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publicado em: Environ Mol Mutagen. 2022 Aug;63(Suppl 1):88. (Abstracts from the 13th International Conference on Environmental Mutagens and 53rd Annual Meeting of the Environmental Mutagenesis and Genomics Society). https://onlinelibrary.wiley.com/toc/10982280/2022/63/S1 Human exposure to titanium dioxide nanomaterials(TiO2NMs) occurs particularly by ingestion, due to food/food contact materials and consumer products. However, the possibility of adverse effects in gastrointestinal tract is unclear. Aiming to study the impact of digestion on the NMs’ properties and their cellular/molecular effects, two human intestinal cell lines were used, Caco-2 and HT29-MTX-E12. After exposure to TiO2NMs(NM-102, NM-103, NM-105), undigested or subjected to standardized static in vitro digestion method (mimicking human digestion), the cells were analyzed for toxicity, genotoxicity, reactive oxygen species, NM uptake and intestinal translocation. We showed that in vitro digestion of TiO2NMs may increase their toxicity and DNA-damaging effect, depending on the NM, more relevant for the rutile/anatase NM-105, possibly due to its smaller hydrodynamic size in the cellular medium. Effects on chromosomal integrity were seen in HT29-MTX-E12 cells, for all tested TiO2NMs, especially after digestion. Internalization into early endosomes was confirmed for NM-103 and NM-105, before and after digestion, in monolayers of both cell lines, and at the apical membrane of polarized Caco-2 cells. The internalized NMs accumulated in late endosomes/multivesicular bodies, partially transversing the basolateral membrane of polarized Caco-2 cells without changing transepithelial electrical resistance or epithelial marker abundance. These results suggest that part of the TiO2NMs can be transcytosed through colonic epithelia without disrupting intestinal barrier integrity. Overall, the biological outcomes from TiO2NMs interaction with intestinal cells were more pronounced after digestion, highlighting its relevance in the hazard assessment of ingested NMs. Funded by FCT/MCTES through the projects: PTDC/SAU-PUB/29481/2017, UIDB/00009/2020; UIDP/00009/2020;UIDP/50017/2020+UIDB/50017/2020+LA/P/ 0094/2020; CEECIND/03143/2017 (LG), 2020.07168.BD (NV). info:eu-repo/semantics/publishedVersion

Nanomaterials(NMs) provide a basis for key enabling technologies, in view of their potential to improve many products and processes, namely in food and feed industry. That is the case of titanium dioxide NMs(TiO2 NMs), presenting beneficial properties for a broad range of innovative applications such as food additives, toothpaste, pharmaceuticals, food products, etc., that may drive ingestion. The oral exposure can occur directly, by consumption of products/pharmaceuticals or foods containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or environmental sources. As such, the gastrointestinal tract is the first site of contact of the ingested NMs, allowing a systemic exposure if the intestinal barriers is surpassed. This work aimed to investigate how the digestion process affects the physicochemical properties of three different TiO2 NMs(NM-102, NM-103 and NM-105) and their toxic effects on intestinal cells. After undergoing digestion through the standardized static INFOGEST 2.0 in vitro digestion method, the cytotoxicity of the TiO2 NMs was determined in Caco-2 and HT29-MTX-E12 intestinal cells, using the MTT assay. Furthermore, the cytokinesis-blocked micronucleus assay was used to investigate their genotoxicity in both cell lines in order to predict their carcinogenic potential. The results showed that, for one TiO2 NM(NM-105), the digestion caused changes in the hydrodynamic size of the NM and a more pronounced toxicity in HT29-MTX-E12 intestinal cells, as compared to the undigested one. The micronucleus assay suggests effects on the chromosomal integrity in the HT29-MTXE12 cells, for all the tested TiO2 NM especially after the in vitro digestion. Overall, we conclude that including the digestion prior to the in vitro bioassays for the safety evaluation of ingested NMs, allows integrating the physiological modifications that the NMs suffer in the organism, contributing to an improved hazard assessment of ingested NMs. The authors acknowledge Joint Research Centre(JRC, Ispra, Italy) for providing the NMs and funding by the Portuguese Foundation for Science and Technology FCT/MCTES through national funds(PIDDAC), PTDC/SAU-PUB/29481/2017, Toxicogenomics(UIDB/00009/2020; UIDP/00009/2020), iMed.ULisboa(UIDB/04138/2020 and UIDP/04138/2020) and CESAM(UIDP/50017/2020+UIDB/50017/2020. L. Gonçalves Principal Researcher grant (CEECIND/03143/2017). FCT/MCTES PhD studentship 2020.07168.BD was attributed to NV. N/A

Cellulose 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. FCT/MCTES through national funds (PTDC/SAU-PUB/29481/2017; PTDC/SAUPUB/ 32587/2017) and (UIDB/00009/2020; UIDP/00009/2020); FCT/MCTES PhD studentship 2020.07168.BD was attributed to NV info:eu-repo/semantics/publishedVersion

The 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. Work funded by FCT/MCTES through national funds (PIDDAC) PTDC/SAU-PUB/29481/2017, PTDC/SAU-PUB/32587/2017, and UIDB/00009/2020; UIDP/00009/2020). NV holds a FCT PhD Scholarship grant 2020.07168.BD. N/A

