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- Differentially expressed microRNAs in human bronchial epithelial cells after exposure to nanofibrillar, microfibrillar or nanocrystalline cellulosePublication . Ventura, Célia; Vieira, Luís; Silva, Catarina; Louro, Henriqueta; Silva, Maria JoãoIn recent years, there has been a growing interest in nanocellulose, aninnovative engineered nanomaterial with physicochemical propertiesthat give it an enormous potential for use in awide varietyof industrialand biomedical applications. This expanding use is raising concern sabout its potential effects on human health after occupational,environmental or consumer exposure. To date, several studies aboutthe potential nanocellulose toxicity have been performed, mainly invitro, some indicating its biocompatibility, others suggesting geno-toxic or inflammatory effects, depending on the nanocellulose specificphysicochemical properties [1]. Nevertheless, to our knowledge, nostudy has addressed nanocellulose epigenotoxicity by analyzing itseffects at the microRNA expression level. Thus, this study aimed atidentifying the differentially expressed microRNAs (DEmiRNAs) in human bronchial epithelial cells (BEAS-2B cells), after 24 h exposure tothree different types of nanocellulose, two fibrillar (CNF and CMF;cellulose nanofibrils and cellulose microfibrils) and one crystalline(CNC; cellulose nanocrystals) derived fromEucaliptus globuluskraftpulp. For this purpose, microRNAs extracted from exposed and non-exposed cells were sequenced on a NextSeq 550 (Illumina). DEmiRNAswere obtained using sRNAtoolbox, and only DEmiRNAs identified atleast by two different methods were considered for further analysis.The results showed that both CNF and CMF did not change microRNAexpression on BEAS-2B cells, as they did not induce any statisticallysignificant (FDR⍰0.05) DEmiRNA as compared to non-exposed cells.By contrast, CNC induced the over- and under-expression of 22 and 30microRNAs, respectively. The ongoing bioinformatics study about theBEAS-2B cellular pathways that are enriched with these DEmiRNAswill give mechanistic insights that might help predicting its toxico-logical outcomes. Moreover, the profile of DEmiRNAs identified afterexposure to CNC will be further investigated in order to explore itspotential use as an effect biomarker for human biomonitoring studies.
- Safety and efficacy of a feed additive consisting of endo‐1,4‐β‐d‐mannanase produced by Thermothelomyces thermophilus DSM 33149 (Natupulse® TS/TS L) for chickens and turkeys for fattening, minor poultry species for fattening and ornamental birds (BASF SE)Publication . Bampidis, Vasileios; Azimonti, Giovanna; Bastos, Maria de Lourdes; Christensen, Henrik; Durjava, Mojca; Dusemund, Birgit; Kouba, Maryline; López‐Alonso, Marta; López Puente, Secundino; Marcon, Francesca; Mayo, Baltasar; Pechová, Alena; Petkova, Mariana; Ramos, Fernando; Sanz, Yolanda; Villa, Roberto Edoardo; Woutersen, Ruud; Brantom, Paul; Herman, Lieve; Louro, Henriqueta; Anguita, Montserrat; Brozzi, Rosella; Galobart, Jaume; Innocenti, Matteo Lorenzo; Ortuño, Jordi; Pettenati, Elisa; Pizzo, Fabiola; Revez, JoanaFollowing a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of endo-1,4-β-d-mannanase produced by Thermothelomyces thermophilus DSM 33149 (Natupulse® TS/TS L) as a zootechnical feed additive for chickens and turkeys for fattening, minor poultry species for fattening and ornamental birds. The additive under assessment, Natupulse® TS/TS L, does not pose any safety concern regarding the production strain. The FEEDAP Panel concluded that the additive is tolerated by chickens for fattening, and this conclusion can be extrapolated to all poultry for fattening. Due to the lack of reliable data regarding the potential of the additive to induce chromosomal damage, the FEEDAP Panel cannot conclude on the safety of the additive for the target species and on the safety for consumer. The use of the additive in animal nutrition is considered safe for the environment. The additive is considered not to be irritant to the skin and eyes but is considered a respiratory sensitiser, although exposure by inhalation is unlikely. The Panel could not conclude on the potential of the additive to be a skin sensitiser. Due to the lack of reliable data, the FEEDAP Panel considered that a potential of the additive to induce chromosomal damage in exposed unprotected users cannot be excluded. Consequently, exposure of users should be minimised. The Panel concluded that the additive Natupulse® TS/TS L has the potential to be efficacious in chickens for fattening at the proposed conditions of use and this conclusion can be extrapolated to turkeys for fattening, minor poultry species for fattening and ornamental birds.
- Analysis of the In Vitro Toxicity of Nanocelluloses in Human Lung Cells as Compared to Multi-Walled Carbon NanotubesPublication . 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 JoãoCellulose 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.
- Genotoxicity in peripheral blood leukocytes and reticulocytes of e-waste management workersPublication . Aimonen, K.; Silva, Maria João; Tavares, Ana; Moreira, Rodrigo; Louro, Henriqueta; Catalán, J.; Duca, R.; Godderis, L.; Mahiout, S.; Martins, C.; Martinsone, I.; Matisane, L.; Namorado, S.; Van Nieuwenhuyse, A.; Pinhal, Hermínia; Porras, S.; Remes, J.; Verdonck, J.; Viegas, S.; Scheepers, P.; Santonen, T.The European Commission has recently adopted a new Circular Economy Action Plan, which recognizes the critical role of material circulation in achieving sustainable development. The waste management sector will play a pivotal role in this, and an increase in the number of workers involved in waste recycling is expected. However, a recent multi-centric study conducted as part of the European Human Biomonitoring Initiative (HBM4EU) highlights potential occupational health risks associated with e-waste management. This study reports the exposure of e-waste workers from six European countries to metals and the early genotoxic effects from exposure to a wide array of toxic chemicals (and their mixtures) in the occupational environment. The results showed that e-waste workers are exposed to higher levels of hazardous metals such as lead (Pb), cadmium (Cd), mercury (Hg), and chromium (Cr) than controls. Significantly higher levels were detected in the post-shift urine and blood samples of different subcategories of e-waste workers. Especially the level of Pb was elevated in urine and blood samples among all worker categories, and the highest values were detected in battery recycling workers. Genotoxic effects were assessed by the micronucleus frequencies in peripheral blood lymphocytes (MNPBL) and reticulocytes (MNRET). MNPBL were analyzed in 95 workers and 50 controls, and MNRET in 82 workers and 41 controls. While there were no statistically significant differences between all workers and controls, the subgroup of battery recycling workers showed significantly higher frequency of MNPBL than controls or other exposed subgroups such as workers handling white goods, metals and plastics or miscellaneous e-waste. The highest MNPBL and MNRET frequencies were observed in workers handling brown goods, but the difference to controls, or to the other worker categories, was not statistically significant. Post-shift urinary levels of studied metals did not show positive correlation with the micronucleus frequencies, which might be partially explained by the heterogeneity of activities considered and related exposure levels found. In addition to metals, the study also measured exposure to flame retardants, phthalates, and polychlorinated biphenyls (PCBs) and, thereby, correlations between exposure to those compounds and effect biomarkers will be further explored. Overall, the study highlights the need to raise awareness of potential hazards and improve risk management measures in the e-waste management sector. The micronucleus results provide valuable new information on early biological effects from occupational exposures during e-waste management that also contribute to identifying worker groups that are at higher risk of adverse health effects.
- Safety assessment of a new matrix metalloproteinase inhibitor extracted from Lupinus albus seeds, through the analysis of its cytotoxic and genotoxic propertiesPublication . Bischoff, Nicolaij; Mota, J.; Louro, Henriqueta; Ventura, Célia; Ferreira, J.R.B.; Lima, A,; Silva, Maria JoãoColorectal cancer (CRC) is the third most common cancer worldwide, being highly metastatic and mostly resistant to anticancer treatment. Especially due to its high incidence in young people and the lack of adequate treatment, CRC is an increasing health risk for future generations. Since death of most colon cancer patients is due to metastatic disease, rather than the primary tumors itself, the mechanisms in the metastatic progression of CRC are intensively being studied. Subgroups of matrix metalloproteinase´s (MMP´s), especially gelatinases (MMP-2, MMP-9) have been identified to be frequently involved in the metastatic process, which makes them a useful drug target. On the other hand, experimental, epidemiological and clinical studies have shown that the consumption of legumes significantly reduces the incidence of several tumor types, including CRC. These effects are generally associated with the inhibitory action of legume seeds on MMP’s. MMP play a key role in cellular homeostasis due to their ability to initiate, enhance or down-regulate signal cascades, involving cell growth, inflammation, cytokine and growth-factor release. Overexpression of MMPs results in matrix degradation and is strongly associated with cancer cell invasion and metastasis. Deflamin is a novel matrix metalloproteinase inhibitor (MMPI) extracted from the seeds of Lupinus albus, which exhibits anti-inflammatory properties, particular at the gastrointestinal level and has been pointed as a promising cancer preventive agent. It shows especially high inhibitory activity against MMP-2 and MMP-9. This makes deflamin a great candidate to become a valuable anti-inflammatory nutraceutical agent, as well as a useful asset in the treatment of inflammatory bowel diseases (IBD). However potential secondary adverse effects must be avoided and for that purpose an early evaluation of its potential toxic effects to human cells is needed. This work is aimed at contributing to the safety evaluation of deflamin through the analysis of cytotoxic and genotoxic properties of the purified deflamin and a Lupinus albus extract in Caco-2 cells. Furthermore, its bioavailability and transport via a Caco-2 monolayer system will be tested to get further insights on possible uptake scenarios in the human intestine. The cytotoxic effects are analyzed by assessing cell viability (MTT assay), following Caco-2 cells exposure to a concentration range (5-640 µg/ml) of the pure deflamin or the extract during 24, 48 and 72 hours. Genotoxicity is assessed by the alkaline comet assay and the cytokinesis-block micronucleus assay. The effects on the intestinal barrier are analyzed by measuring the transepithelial resistance (TER) in a differentiated Caco-2 monolayer upon apical exposure to deflamin or the extract. Preliminary data suggests that neither the pure deflamin nor the extract display cytotoxicity in the tested concentration range. The results of the genotoxicity studies will be presented and discussed, in the light of the future application of deflamin as a chemopreventive agent, ensuring the absence of adverse side effects.
- Developing human biomonitoring as a 21st century toolbox within the European exposure science strategy 2020-2030Publication . Zare Jeddi, Maryam; Hopf, Nancy B.; Louro, Henriqueta; Viegas, Susana; Galea, Karen S.; Pasanen-Kase, Robert; Santonen, Tiina; Mustieles, Vicente; Fernandez, Mariana F.; Verhagen, Hans; Bopp, Stephanie K.; Antignac, Jean Philippe; David, Arthur; Mol, Hans; Barouki, Robert; Audouze, Karine; Duca, Radu-Corneliu; Fantke, Peter; Scheepers, Paul; Ghosh, Manosij; Van Nieuwenhuyse, An; Lobo Vicente, Joana; Trier, Xenia; Rambaud, Loïc; Fillol, Clémence; Denys, Sebastien; Conrad, André; Kolossa-Gehring, Marike; Paini, Alicia; Arnot, Jon; Schulze, Florian; Jones, Kate; Sepai, Ovnair; Ali, Imran; Brennan, Lorraine; Benfenati, Emilio; Cubadda, Francesco; Mantovani, Alberto; Bartonova, Alena; Connolly, Alison; Slobodnik, Jaroslav; Bruinen de Bruin, Yuri; van Klaveren, Jacob; Palmen, Nicole; Dirven, Hubert; Husøy, Trine; Thomsen, Cathrine; Virgolino, Ana; Röösli, Martin; Gant, Tim; von Goetz, Natalie; Bessems, JosHuman biomonitoring (HBM) is a crucial approach for exposure assessment, as emphasised in the European Commission’s Chemicals Strategy for Sustainability (CSS). HBM can help to improve chemical policies in five major key areas: (1) assessing internal and aggregate exposure in different target populations; 2) assessing exposure to chemicals across life stages; (3) assessing combined exposure to multiple chemicals (mixtures); (4) bridging regulatory silos on aggregate exposure; and (5) enhancing the effectiveness of risk management measures. In this strategy paper we propose a vision and a strategy for the use of HBM in chemical regulations and public health policy in Europe and beyond. We outline six strategic objectives and a roadmap to further strengthen HBM approaches and increase their implementation in the regulatory risk assessment of chemicals to enhance our understanding of exposure and health impacts, enabling timely and targeted policy interventions and risk management. These strategic objectives are: 1) further development of sampling strategies and sample preparation; 2) further development of chemical-analytical HBM methods; 3) improving harmonisation throughout the HBM research life cycle; 4) further development of quality control / quality assurance throughout the HBM research life cycle; 5) obtain sustained funding and reinforcement by legislation; and 6) extend target-specific communication with scientists, policymakers, citizens and other stakeholders. HBM approaches are essential in risk assessment to address scientific, regulatory and societal challenges. HBM requires full and strong support from the scientific and regulatory domain to reach its full potential in public and occupational health assessment and in regulatory decision-making.
