Cellular and Molecular Mechanisms of Toxicity of Ingested Nanomaterials

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Investigating ingested nanomaterials’ safety – the case of TiO2 and innovative nanocelluloses.

Publication . Vital, Nádia; Silva, Maria João; Louro, Henriqueta

Introduction: The development of nanomaterials(NMs)-based technologies led to their increased use in key sectors and products related to food, food contact materials and feed. Many available products have NMs, as intentional constituents or contaminants from process ou food packaging release, such as silicon or titanium dioxide(TiO2) NMs. Others are being developed, like nanocelluloses(CNMs; doi:10.3390/nano12193375). However, it is recognised that the NMs’ specific physicochemical properties, conferring them unique benefi cial characteristics, can also elicit nano-bio interactions leading to toxic potential. Also, their dynamic behaviour in the surrounding matrix, may lead to secondary features determining the toxicological outcomes. Recognizing that processes like intake or digestion may modify the NMs’ characteristics leading to unexpected toxicity in human cells, EFSA included the use of in vitro digestion models in their specific guidelines concerning risk assessment of nanomaterials for food and feed(DOI:10.2903/j.efsa.2021.6768). Methodology: With the aim to contribute to the safety assessment of NMs, intestinal cell models (Caco-2 and HT29-MTX-E12 cells) were exposed to TiO2 NMs or innovative CNMs. Additionally, samples submitted previously to in vitro simulation of human digestion were used, and the genotoxicity(comet and micronucleus assays) was investigated with and without the digestion process. Results: After TiO2 NMs’ exposure, the micronucleus assay, an indicator of cancer risk, suggested eff ects on the chromosomal integrity in the HT29-MTX-E12 cells, for all the tested TiO2 NMs, especially after the in vitro digestion. Upon exposure to the two CNMs, no chromosomal damage was observed in the micronucleus assay, but the comet assay revealed DNA damage in the same cells, after 3h and 24h exposure, an effect slightly more relevant after the digestion of the cellulose nanofibril. Conclusion: Overall, the results show diff erent outcomes when using different NMs, and with/without digestion. Thus, it is important to consider the primary and secondary NMs’ characteristics determining the adverse eff ects, taking into account the human digestion for nanosafety assessment.

2023-09-04Conference object

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Nanotoxicologia e Nanosegurança

Publication . Louro, Henriqueta

Sobre avaliação de riscos dos nanomateriais.

2023-06-28Conference object

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In Vitro Cytotoxicity and Genotoxicity Assessment of Novel Cellulose Nanomaterials using intestinal cells

Publication . Vital, Nádia; Silva, Maria João; Kranendonk, M.; Louro, Henriqueta

Cellulose nanomaterials (CNMs) have been investigated for several applications, including in food and food packaging (e.g. as candidates for zero-calorie filler/thickener/stabilizers; as substitutes of petroleum-based food packaging materials). The widening of these applications will lead to human exposure via oral route, and potentially, to adverse health outcomes. To contribute to the CNMs safety evaluation, the aim of this study was to analyse the in vitro cytotoxicity and genotoxicity of two new micro/nanofibrillated celluloses (CMF/CNFs), using the HT29-MTX-E12 human intestinal cell model. CNMs were synthetized from industrial Eucalyptus globulus kraft and their physicochemical properties were characterized. Upon cells exposure to 3.1 - 200 μg/mL of CNMs during 24 h, the cytotoxicity was evaluated by the MTT and clonogenic assays, and the genotoxicity by the cytokinesis block micronucleus (CBMN) and comet assays. None of the CNMs was cytotoxic in the concentration-range tested. Concerning genotoxicity assessment, CMF induced a significant level of DNA damage (comet assay) in cells exposed for 3h to 25, 50 and 100 µg/mL and for 24h, to 50 µg/mL, compared with controls. No increases were observed with the FPG-modified comet assay compared with negative control. Cells treatment with the CNF for 3h significantly increased DNA damage at 14.3, 25, 50 µg/mL while a 24h treatment produced significant damage at 50 µg/mL, compared with control. For the latter concentration, induction of oxidative DNA damage was observed for both time points. In contrast, no increase in chromosomal damage was observed using the CBMN assay upon 52h of exposure. To our knowledge, this is the first study in which CNMs were evaluated for their genotoxic effects using the HT29MTX-E12 cell model, relevant for their potential ingestion. Our findings show that cytotoxicity, the endpoint generally used to assess their biocompatibility, is not sufficient to assess their safety to humans. Ongoing studies including the in vitro simulation of human digestion will allow a more comprehensive assessment of CNMs safety. This should be done at an early stage of their development, to ensure their sustainable and innovative application in food technology.

2023-05Conference object

Open access

Effects of TiO2 Nanoparticles on the Genome-Wide Methylation of Human Epithelial Intestinal Cells

Publication . Valente, Ana; Vieira, Luís; Silva, Catarina; Louro, Henriqueta; Silva, Maria João; Ventura, Célia

Introduction: Titanium dioxide nanoparticles (TiO2NP) have multiple applications in industry (e.g., engineering, cosmetics, food additives), and biomedicine (e.g., targeted drug delivery and biosensing). Food-grade TiO2 (E171) is applied as a food additive to whiten and improve the opacity of food products, while also having the ability to enhance its flavour. In 2021, EU member states banned E171 from all food products, since there is doubts about its genotoxicity. Nevertheless, the ingestion of TiO2NPs may still occur through to other sources, such as contaminated food or water, consumer products (e.g., toothpaste and lipstick) or pharmaceutics. To date, there is some in vitro evidence that TiO2NP may induce changes in DNA methylation. However, very few studies were performed, and none used genome-wide approaches to identify possible differentially methylated genes induced by TiO2NP exposure, and its impact on molecular pathways. Methodology: Caco-2 epithelial intestinal cells were exposed to 14 μg/ml of anatase, rutile or brookite phase TiO2NP for 24h. Genomic DNA was extracted from exposed and non-exposed cells. DNA libraries were generated using the Premium Reduced Representative Bisulfite Sequencing (RRBS) kit (Diagenode) and sequenced on the NextSeq 550 system (Illumina) using 100 bp paired reads. The Galaxy platform was used for read treatment and mapping, methylation calling and assessing of differentially methylated regions between exposed and non-exposed cells. Pathway analysis was performed using Reactome, and gene ontology analysis with the ClueGO plugin in Cytoscape. Results: Significant differential methylation (p ≤ 0.05) of 92 genes (21 hyper- and 71 hypo-methylated), 70 genes (12 hyper- and 58 hypo-methylated) and 88 genes (21 hyper- and 67 hypo-methylated) was observed for the anatase, rutile and brookite phase TiO2NP, respectively. Functional pathway analysis of these methylation changes identified several relevant cellular pathways that may be altered by exposure, such as G alpha signalling events, being some associated to colon cancer. Conclusions: All types of TiO2NP induce changes in genome methylation of intestinal cells, which may affect cell proliferation, differentiation and survival. Moreover, although some dysfunctional pathways are shared between the three TiO2NP, many are type-specific, suggesting different molecular mechanisms of action for each TiO2NP.

2023-11-17Conference object

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Investigation of In Vitro Cytotoxicity and Genotoxicity of Novel Cellulose Nanomaterials in two intestinal cell models

Publication . Vital, Nádia; Silva, Maria João; Kranendonk, Michel; Louro, Henriqueta

Cellulose nanomaterials (CNMs) have been developed for applications in multiple food-related products, as food additives (e.g. stabilizers or thickeners), non-caloric fiber sources, or substitutes for petroleum-based food packaging materials(1,2). This work aimed to contribute for the safety assessment of two micro/nanofibrillated celluloses (CMF/CNFs), synthetized from industrial Eucalyptus globulus kraft. Based on the European Food Safety Authority nano guidance(3), the in vitro cyto- and genotoxic effects were investigated using the Caco-2 and HT29-MTX-E12 human intestinal cell models. To incorporate the effect of the digestion process on the toxicological outcomes, a harmonized protocol for in vitro simulation of human digestion was used(3). After exposure of cells to digested and undigested CNMs samples (concentrations of 3.1 to 200 μg/mL), the cytotoxicity was evaluated by the MTT and clonogenic assays, and the genotoxicity by the cytokinesis block micronucleus (CBMN; OCDE TG 487(4)) and comet assays. No cytotoxic effects could be attributed to CNMs exposure, with and without digestion, regardless of the cell line used. No chromosomal damage was detected in the two cell lines exposed to each CNMs for 52h, using the micronucleus assay. Conversely, the comet assay revealed the induction of DNA damage in HT29-MTX-E12 cells, after 3h and 24h of exposure to the two CNMs, without significant contribution of oxidative DNA damage. Additionally, in the same cell line, a mild increase in DNA damage, was observed after exposure to the digested CNF comparatively to not digested CNF, after 3h exposure. To our knowledge, this is the first study in which CNMs were evaluated for their genotoxic effects using the CBMN and comet assays in Caco-2 and HT29MTXE12 cell models. Our findings show that cytotoxicity, the endpoint generally used to assess their biocompatibility, is not sufficient to assess their safety to humans. Ongoing studies will contribute to a more comprehensive early-stage assessment of CNMs safety, towards sustainable and innovative application in food technology.

2023-09-20Conference object

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