Browsing by Author "Vital, Nádia"
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- 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.
- Analysis of the in vitro cytotoxicity and genotoxicity of cellulose nanomaterials in intestinal cellsPublication . Vital, Nádia; Pinto, Fátima; Kranendonk, Michel; Silva, Maria João; Louro, HenriquetaCellulose nanomaterials(CNMs) are recognized as promising bionanomaterials due to their natural and renewable source and attractive properties, with potential applications in multiple food-related products, as zero-calorie filler/thickener/stabilizer, or as substitutes of petroleum-based food packaging materials. These applications in food lead to human exposure through ingestion and their biopersistence raise concerns about their potential impact on the gastrointestinal tract. The present study aimed to investigate the in vitro cytotoxicity and genotoxicity of two types of micro/nanofibrillated celluloses(CMF/CNFs) using a human intestinal cell line. CMF and CNF were synthetized from industrial bleached Eucalyptus globulus kraft and their physicochemical properties were previously characterized. Following Caco-2 cells exposure to a concentration-range of CMF or CNF, the cytotoxicity was assessed by the MTT and clonogenic assays, while genotoxicity was assessed by the cytokinesis block micronucleus assay(CBMN). Outcomes indicated no cytotoxic or genotoxic effects upon exposure of Caco-2 cells to these CNMs for the tested concentration-range, suggesting their biocompatibility. This is the first study using the CBMN assay after Caco-2 cells exposure to CNMs. The results of ongoing studies using in vitro simulation of human digestion will allow a more comprehensive assessment of CNMs safety. By incrementing the knowledge on the cellular effects of novel CNMs in the human intestine, we expect to contribute to their safety assessment at an early-stage of their technological development towards the sustainable innovation in food technology.
- Analysis of the In Vitro Toxicity of Nanocelluloses in Human Lung Cells as Compared to Multi-Walled Carbon NanotubesPublication . Pinto, Fátima; Lourenço, Ana Filipa; Pedrosa, Jorge F.S.; Gonçalves, Lídia; Ventura, Célia; Vital, Nádia; Bettencourt, Ana; Fernandes, Susete N.; da Rosa, Rafaela R.; Godinho, Maria Helena; Louro, Henriqueta; Ferreira, Paulo J.T.; Silva, Maria JoãoCellulose micro/nanomaterials (CMNM), comprising cellulose microfibrils (CMF), nanofibrils (CNF), and nanocrystals (CNC), are being recognized as promising bio-nanomaterials due to their natural and renewable source, attractive properties, and potential for applications with industrial and economical value. Thus, it is crucial to investigate their potential toxicity before starting their production at a larger scale. The present study aimed at evaluating the cell internalization and in vitro cytotoxicity and genotoxicity of CMNM as compared to two multi-walled carbon nanotubes (MWCNT), NM-401 and NM-402, in A549 cells. The exposure to all studied NM, with the exception of CNC, resulted in evident cellular uptake, as analyzed by transmission electron microscopy. However, none of the CMNM induced cytotoxic effects, in contrast to the cytotoxicity observed for the MWCNT. Furthermore, no genotoxicity was observed for CNF, CNC, and NM-402 (cytokinesis-block micronucleus assay), while CMF and NM-401 were able to significantly raise micronucleus frequency. Only NM-402 was able to induce ROS formation, although it did not induce micronuclei. Thus, it is unlikely that the observed CMF and NM-401 genotoxicity is mediated by oxidative DNA damage. More studies targeting other genotoxicity endpoints and cellular and molecular events are underway to allow for a more comprehensive safety assessment of these nanocelluloses.
