assessment. Additionally, potential inflammatory and epigenetic effects are being evaluated. These results contribute to the weight of evidence of nanocelluloses biological effects and knowledge of the underlying molecular mechanisms. Such information will drive the synthesis of the safest nanocelluloses,thus minimising potential negative impacts of their use on human and environmental health.
- Nucleoplasmic bridges in alveolar cells induced by nanomaterial exposurePublication . Ventura, Célia; Matos, Paulo; Pereira, Joana F.S.; Marques, Bárbara; Sousa-Uva, António; Silva, Maria JoãoIntroduction: Multi-walled carbon nanotubes (MWCNT) are widely used engineered nanomaterials in industrial and biomedical applications. Yet, MWCNT inhalation, particularly in the occupational settings, may induce pulmonary adverse effects as acute or persistent pulmonary inflammation, interstitial fibrosis, granuloma, and bronchiolar or bronchioloalveolar hyperplasia, and the MWCNT-7 (Mitsui-7) has been classified as possibly carcinogenic to humans. In this work, we analyse the in vitro genotoxicity of MWCNT-7 using the cytokinesis-block micronucleus assay to evaluates the ability of MWCNT-7 to affect the viability of alveolar epithelial cells (necrosis, apoptosis), its mitotic status (mononucleated, binucleated, multinucleated) and its chromosomal damage or instability status (presence of micronuclei, nucleoplasmic bridges and nuclear buds).
- Signaling Pathways Driving Aberrant Splicing in Cancer CellsPublication . Gonçalves, Vânia; Pereira, Joana F.S.; Jordan, PeterAberrant profiles of pre-mRNA splicing are frequently observed in cancer. At the molecular level, an altered profile results from a complex interplay between chromatin modifications, the transcriptional elongation rate of RNA polymerase, and effective binding of the spliceosome to the generated transcripts. Key players in this interplay are regulatory splicing factors (SFs) that bind to gene-specific splice-regulatory sequence elements. Although mutations in genes of some SFs were described, a major driver of aberrant splicing profiles is oncogenic signal transduction pathways. Signaling can affect either the transcriptional expression levels of SFs or the post-translational modification of SF proteins, and both modulate the ratio of nuclear versus cytoplasmic SFs in a given cell. Here, we will review currently known mechanisms by which cancer cell signaling, including the mitogen-activated protein kinases (MAPK), phosphatidylinositol 3 (PI3)-kinase pathway (PI3K) and wingless (Wnt) pathways but also signals from the tumor microenvironment, modulate the activity or subcellular localization of the Ser/Arg rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) families of SFs.
- 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.