Browsing by Author "Rolo, Dora"
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- Adverse Outcome Pathways (AOPs) development, a tool for predictive nanotoxicologyPublication . Rolo, Dora; Louro, HenriquetaNanomaterials (NMs) have the potential to improve novel and useful wide applications in electronics, chemicals, environmental protection, biological medicine, food and others. Therefore, NMs rapid proliferation presents a dilemma to regulators regarding hazard identification, with increased concerns for public health. Predictive nanotoxicology describes a multidisciplinary approach to NMs evaluation that uses a set of in vitro and in silico methods to forecast the effects on biological systems. This approach offers advantages to traditional hazard assessment methods, such as reducing the reliance on animal studies, associated costs and ethical issues. It may be used with several applications in environmental and human health risk assessment and NMs hazard identification, as well as for regulation. The Adverse Outcome Pathways (AOPs) are the central element of a toxicological knowledge framework, promoted by member countries through OECD, built to support chemical risk assessment based on mechanistic reasoning. AOPs describes a logical sequence of causally linked events at different levels of biological organisation, which follows exposure and leads to an adverse health effect in humans or wildlife. The integrative analysis of the cellular and molecular mechanisms of nanotoxicity towards a definition of key events, may lead to adverse outcomes, driving a sequential line and defining an AOP landscape. Each defined AOP is available for crossing data, linking known and unknown landscapes. Since the biological effects that relate to possible genotoxicity and increased risk of cancer due to NMs exposure are under analysis, the development and assessment of AOPs are important novel strategic tools for predictive nanotoxicology.
- Adverse Outcome Pathways Associated with the Ingestion of Titanium Dioxide Nanoparticles - A Systematic ReviewPublication . Rolo, Dora; Assunção, Ricardo; Ventura, Célia; Alvito, Paula; Gonçalves, Lídia; Martins, Carla; Bettencourt, Ana; Jordan, Peter; Vital, Nádia; Pereira, Joana; Pinto, Fátima; Matos, Paulo; Silva, Maria João; Louro, HenriquetaTitanium dioxide nanoparticles (TiO2-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO2-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO2-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO2-NPs in food, announced by the European Food Safety Authority (EFSA).
- 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.
- 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.
- Aula 9: Vias de Efeitos Adversos e Avaliação de RiscoPublication . Rolo, Dora; Silva, Maria João; Louro, HenriquetaSobre Vias de Efeitos Adversos e Avaliação de Risco.
- Biological effects of ingested nanomaterials and potential adverse outcomes for human healthPublication . Vieira, Adriana; Gramacho, Ana Catarina; Rolo, Dora; Vital, Nádia; Pereira, Joana; Matos, Paulo; Jordan, Peter; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana F.; Silva, Maria João; Louro, HenriquetaThe technology based on manufactured nanomaterials (NMs) has been pointed as key enabling technology, due to its potential to improve many products and processes, namely in agriculture, food and feed industry, leading to an exponential growth. Many products, already available, have NMs, such titanium dioxide NMs used as food additives, and many others are in development. Oral exposure may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or even through concentration in the food chain due to environmental accumulation. Therefore, the gastrointestinal tract appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. One major concern for public health is that NMs may produce biological effects, such as genotoxicity that are associated with increased risk of cancer. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the test systems. This work aimed to investigate the nano-bio interactions of titanium dioxide NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential adverse impacts on human health. The results of the NMs uptake by intestinal cells, as well as their cytotoxic and genotoxic effects will be presented. This nanotoxicology approach may be incorporated at early-stage in the development of new NMs for food industry, in a “safe-by-design” approach that will enable safety to keep pace with innovation.
- Cell culture process. Virtual Lab videoPublication . Pinto, Fátima; Rolo, Dora; Vital, Nádia; Ventura, Célia; Moreira, Rodrigo; Cadete, João; Louro, Henriqueta; Silva, Maria JoãoCell Culture Process. Virtual Lab video.
- Cellular and molecular mechanisms of toxicity of ingested nanomaterialsPublication . Gramacho, Ana Catarina; Rolo, Dora; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia M.; Bettencourt, Ana; Alvito, Paula; Pereira, Joana; Jordan, Peter; Silva, Maria João; Louro, HenriquetaThe technology based on manufactured nanomaterials (NMs) has been pointed as key enabling technology, due to its potential to improve many products and processes, namely in agriculture, food and feed industry. Many of such products, already available, have NMs such as titanium dioxide nanomaterials (TiO2) and the oral exposure may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or even through concentration in the food chain 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. One major concern for public health is that NMs may produce adverse outcomes (AO) such as genotoxic effects that are associated with increased risk of cancer. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the test systems. INSA has previously shown that NMs with the same chemistry, but differing in primary properties may yield different biological effects. Conversely, the NMs properties are context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining toxicological outcomes. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. INGESTnano project aims to investigate the nano-bio interactions of NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential negative impacts on human health with special reference to organ-specific cells. TiO2 has been selected as case-study to setup a workflow for addressing nanosafety concerns that may be in the future applied to other NMs to which GIT may be exposed. It is expected that this project will contribute to the safety evaluation of the TiO2 ingested, by elucidating key events (KE) elicited by these NMs and linking exposure to AO.
- Cellular and Molecular Mechanisms of Toxicity of Ingested Titanium Dioxide NanomaterialsPublication . Vieira, Adriana; Gramacho, Ana; Rolo, Dora; Vital, Nádia; Silva, Maria João; Louro, HenriquetaAn exponential increase in products containing titanium dioxide nanomaterials (TiO2), in agriculture, food and feed industry, lead to increased oral exposure to these nanomaterials (NMs). Thus, the gastrointestinal tract (GIT) emerges as a possible route of exposure that may drive systemic exposure, if the intestinal barrier is surpassed. NMs have been suggested to produce adverse outcomes, such as genotoxic effects, that are associated with increased risk of cancer, leading to a concern for public health. However, to date, the differences in the physicochemical characteristics of the NMs studied and other variables in the test systems have generated contradictory results in the literature. Processes like human digestion may change the NMs characteristics, inducing unexpected toxic effects in the intestine. Using TiO2 as case-study, this chapter provides a review of the works addressing the interactions of NMs with biological systems in the context of intestinal tract and digestion processes, at cellular and molecular level. The knowledge gaps identified suggest that the incorporation of a simulated digestion process for in vitro studies has the potential to improve the model for elucidating key events elicited by these NMs, advancing the nanosafety studies towards the development of an adverse outcome pathway for intestinal effects.
- 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.