The technology based on manufactured nanomaterials (NMs) has been pointed as key enabling technology, due to its potential to improve many products and processes, namely in agriculture, food and feed industry, leading to an exponential growth. Many products, already available, have NMs, such titanium dioxide NMs used as food additives, and many others are in development. Oral exposure may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of foods contaminated with NMs released from food-contact materials or even through concentration in the food chain due to environmental accumulation. Therefore, the gastrointestinal tract appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. One major concern for public health is that NMs may produce biological effects, such as genotoxicity that are associated with increased risk of cancer. Although NMs have been extensively investigated in recent years, the studies have generated contradictory results, possibly due to differences in the physicochemical properties of the NMs studied and to other variables in the test systems. This work aimed to investigate the nano-bio interactions of titanium dioxide NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential adverse impacts on human health. The results of the NMs uptake by intestinal cells, as well as their cytotoxic and genotoxic effects will be presented. This nanotoxicology approach may be incorporated at early-stage in the development of new NMs for food industry, in a “safe-by-design” approach that will enable safety to keep pace with innovation. This work was funded by FCT/MCTES through national funds (PIDDAC), PTDC/SAUPUB/29481/2017 and co-funded by UIDP/00009/2020 (Centre for Toxicogenomics and Human Health – ToxOmics, FCT), iMed.ULisboa (Pest-UID/DTP/04138/2018) and CESAM (UID/AMB/50017/2019). info:eu-repo/semantics/publishedVersion

Titanium dioxide nanomaterials (TiO2) have been frequently applied as food additives, in pharmaceuticals and in personal care products, such as toothpastes. Despite some regulators like EFSA concluded that the absorption of orally administered TiO2 is low, and that the use of TiO2 as a food additive does not raise a genotoxic concern, the presence of TiO2 in human organs was recently reported. This exposure may lead to adverse outcomes and has been poorly investigated. Furthermore, many of the biological effects of TiO2 described in the literature often overlook adequate physicochemical properties and their modification due to NMs interaction with the surrounding physiological matrices happening, e.g, during digestion. This work aimed to investigate in intestinal cells, the cyto- and genotoxic effects of TiO2 after the simulation of the human digestive process using the standardized INFOGEST in vitro digestion method, to better understand their potential negative impacts on the gastrointestinal tract. The TiO2 were characterized before and after digestion using DLS, zeta potential and TEM-EDS. The digestion product was used for cytotoxicity (MTT) and genotoxicity (comet, micronucleus) assays in two types of intestinal cells (Caco-2 and mucus secreting HT29-MTX cells). The results of the cytotoxicity and genotoxicity assays are discussed in view of the TiO2 secondary characteristics, to further understand the potential adverse intestinal outcomes in light of the transformation they suffer during digestion. Funded by FCT/MCTES through national funds (PIDDAC), PTDC/SAU-PUB/29481/2017 and co-funded by UIDB/00009/2020+UIDP/00009/2020 (Centre for Toxicogenomics and Human Health – ToxOmics, FCT), iMed.ULisboa (PestUID/DTP/04138/2019) and CESAM (UID/AMB/50017/2019). N/A

The exponential development of nanomaterials (NMs) contrasts with insufficient risk assessment for human health and the environment, leading to concerns for public health. Their potential to improve many products and processes, namely in agriculture, food and feed industry, has led to increased use of NMs, such as titanium dioxide nanomaterials (TiO2). In fact, oral exposure to TiO2 may occur either directly, through the consumption of products/pharmaceuticals containing NMs, or indirectly, through the ingestion of food/water contaminated due to environmental accumulation. Therefore, the gastrointestinal tract (GIT) appears to be a probable route of exposure to NMs and may lead to systemic exposure if the body barriers are surpassed. Since NMs primary physicochemical properties may define nano-bio interactions1, NMs with the same chemistry but with different shape, diameter, length, surface charge or functionalization may lead to different toxicities. Conversely, since the GIT is chemically and physically complex, ingested NMs will pass through different environments prior to their intestinal uptake, affecting their physicochemical properties, so that these secondary physicochemical properties should be considered while evaluating their safety in the food chain. The aim of this work was to investigate the nano-bio interactions of three TiO2 (NM-102, NM-103, NM-105, from Joint Research Centre, Ispra) in the context of intestinal tract and digestion processes. As alternative to animal testing, an in vitro harmonized digestion method2 was used for simulating the human digestion of NMs and their characteristics were studied in acellular and intestinal (Caco-2) cells context. The NMs were characterized using dynamic light scattering (DLS, for size distribution), electrophoretic light scattering (zeta potential), transmission electron microscopy (TEM) for morphological characterization and X-ray energy dispersive spectrometry (EDS) for elemental chemical analysis. TiO2 samples were labelled directly with a fluorescent probe and used for uptake studies with confocal microscopy (CM). In parallel, a co-culture model of Caco-2 with mucous-secreting HT29-MTX was initiated and exposed to TiO2 to ascertain epithelial barrier translocation. The results did not show major differences in the NMs’ pH, osmolality, charge signal or mean size, except for NM-103. TEM and TEM –EDS analysis showed that it is possible to identify primary particles after digestion process and preliminary CM results suggest that some of the tested TiO2 nanomaterials can be uptake by the cells, suggestive of potential subcellular effects that warrant further investigation. References [1] H. Louro, A. Saruga, J. Santos, M. Pinhão, M.J. Silva, Toxicol. In Vitro. 56 (2019)172–183. [2] A. Brodkorb, L.Egger, M. Alminger, P. Alvito et al. Nat Protoc. 2019 Apr;14(4):991-1014. Funded by FCT/MCTES through national funds (PIDDAC), PTDC/SAU-PUB/29481/2017 and co-funded by UID/BIM/00009/2013 (Centre for Toxicogenomics and Human Health – ToxOmics, FCT), iMed.ULisboa (Pest-UID/DTP/04138/2018) and CESAM (UID/ AMB/50017/2019). info:eu-repo/semantics/publishedVersion