Toxicological profile of cellulose nanomaterials in human gastrointestinal cells

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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

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Analysis of the in vitro cytotoxicity and genotoxicity of cellulose nanomaterials in intestinal cells

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

Cellulose 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.

2022-09Conference object

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Cellular and Molecular Mechanisms of Toxicity of Ingested Titanium Dioxide Nanomaterials

Publication . Vieira, Adriana; Gramacho, Ana; Rolo, Dora; Vital, Nádia; Silva, Maria João; Louro, Henriqueta

An exponential increase in products containing titanium dioxide nanomaterials (TiO2), in agriculture, food and feed industry, lead to increased oral exposure to these nanomaterials (NMs). Thus, the gastrointestinal tract (GIT) emerges as a possible route of exposure that may drive systemic exposure, if the intestinal barrier is surpassed. NMs have been suggested to produce adverse outcomes, such as genotoxic effects, that are associated with increased risk of cancer, leading to a concern for public health. However, to date, the differences in the physicochemical characteristics of the NMs studied and other variables in the test systems have generated contradictory results in the literature. Processes like human digestion may change the NMs characteristics, inducing unexpected toxic effects in the intestine. Using TiO2 as case-study, this chapter provides a review of the works addressing the interactions of NMs with biological systems in the context of intestinal tract and digestion processes, at cellular and molecular level. The knowledge gaps identified suggest that the incorporation of a simulated digestion process for in vitro studies has the potential to improve the model for elucidating key events elicited by these NMs, advancing the nanosafety studies towards the development of an adverse outcome pathway for intestinal effects.

2022Book part

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Oral Exposure to TiO2 and Cellulose Nanomaterials: review of hazard identification in the adverse outcome pathway landscape

Publication . Rolo, Dora; Vital, Nádia; Silva, Maria Raquel; Louro, Henriqueta

Several 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.

2022-09-24Conference object

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