Browsing by Author "Kranendonk, Michel"
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- 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.
- 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.
- Cytotoxicity Assessment of Novel Cellulose Nanomaterials on Intestinal CellsPublication . Vital, Nádia; Pinto, Fátima; Rolo, Dora; Pedrosa, Jorge; Ferreira, Paulo J.T.; Kranendonk, Michel; Silva, Maria João; Louro, HenriquetaCellulose nanomaterials (CNMs) emerged as an important group of nanomaterials with potential applications in multiple food-related products, as zero-calorie filler/thickener/stabilizer, or as a substitute for petroleum-based food packaging materials. Human oral exposure to CNMs is increasing, but little is known about the potential adverse biological impact of CNMs on human gastrointestinal tract. To contribute to the development of innovative CNMs for the food sector and to their early-stage safety assessment, in this work new CNMs were prepared, and their cytotoxic effects were investigated in human intestinal cell lines. Two cellulose micro/nanofibrillated (CNFs), were synthetized from industrial bleached Eucalyptus globulus kraft pulp using different pretreatments (enzymatic and TEMPO-mediated oxidation) followed by a high-pressure homogenization process. Potential cytotoxic effects were evaluated by the MTT assay using two human intestinal cell models (Caco-2 and HT29-MTX-E12). Since in humans the digestion process may modify the physicochemical properties of NMs, potentially influencing biological outcomes, the CNFs were subjected to a harmonized in vitro digestion method before cytotoxicity testing. No cytotoxic effect was observed after 24h exposure to the undigested CNFs in the concentration-range tested (3.1 µg/mL-200 µg/mL), irrespectively of the cell line used. Similar results were obtained for the digested CNFs for concentrations up to 14.3 µg/mL. The observation that the in vitro digestion mixture was cytotoxic by itself for concentrations above 7.6 % (v/v) (i.e., equivalent to CNMs exposure > 14.3 µg/mL) impaired cytotoxicity assessment at higher CNFs concentrations. Complementary cytotoxicity assays and future optimization of the in vitro digestion procedure to reduce its toxicity are underway, to refine the assessment of CNFs cytotoxicity, particularly after digestion. Furthermore, genotoxicity studies will increase the knowledge on the cellular effects of CNMs in the human intestine, contributing to the safety assessment of CNMs early in its development stage, towards sustainable innovation of nanomaterials, thereby protecting human health.
- 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.
- 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.
- Toxicological assessment of cellulose nanomaterials: focus on oral exposurePublication . Vital, Nádia; Kranendonk, Michel; Louro, HenriquetaAbout cellulose nanomaterials (CNMs).
- Toxicological Assessment of Cellulose Nanomaterials: Oral ExposurePublication . Vital, Nádia; Ventura, Célia; Kranendonk, Michel; Silva, Maria João; Louro, HenriquetaCellulose nanomaterials (CNMs) have emerged recently as an important group of sustainable bio-based nanomaterials (NMs) with potential applications in multiple sectors, including the food, food packaging, and biomedical fields. The widening of these applications leads to increased human oral exposure to these NMs and, potentially, to adverse health outcomes. Presently, the potential hazards regarding oral exposure to CNMs are insufficiently characterised. There is a need to understand and manage the potential adverse effects that might result from the ingestion of CNMs before products using CNMs reach commercialisation. This work reviews the potential applications of CNMs in the food and biomedical sectors along with the existing toxicological in vitro and in vivo studies, while also identifying current knowledge gaps. Relevant considerations when performing toxicological studies following oral exposure to CNMs are highlighted. An increasing number of studies have been published in the last years, overall showing that ingested CNMs are not toxic to the gastrointestinal tract (GIT), suggestive of the biocompatibility of the majority of the tested CNMs. However, in vitro and in vivo genotoxicity studies, as well as long-term carcinogenic or reproductive toxicity studies, are not yet available. These studies are needed to support a wider use of CNMs in applications that can lead to human oral ingestion, thereby promoting a safe and sustainable-by-design approach. View Full-Text