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- 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
- 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.
- Evaluation of the cyto- and genotoxicity of two types of cellulose nanomaterials using human intestinal cells and in vitro digestion simulationPublication . Vital, Nádia; Cardoso, Maria; Kranendonk, Michel; Silva, Maria Joao; Louro, HenriquetaEmerging cellulose nanomaterials (CNMs) may have commercial impacts in multiple sectors, being their application particularly explored in the food sector. Thus, their potential adverse effects in the gastrointestinal tract should be evaluated before marketing. This work aimed to assess the safety of two CNMs (CNF–TEMPO and CMF–ENZ) through the investigation of their cytotoxicity, genotoxicity (comet and micronucleus assays), and capacity to induce reactive oxygen species in human intestinal cells, and their mutagenic effect using the Hprt gene mutation assay. Each toxicity endpoint was analysed after cells exposure to a concentration-range of each CNM or to its digested product, obtained by the application of a standardized static in vitro digestion method. The results showed an absence of cytotoxic effects in intestinal cells, up to the highest concentration tested (200 µg/mL or 25 µg/mL, for non-digested and digested CNMs, respectively). Of note, the cytotoxicity of the digestion control limited the top concentration of digested samples (25 µg/mL) for subsequent assays. Application of a battery of in vitro assays showed that CNF–TEMPO and CMF–ENZ do not induce gene mutations or aneugenic/clastogenic effects. However, due to the observed DNA damage induction, a genotoxic potential cannot be excluded, even though in vitro digestion seems to attenuate the effect. The lowest digested CNF–TEMPO concentration induced chromosomal damage in Caco-2 cells, leading to an equivocal outcome. Ongoing research on epigenotoxic effects of these CNMs samples may strengthen the lines of evidence on their safety when ingested, paving the way for their innovative application in the food industry.
- 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.