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- Early-stage nanosafety assessment as a critical tool for innovative nanomaterials developmentPublication . Louro, Henriqueta; Ventura, Célia; Rolo, Dora; Vital, Nádia; Pinto, Fátima; Silva, Maria JoãoThe perspectives of innovation through the use of nanomaterials (NMs) in key sectors such as agriculture, food industry, medicine, energy, environment, and electronics, has exponentially increased their development, production, and application. However, a major concern for public health is that some materials for long being considered safe for humans, e.g., titanium dioxide or cellulose fibers, can acquire different properties at the nanoscale that, despite being more attractive for industrial applications, may also elicit nano-bio interactions and toxic effects. Furthermore, their physicochemical properties can be influenced by the surrounding matrix or by physiological processes, such as digestion or inhalation, that modify their primary physicochemical properties. These secondary features may also influence the NMs toxicity and associated adverse health outcomes, such chronic inflammation and/or cancer. Therefore, the safety assessment of NMs must be conducted early in their development process and follow the nanotoxicology principles, in order to unveil the most relevant physicochemical characteristics that determine their potential adverse effects. In this work, the nanotoxicological investigation for the case studies of titanium dioxide NMs and nanocelluloses are presented. They illustrate the establishment of relationships between NMs characteristics and their toxicological properties and how they may direct the synthesis of innovative and safer NMs. If such tool is used at an early stage of NMs or product development, it moves industry towards a safe and sustainable by design (SSBD) approach that will enable safety to keep pace with innovation for the benefit of citizens. The Portuguese ISO/CEN Technical Commission for Nanotechnologies (CT194) is acknowledged for its role in bridging the gap between science and industry.
- Oral Exposure to TiO2 and Cellulose Nanomaterials: review of hazard identification in the adverse outcome pathway landscapePublication . Rolo, Dora; Vital, Nádia; Silva, Maria Raquel; Louro, HenriquetaSeveral products in the global market have been improved using titanium dioxide nanomaterials (TiO2 NMs), and many other NMs under development, e.g., cellulose NMs(CNMs), with potential for use in agriculture, food, and feed industries. Despite being considered key enabling technologies, the exponential use of NMs in food technology leads to concerns about adverse health outcomes upon ingestion, such as potential genotoxicity and cancer of the gastrointestinal tract (GIT) due to their bioaccumulation. The aim of this study, considering oral exposure to TiO2 NMs and CNMs as case studies, was to explore the knowledge about these NMs’ cellular and molecular mechanisms of action that may be central to their predicted adverse outcomes pathways at the GIT. For this purpose, literature reviews were setup to target the hazard of these NMs in the GIT context, directed to identify the molecular initiating event (MIE) and key events (KE) that mediate potential genotoxic and carcinogenic effects, thus contributing to Adverse Outcome Pathways (AOP) landscape. From the review of in vitro/in vivo studies, the suggested MIE involves the cellular uptake by intestinal cells and effects at lysosomal level. Several possible KE like inflammation, persistent cell injury/cell death, ROS generation, and DNA damage that may mediate the formation of adenomas/carcinomas were identified for TiO2 NMs; the information for CNMs is scarce. Some knowledge gaps were also identified, opening new avenues for more mechanistic research that will feed into AOPs.
