Research Institute for Medicines

Funder

Organizational Unit

Publications

Analysis of the In Vitro Toxicity of Nanocelluloses in Human Lung Cells as Compared to Multi-Walled Carbon Nanotubes

Publication . 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 João

Cellulose 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.

2022-04-22Journal article

Open access

Oral Exposure to Titanium dioxide nanoparticles: a Systematic Literature Review as a tool to develop Adverse Outcome Pathway landscapes and supporting in vitro assays

Publication . Rolo, Dora; Silva, Maria João; Louro, Henriqueta

Despite being considered key enabling technologies, the exponential use of nanoparticles in food technology leads to concerns about adverse health outcomes upon ingestion. The use of Titanium dioxide nanoparticles (TiO2-NPs) as a food additive was considered no longer safe by the European Food Safety Authority (EFSA) in 2022, and the European Commission announced the decision to ban its use. Nevertheless, other products containing TiO2-NPs, such as pharmaceuticals, personal hygiene, or cosmetics, that are not covered in the food regulation, may lead to ingestion of TiO2-NPs. In this regard, further research is needed. A valuable tool is the establishment of Adverse Outcome Pathways (AOPs) landscapes, which contributes to support risk assessment and may drive policy decisions. AOPs describes a sequence of causally linked events at different levels of biological organization leading to adverse health effects. Our main goal was to further understand the molecular and cellular mechanisms, triggered after the ingestion of TiO2-NPs. A systematic literature review was performed, integrating information produced on this topic and provide data for a standardized assessment of the evidence. AOP landscapes were proposed in order to identify mechanisms that mediate adverse outcomes, and additional in vitro assays were performed. Our in vitro findings suggest that part of the ingested TiO2-NPs can be transcytosed through colonic epithelia without disrupting intestinal barrier integrity. These results are consistent with our AOPs proposal where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. Based on a mechanistic reasoning, this study contributes to increase the understanding of ingested TiO2-NPs kinetics, their bioavailabity to induce systemic effects crossing the intestinal barrier and potential fate in Humans. Overall, the findings further support a limitation of the use of TiO2-NPs in food, as announced by EFSA.

2022-11-16Lecture

Restricted access

Titanium dioxide nanomaterials - induced DNA damage in intestinal cells following simulated in vitro digestion

Publication . Vieira, Adriana; Rolo, Dora; Vital, Nádia; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana; Silva, Maria João; Louro, Henriqueta

Introduction: The increased use of titanium dioxide nanomaterials (TiO2) in food products has raised oral exposure to those nanomaterials, with subsequent risks to human health, particularly genotoxicity and, ultimately, cancer development. In humans, the digestion process may modify the physicochemical properties of TiO2, thereby shaping the potential biological outcomes. Thus, such process should be considered when assessing their hazard upon oral exposure. This work aimed to investigate the genotoxic effects of three TiO2 (NM-102, NM-103 and NM-105, JRC repository) after the simulation of the human digestive process using the standardized INFOGEST in vitro digestion method. The secondary physicochemical properties and DNA damage levels, using the comet assay, were analysed in two intestinal cell lines exposed for 24h to digested or undigested TiO2. Results: An increase in the level of DNA strand breaks in two intestinal cell lines(Caco-2 and HT29-MTX-E12) was observed after exposure to digested NM-105, concomitantly with a decrease in its hydrodynamic size, comparatively to the undigested nanomaterial. Moreover, the digested NM-103 induced DNA damage in Caco-2 cells whereas the undigested nanomaterial did not. The FPG-modified comet assay also revealed an increase in oxidative DNA lesions upon treatment of Caco-2 with NM-103 and HT29-MTX-E12 with NM-102. Conclusions: One of the digested TiO2(NM-105) can be classified as potentially genotoxic in both cell lines, while the digested NM-103 induced an equivocal genotoxic response in Caco-2 cells. Therefore, the digestion simulation is of relevance to investigate the potential genotoxic effects of ingested nanomaterials.

2021-04-20Conference object

Restricted access

Titanium Dioxide Nanoparticles molecular effects: Internalization in the Human Intestinal Epithelium

Publication . Rolo, Dora; Pereira, Joana FS; Matos, Paulo; Gonçalves, Lídia; Bettencourt, Ana Francisca; Jordan, Peter; Silva, Maria João; Louro, Henriqueta

The use of titanium dioxide nanoparticles (TiO2-NPs) as food additive and in food packaging demands a thorough assessment of their potential risk for human health, specifically with regard to gastrointestinal tract. Using intestinal cells, we analyzed the mechanisms by which digested TiO2-NPs (NM-102, NM-103 and NM-105, JRC repository) translocate through the intestinal epithelium layer, as compared to non-digested particles. Human digestion of TiO2-NPs was simulated using the INFOGEST in vitro harmonized digestion method. Caco-2 cells were grown as polarized cell monolayer for exposure and differentiation was evaluated by TEER. The translocation of TiO2-NPs, tagged with alizarin red, through the cell barrier was analysed by confocal microscopy, using colocalization with antibodies against specific endosomal compartments. The internalization of the TiO2-NPs was confirmed for the three TiO2-NPs tested, both before and after digestion simulation. The smallest TiO2-NPs were internalized into EEA1-positive early- endosomes and accumulated in late endosomes (Rab7), with only a small fraction following the degradative pathway to the lysosome (LAMP1). The data suggested that at least part of the TiO2-NPs could be redirected to the secretory pathway. Consistently, fluorescence passing from the apical to the basolateral chamber was observed, without disruption of the intestinal barrier function. The changes in cell function or signal transduction pathways are being studied and possible consequences to human gastrointestinal tract arediscussed. We thank the support from P. Alvito, C. Martins and R. Assunção (INSA,Lisbon, Portugal).

2021-09-23Conference object

Restricted access

Ingested nanomaterials: impact of digestion process in the physicochemical characteristics and biological consequences in intestinal cells

Publication . 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, Henriqueta

Nanomaterials(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.

2021-11-11Conference object

Restricted access