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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. FCT/MCTES projects PTDC/SAU-PUB/29481/2017; PTDC/SAU-PUB/32587/2017; UIDB/00009/2020; UIDP/ 00009/2020. Nádia Vital holds a FCT/MCTES PhD Scholarship grant (2020.07168.BD). N/A

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

About Investigation of the genotoxicity of digested titanium dioxide nanomaterials in human intestinal cells This work was funded by FCT/MCTES through national funds (PIDDAC), PTDC/SAU-PUB/29481/2017 and co-funded by UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health – ToxOmics, FCT), iMed.ULisboa (PestUID/DTP/04138/2018) and CESAM (UID/AMB/50017/2019). N/A

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

publicado em: Environ Mol Mutagen. 2021;62(S1):42 (P11). https://onlinelibrary.wiley.com/doi/epdf/10.1002/em.22458 The use of titanium dioxide nanoparticles (TiO2-NPs) as food additive and in food packaging demands a thorough assessment of their potential risk for human health, specifically with regard to gastrointestinal tract. Using intestinal cells, we analyzed the mechanisms by which digested TiO2-NPs (NM-102, NM-103 and NM-105, JRC repository) translocate through the intestinal epithelium layer, as compared to non-digested particles. Human digestion of TiO2-NPs was simulated using the INFOGEST in vitro harmonized digestion method. Caco-2 cells were grown as polarized cell monolayer for exposure and differentiation was evaluated by TEER. The translocation of TiO2-NPs, tagged with alizarin red, through the cell barrier was analysed by confocal microscopy, using colocalization with antibodies against specific endosomal compartments. The internalization of the TiO2-NPs was confirmed for the three TiO2-NPs tested, both before and after digestion simulation. The smallest TiO2-NPs were internalized into EEA1-positive early- endosomes and accumulated in late endosomes (Rab7), with only a small fraction following the degradative pathway to the lysosome (LAMP1). The data suggested that at least part of the TiO2-NPs could be redirected to the secretory pathway. Consistently, fluorescence passing from the apical to the basolateral chamber was observed, without disruption of the intestinal barrier function. The changes in cell function or signal transduction pathways are being studied and possible consequences to human gastrointestinal tract arediscussed. We thank the support from P. Alvito, C. Martins and R. Assunção (INSA,Lisbon, Portugal). Funded by FCT/MCTES-PIDDAC,PTDC/SAU-PUB/29481/2017, iMed.ULisboa (UID/DTP/04138/2019; UIDB/04138/2020); ToxOmics(UIDB/00009/2020; UIDP/00009/2020) and CEECIND/03143/2017 (L. Gonçalves). 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

Introduction: The increased use of titanium dioxide nanoparticles (TiO2-NPs) as a food additive demands a thorough assessment of their potential risk for human health. Via oral exposure they may lead to adverse local or systemic outcomes, and few studies have focused on the cellular internalization mechanisms (endocytosis) of TiO2-NPs. The objective was to analyze the mechanisms by which TiO2-NPs (NM-102, NM-103 and NM-105, JRC repository) translocate by the intestinal epithelium layer, using monolayers of human intestinal cell lines (Caco-2 and HT29-MTX), as well as polarized Caco-2 cells, and co-cultures of both cells. Results: We evaluated cell differentiation by transepithelial resistance measurements and the translocation of TiO2-NPs tagged with alizarin through the intestinal barrier by confocal microscopy and we confirmed the internalization of the TiO2-NPs in both cell line models. Co-localization studies suggested that the smallest TiO2-NPs were internalized into EEA1-positive early-endosomes and accumulate in late endosomes (Rab7), with only a small fraction following the degradative pathway to the lysosome (LAMP1). This suggested that at least part of the TiO2-NPs could be redirected to the secretory pathway. Consistently, we detected fluorescence passing from the apical (AP) to the basolateral (BL) chamber, depending on the characteristics of cell model and TiO2-NPs tested. Conclusions: Small TiO2-NPs were endocytosed by Caco-2 cells, with an increase in particle diameter suggesting intracellular aggregation, whereas larger agglomerates deposited mainly extracellularly. Following endocytosis, TiO2 NPs were trafficked through different intracellular compartments including early and late endosomes/endo-lysosomes, with part being subjected to AP to BL transport. Summary: TiO2-NPs may be translocated across the intestinal epithelium layer; This study contributes to a better mechanistic understanding of in vitro TiO2-NPs kinetics as well as their potential fate and effects on humans; It is expected that such an approach will reduce uncertainties in the hazard assessment of ingested TiO2-NPs for human health. Work funded by FCT/MCTES through national funds (PIDDAC), PTDC/SAU-PUB/29481/2017 iMed.ULisboa (UID/DTP/04138/2018), and co-funded by ToxOmics(UIDB/00009/2020; UIDP/00009/2020). LG has a Principal Researcher Grant CEECIND/03143/2017. N/A

Introduction: 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. FCT/MCTES (PTDC/SAU-PUB/29481/2017); ToxOmics(UIDB/00009/2020); iMed.ULisboa(UIDB/04138/2020, CEECIND/03143/2017); CESAM (UIDP/50017/2020+UIDB/50017/2020). 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 increased use of titanium dioxide nanoparticles (TiO2NPs) as a food additive demands a deep assessment of their potential risk for human health, including their abilities to cross biological barriers. In vitro models of the intestinal barrier are being increasingly used to evaluate NPs exposure risk. Most of these studies have focused on standard monoculture models of Caco-2 monolayers. However, they exhibit several limitations such as the lack of mucus layer and a low paracellular permeability. We aim to study TiO2NPs with an in vitro model of intestinal barrier using co-culture of two types of cells: absorptive Caco-2 and mucus-secreting HT29-MTX. This co-culture confers more physiological intestinal epithelium-like properties to the model, such as mucus secretion and tight junction formation, allowing a more adequate investigation of the cellular effects elicited by NPs. Due to the multiple variables and parameters playing a part when the model's complexity is increased, we characterized the robustness of this model by evaluating cell differentiation by confocal microscopy and Western blot while monitoring epithelial barrier formation, through measurement of both transepithelial resistance (TEER)and paracellular permeability (Lucifer yellow). An optimized model of the intestinal barrier will be used to better understand the uptake, adhesion and localization of TiO2NPs, both directly and after the simulation of the human digestive process using the harmonized in vitro digestion protocol. Preliminary data shows that these complex models can add valuable information to study the potential negative impacts and genotoxicity of TiO2NPs on human health. Funded by FCT/MCTES through national funds (PIDDAC), PTDC/SAU-PUB/29481/2017 and co-funded by ToxOmics (UIDB/00009/2020) 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

3 páginas, 1 figura, 2 tablas -- PAGS nros. 2328-2320, A total of 91 of 1,480 invasive isolates (6.1%) collected from adults in Barcelona, Spain, in the period of 1994 to 2008 were of serogroup 6 (6B, 47 isolates; 6A, 28; and 6C, 16). Throughout this period, serotype 6B (Spain6B-ST90) decreased, and serotype 6A remained stable. An increase in serotype 6C (ST224) in the 2004-2008 period was observed, This study was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (PI060647, PI081922, and PI091904) and by CIBER de Enfermedades Respiratorias (CIBERES; grant CB06/06/0037), a project run by the Instituto de Salud Carlos III. D.R. was supported by a grant from IDIBELL (Bellvitge Biomedical Research Institute) and an “Agustí Pumarola” scholarship from the Societat Catalana de Malalties Infeccioses and the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica