Browsing by Author "Gramacho, Ana Catarina"
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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.
- 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.
- 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, 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.
- Challenges of the Application of In Vitro Digestion for Nanomaterials Safety AssessmentPublication . Vital, Nádia; Gramacho, Ana Catarina; Silva, Mafalda; Cardoso, Maria; Alvito, Paula; Kranendonk, Michel; Silva, Maria Joao; Louro, HenriquetaConsidering the increase in the production and use of nanomaterials (NM) in food/feed and food contact materials, novel strategies for efficient and sustainable hazard characterization, especially in the early stages of NM development, have been proposed. Some of these strategies encompass the utilization of in vitro simulated digestion prior to cytotoxic and genotoxic assessment. This entails exposing NM to fluids that replicate the three successive phases of digestion: oral, gastric, and intestinal. Subsequently, the resulting digestion products are added to models of intestinal cells to conduct toxicological assays, analyzing multiple endpoints. Nonetheless, exposure of intestinal cells to the digested products may induce cytotoxicity effects, thereby posing a challenge to this strategy. The aim of this work was to describe the challenges encountered with the in vitro digestion INFOGEST 2.0 protocol when using the digestion product in toxicological studies of NM, and the adjustments implemented to enable its use in subsequent in vitro biological assays with intestinal cell models. The adaptation of the digestion fluids, in particular the reduction of the final bile concentration, resulted in a reduced toxic impact of digestion products.
- Evaluation of the cytotoxicity and genotoxicity of ingested titanium dioxide nanomaterials in intestinal cellsPublication . Gramacho, Ana Catarina; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia; Simão Bettencourt, Ana; Paula, Alvito; Silva, Maria João; Louro, HenriquetaMany products already available, namely in agriculture and food, contain nanomaterials (NMs) and thus human ingestion of these compounds is probable through consumer products or food chain. 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 tested systems. Furthermore, the NMs properties have been recognized as being context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining the toxicological outcome. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. This work aimed to investigate the nano-bio interactions of titanium dioxide NMs (from Joint Research Centre, Ispra) in the context of intestinal tract and digestion processes, to better understand key events that may be linked to an adverse outcome pathway (AOP). In vitro for digestion was simulated and the NMs secondary properties in the intestinal cell moiety were characterized after this process. The cytotoxic and genotoxic effects of digested NMs were determined after the in vitro exposure of human intestinal cells (Caco-2). In addition, the FPG-comet assay was used to analyze oxidative DNA lesions. The digestion products without the NM showed cytotoxic effects above the concentration of 10% in cell culture medium, leading to the need to reduce its concentration below this level. Therefore, initial dose-range studies set a working range of NM concentrations of 0.14 up to 14 µg/ml (0.5-4.5 % of digestion product), which relates also with the predicted levels of exposure of human intestinal cells in real life conditions. Under these conditions, the preliminary results suggest that the tested titanium dioxide NMs do not yield cytotoxic or genotoxic effects upon 24h of exposure of Caco-2 cells, directly after the digestion process. Future studies will investigate the subcellular localization of NMs, integrity of cell junctions, activation of stress signaling pathways and secretion of inflammatory cytokines, to allow an integrated approach to potential adverse effects of the NMs. By elucidating key events elicited by NMs, linking exposure to adverse outcomes, it is expected to contribute to the safety evaluation of NMs within an AOP landscape.
- Ingested nanomaterials: effects of titanium dioxide in human cellsPublication . Gramacho, Ana Catarina; Silva, Maria João; Louro, HenriquetaNowadays, the consumer products containing nanomaterials (NMs) are numerous. Among these, titanium dioxide nanomaterials (TiO2) are the most frequently applied, used as food additive, pharmaceuticals, toothpastes and many more. The wide commercialization of consumer products containing TiO2 contributes to a substantial increase of human exposure to this compound, which is worrying for public health, since the existent studies generated contradictory results about TiO2 safety. The fact that NMs are dependent of the context (surrounding matrix) can contribute to explain the contradictory results reported in the literature. Furthermore, it has been hypothesized that NMs physicochemical properties may define bio-nano interactions, which suggest that NMs with the same chemistry but with different shape, diameter, length, surface charge or functionalization may lead to different toxicities (Louro et al., 2019). This work aimed to assess whether the secondary physicochemical characteristics of three different TiO2, i.e., the characteristics after the digestion process, produce different biological effects in intestinal cells. The three NMs were obtained from the Joint Research Centre, Ispra, Italy (NM-102, NM-103 and NM-105), and display different charge, size and crystal structure, coated and uncoated, as characterized by the producer (Rasmussen et al., 2014). An in vitro digestion process was used to mimic human digestion (Brodkorb et al., 2019) and the resulting product was used for cytotoxicity assessment in a human intestinal cell line (Caco-2), in comparison to undigested NMs. In addition, cellular uptake was investigated using confocal microscopy. Preliminary results did not evidence a differential biological effect between the three NMs. Following 24h of exposure of Caco-2 cells, all the digested NMs behaved similarly at the concentrations tested (0.14 – 14.3 µg/ml) showing no cytotoxic effects. Likewise, no cytotoxic effects were observed upon exposure to undigested NMs. However, the results showed the nuclear localization of one NM, NM-102, suggesting that it may interact with the genome in cells exposed to this TiO2. Ongoing studies will confirm the subcellular localization of the NMs in exposed intestinal cells and will investigate their genotoxic effects in relation with the secondary properties.
- 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.
- Nanomateriais ingeridos: impacto biológico e relação com as suas características físico-químicasPublication . Gramacho, Ana Catarina; Silva, Maria João; Louro, HenriquetaA utilização de nanomateriais (NMs) em setores variados, designadamente, agricultura e indústria alimentar, encontra-se amplamente disseminada. Assim, muitos produtos alimentares contêm NMs, sendo provável a sua ingestão pelo homem. Torna-se, pois, essencial garantir que esses NMs ingeridos não produzem efeitos adversos, nomeadamente, genotoxicidade que se relaciona com carcinogénese. Neste estudo, pretendemos avaliar uma possível associação entre as propriedades físico-químicas de NMs com a mesma constituição química, dióxido de titânio (TiO2), e os seus efeitos biológicos em células humanas e, por outro lado, identificar as propriedades que determinam a sua toxicidade. Para tal, analisou-se a citotoxicidade de um conjunto de quatro NMs de TiO2 tendo-se observado que apenas um deles induzia morte celular. Relativamente aos efeitos genotóxicos, apesar de nenhum NM ter induzido alterações cromossómicas (ensaio do micronúcleo), observou-se que apenas os NMs na forma cristalina anatase (mas não rutilo) induziam efeitos lesivos no DNA (ensaio do cometa). Neste último ensaio, verificou-se ainda a existência de uma correlação inversa entre o tamanho do NM em meio de cultura e a sua genotoxicidade. Futuramente, investigaremos o efeito do processo de digestão nas características destes NMs, bem como nos seus efeitos genotóxicos in vitro, visando a tradução dos resultados em potenciais implicações na saúde humana.