- Global DNA methylation and Reactive Oxygen Species assessment after in vitro exposure to novel cellulose nanomaterials on intestinal cellsPublication . Vital, Nádia; Ventura, Célia; Kranendonk, M.; Silva, Maria João; Louro, HenriquetaInnovative cellulose nanomaterials (CNMs) find applications in multiple food-related products. However, it is recognized that nanomaterials (NMs) may cause adverse effects frequently by biological mechanisms that include production of intrinsic reactive oxygen species (ROS), or epigenetic alterations, by changes in gene expression through alterations in DNA methylation. To contribute to the safety assessment of two types of micro/nanofibrillated celluloses (CMF/CNFs) derived from industrial Eucalyptus globulus kraft, intracellular ROS levels and global DNA methylation patterns were evaluated using human intestinal cell models exposed to these CNMs. A harmonized protocol for in vitro simulation of human digestion was applied to incorporate the effect of potential changes in the physicochemical properties of CNMs due to the digestion process. After exposure of intestinal cells to digested and undigested CNMs samples (concentrations from 3.1 to 200 μg/mL, 3h and 24h), ROS levels were determined using the 2,7-dichlorofluorescein diacetate probe and fluorescence detection. The global DNA methylation was assessed after 24h exposure, using the methylated DNA Quantification Kit(colorimetric, Abcam) to quantify the levels of methylated cytosines (5-methylcytosine) in CpG dinucleotides. No increased ROS were observed after exposure to CNMs with and without digestion. Conversely, the preliminary results showed significant increased methylation of DNA after exposure to the CMF, without digestion, as compared to the negative control. No effect could be observed for digested sample, possibly due to the high background of the digestion product per se. Our preliminary findings seem to suggest the occurrence of biological effects of CNMs in intestinal cells that require further investigation, as may lead to a toxic impact on gastrointestinal cells, of concern for human health.
- Investigation of In Vitro Cytotoxicity and Genotoxicity of Novel Cellulose Nanomaterials in two intestinal cell modelsPublication . Vital, Nádia; Silva, Maria João; Kranendonk, Michel; Louro, HenriquetaCellulose 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.
- 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
- Normalização em Nanotecnologias SC1 – Terminologia e NomenclaturaPublication . Vital, NádiaDefinição e desenvolvimento de terminologia e nomenclatura inequívoca e uniforme no domínio das nanotecnologias, para facilitar a comunicação e promover o entendimento comum, entre as organizações e os indivíduos na indústria e aqueles que com eles interagem.
- Titanium dioxide nanomaterials - induced DNA damage in intestinal cells following simulated in vitro digestionPublication . Vieira, Adriana; Rolo, Dora; Vital, Nádia; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana; Silva, Maria João; Louro, HenriquetaIntroduction: 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.
- In Vitro Cytotoxicity and Genotoxicity Assessment of Novel Cellulose Nanomaterials using intestinal cellsPublication . Vital, Nádia; Silva, Maria João; Kranendonk, M.; Louro, HenriquetaCellulose 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.
- Investigating ingested nanomaterials’ safety – the case of TiO2 and innovative nanocelluloses.Publication . Vital, Nádia; Silva, Maria João; Louro, HenriquetaIntroduction: 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.
- Hazard Assessment of Benchmark Metal-Based Nanomaterials Through a Set of In Vitro Genotoxicity AssaysPublication . Vital, Nádia; Pinhão, Mariana; Yamani, Naouale El; Rundén-Pran, Elise; Louro, Henriqueta; Dušinská, Maria; Silva, Maria JoãoFor safety assessment of nanomaterials (NMs), in vitro genotoxicity data based on welldesigned experiments is required. Metal-based NMs are amongst the most used in consumer products. In this chapter, we report results for three metal-based NMs, titanium dioxide (NM- 100), cerium dioxide (NM-212) and silver (NM-302) in V79 cells, using a set of in vitro genotoxicity assays covering different endpoints: the medium-throughput comet assay and its modified version (with the enzyme formamidopyrimidine DNA glycosylase, Fpg), measuring DNA strand beaks (SBs) and oxidized purines, respectively; the micronucleus (MN) assay, assessing chromosomal damage; and the Hprt gene mutation test. The results generated by this test battery showed that all NMs displayed genotoxic potential. NM-100 induced DNA breaks, DNA oxidation damage and point mutations but not chromosome instability. NM-212 increased the level of DNA oxidation damage, point mutations and increased the MN frequency at the highest concentration tested. NM-302 was moderately cytotoxic and induced gene mutations, but not DNA or chromosome damage. In conclusion, the presented in vitro genotoxicity testing strategy allowed the identification of genotoxic effects caused by three different metal-based NMs, raising concern as to their impact on human health. The results support the use of this in vitro test battery for the genotoxicity assessment of NMs, reducing the use of more expensive, time-consuming and ethically demanding in vivo assays, in compliance with the 3 R’s.