- Are standard genotoxicity tests useful for the safety evaluation of nanomaterials?Publication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Antunes, Susana; Costa, Pedro; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNanomaterials (NMs) are widely used in a diversity of consumer products, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. One of the major concerns is the potential to induce cancer. To analyze in a short term the carcinogenic properties of a compound, genotoxicity assays in mammalian cell lines or animal models are frequently used. In the context of an EU Joint Action, in the present work we have used standard genotoxicity assays (comet, micronucleus and mutation assays) to investigate the effects associated with the exposure to titanium dioxide nanomaterials (TiO2), following standardized dispersion and assay procedures, in three types of human cells and in a mouse model. The results showed slight but significant increases in the frequencies of micronuclei after exposure to some of the NMs, as compared to controls. No clear dose-response relationships could be disclosed. One of the tested TiO2 yielded equivocal results in vitro micronucleus assay and was positive in the comet assay in pulmonary cells. In view of the inconclusive results,it was further analyzed in vivo, using the lacZ transgenic mouse model. It did not induce genotoxic effects in mice, 28 days after injection, despite the accumulation of the NM observed in the mouse liver. Regarding safety assessment, the different genotoxicity observed for closely related NMs, but that differ in some physicochemical characteristics, highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs. The equivocal genotoxicity of the nanosized TiO2 in human cells in vitro was not confirmed in vivo, and therefore the predictive value of these in vitro tests for identifying genotoxic (and potentially carcinogenic) NMs in vivo should be clarified, before extrapolating the conclusions for human health.
- Assessment of the genotoxicity of a titanium dioxide nanomaterial using a combination of in vitro and in vivo assaysPublication . Tavares, Ana; Louro, Henriqueta; Vital, Nádia; Antunes, Susana; Lavinha, João; Silva, Maria JoãoHuman exposure to manufactured nanomaterials such as titanium dioxide (TiO2), often used in sunscreens and cosmetics, has increased worldwide. Their specific properties, such as size and high surface area/mass, render them attractive for many applications, but may also be associated to higher toxicity in biological systems and adverse effects in humans. In the context of EU Joint Action NANOGENOTOX (www.nanogenotox.com), the present work aimed to analyse the potential genotoxic effects of a well-characterized TiO2 nanomaterial, correlating in vitro and in vivo effects. TiO2 dispersions were prepared according to a standardized protocol and were used for exposure of human cells (in vitro) or mice (in vivo). The cytokinesis-block micronucleus assay (OECD guideline 487) was performed in human bronchial epithelial cells (BEAS-2B) and primary cultures of human lymphocytes. Additionally, Comet assay was conducted in BEAS-2B cells. In vivo testing was carried out on a mouse model after exposure of groups of mice intravenously. The mammalian erythrocyte micronucleus test in mouse blood (OECD guideline 474) and comet assay in mouse organs were performed. Concurrent positive chemical controls and a nanoparticle control (ZnO) were included. While the results obtained in BEAS-2B cells showed no induction of micronucleated cells, a significant increase was observed in human lymphocytes at the dose of 125 μg/ml. Exposure of BEAS-2B to TiO2 caused an increase in DNA damage detected by comet assay (3-fold increase, p< 0.006) although no dose-response effect was seen. In mice, there was no genotoxicity in both assays. In summary, using a standardized preparation of nanomaterials, results obtained were mostly negative after TiO2 exposure, in both in vitro and in vivo assays. However, somewhat different genotoxicity outcomes may reflect tissue-specific effects affecting, e.g., cellular uptake of the nanomaterial.
- Aula 8: Efeitos celulares e genéticos dos nanomateriais ingeridosPublication . Vital, Nádia; Louro, Henriqueta; Silva, Maria JoãoSobre os efeitos celulares e genéticos dos nanomateriais ingeridos.
- Avaliação da Segurança de Nanomateriais de Celulose InovadoresPublication . Vital, Nádia; Silva, Maria João; Kranendonk, M.; Louro, HenriquetaIntrodução: Na sociedade atual verifica-se uma preocupação acrescida com a exposição humana a materiais que, embora previamente considerados seguros, poderão revelar-se perigosos para a saúde quando usados à nanoescala. Este poderá ser o caso dos nanomateriais de celulose (CNMs), produzidos de fontes naturais, explorados para diversas aplicações, em particular na indústria alimentar. Podem ser usados como aditivos alimentares (e.g. estabilizadores ou espessantes), fonte de fibra não calórica, ou em embalagens alimentares(1). Antecipa-se um aumento da exposição humana, por ingestão de alimentos contendo CNMs, contaminados através das embalagens alimentares ou por acumulação no ambiente(1). Porém, o conhecimento sobre a sua segurança, em particular os seus efeitos no trato gastrointestinal (GIT), permanece limitado(1). É imperativo estudar os seus potenciais efeitos no organismo após ingestão oral, antes da sua colocação no mercado, avaliando a sua segurança com base nas recomendações da Autoridade Europeia para a Segurança Alimentar(2). Objetivos: Este trabalho visou investigar os potenciais efeitos de dois CNMs no GIT, através da avaliação de potenciais efeitos citotóxicos e genotóxicos em dois modelos celulares intestinais humanos (Caco-2 e HT29-MTX-E12). Metodologia: Estudaram-se duas celuloses micro/nanofibriladas (CNFs) inovadoras, produzidas a partir de polpa kraft branqueada industrial de Eucalyptus globulus. A citotoxicidade foi avaliada pelos ensaios MTT e clonogénico, enquanto a genotoxicidade foi analisada pelos ensaios do micronúcleo e do cometa. Resultados: Não se observou efeitos citotóxicos após exposição aos CNFs nas concentrações testadas, independentemente da linha celular utilizada. Também, não se observaram alterações cromossómicas nas duas linhas celulares expostas a cada CNFs através do ensaio do micronúcleo. Porém, os resultados do ensaio do cometa revelaram a indução de quebras no DNA das células HT29-MTX-E12, após 3h e 24h de exposição para as duas CNFs. Esse efeito foi inconsistente nas células Caco-2 após um tratamento semelhantes com as mesmas amostras. Conclusões: Os resultados obtidos suscitam preocupação sobre a segurança destas CNMs, pois observou-se um efeito genotóxico que deverá continuar a ser investigado, através de testes complementares. Destaca-se a importância de investigar os efeitos genotóxicos, associados a um potencial efeito carcinogénico, para além de uma simples análise da sua citotoxicidade, como é frequentemente utilizada para afirmar a biocompatibilidade de CNMs. Assim, deverá promover-se novas metodologias de produção, uma maior purificação ou uma alteração das propriedades físico-químicas destas CNFs, para reduzir a sua toxicidade, prevenindo, assim, eventuais efeitos nefastos na saúde humana. Referências: 1. Vital N, Ventura C, Kranendonk M, Silva MJ, Louro H. Toxicological Assessment of Cellulose Nanomaterials: Oral Exposure. Nanomaterials [Internet]. 2022 Sep 27; 12(19): 3375 2. EFSA Scientific Committee. Guidance on risk assessment of nanomaterials to be applied in the food and feed chain: human and animal health. EFSA Journal [Internet]. 2021 Aug 3; 19(8):6768
- Avaliação integrada dos efeitos genotóxicos de nanomateriais manufaturados no ratinho transgénico LacZPublication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Costa, Pedro M.; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNum estudo recente, demonstrámos que um nanomaterial de dióxido de titânio na forma cristalina designada anatase, o NM-102 (do repositório do Joint Research Center; Ispra, Itália), induziu um aumento significativo de quebras cromossómicas - detetáveis na forma de micronúcleos - em linfócitos humanos expostos ex vivo(3) não se tendo, porém, observado um efeito dose-resposta. No sentido de prosseguir e aprofundar a avaliação da genotoxicidade do NM-102, o presente estudo teve por objetivo caracterizar os seus efeitos genotóxicos, num modelo animal, utilizando uma abordagem integrada, a qual abrangeu a análise de vários parâmetros de genotoxicidade no mesmo animal.
- 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.