- Effects of TiO2 Nanoparticles on the Genome-Wide Methylation of Human Epithelial Intestinal CellsPublication . Valente, Ana; Vieira, Luís; Silva, Catarina; Louro, Henriqueta; Silva, Maria João; Ventura, CéliaIntroduction: Titanium dioxide nanoparticles (TiO2NP) have multiple applications in industry (e.g., engineering, cosmetics, food additives), and biomedicine (e.g., targeted drug delivery and biosensing). Food-grade TiO2 (E171) is applied as a food additive to whiten and improve the opacity of food products, while also having the ability to enhance its flavour. In 2021, EU member states banned E171 from all food products, since there is doubts about its genotoxicity. Nevertheless, the ingestion of TiO2NPs may still occur through to other sources, such as contaminated food or water, consumer products (e.g., toothpaste and lipstick) or pharmaceutics. To date, there is some in vitro evidence that TiO2NP may induce changes in DNA methylation. However, very few studies were performed, and none used genome-wide approaches to identify possible differentially methylated genes induced by TiO2NP exposure, and its impact on molecular pathways. Methodology: Caco-2 epithelial intestinal cells were exposed to 14 μg/ml of anatase, rutile or brookite phase TiO2NP for 24h. Genomic DNA was extracted from exposed and non-exposed cells. DNA libraries were generated using the Premium Reduced Representative Bisulfite Sequencing (RRBS) kit (Diagenode) and sequenced on the NextSeq 550 system (Illumina) using 100 bp paired reads. The Galaxy platform was used for read treatment and mapping, methylation calling and assessing of differentially methylated regions between exposed and non-exposed cells. Pathway analysis was performed using Reactome, and gene ontology analysis with the ClueGO plugin in Cytoscape. Results: Significant differential methylation (p ≤ 0.05) of 92 genes (21 hyper- and 71 hypo-methylated), 70 genes (12 hyper- and 58 hypo-methylated) and 88 genes (21 hyper- and 67 hypo-methylated) was observed for the anatase, rutile and brookite phase TiO2NP, respectively. Functional pathway analysis of these methylation changes identified several relevant cellular pathways that may be altered by exposure, such as G alpha signalling events, being some associated to colon cancer. Conclusions: All types of TiO2NP induce changes in genome methylation of intestinal cells, which may affect cell proliferation, differentiation and survival. Moreover, although some dysfunctional pathways are shared between the three TiO2NP, many are type-specific, suggesting different molecular mechanisms of action for each TiO2NP.
- Ingested nanomaterials: impact of digestion process in the physicochemical characteristics and biological consequences in intestinal cellsPublication . Vieira, Adriana; Vital, Nádia; Roque, Rossana; Gramacho, Ana Catarina; Rolo, Dora; Gonçalves, Lídia D.; Bettencourt, Ana; Martins, Carla; Assunção, Assunção; Alvito, Paula; Silva, Maria João; Louro, HenriquetaNanomaterials(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.
- AD6.2 WP6 - Sustainability and capacity building. Results of the interaction and surveys with the Environment Protection Agency network and the National Reference Centre on Environment & HealthPublication . Lobo Vicente, Joana; Katsonouri, Andromachi; Hans, Reynders; Campenhout, Karen Van; Tarroja, Elena; Louro, Henriqueta; Isidro, Glória; Silva, Maria João; Bourqui, Martine; von Goetz, NatalieThe aim of this additional deliverable is to analyse the result of the interaction and surveys with the Environment Protection Agency network and the National Reference Centre on Environment & Health. The consultation to both the EPA network and the NRCs was done through a similar targeted survey, with the aim of understanding their perception of the current HBM4EU and their ideas for a future Human Biomonitoring initiative. The online survey also explored if and how their institution would use HBM4EU results in their work and it also explored the focus that their institution considered a future initiative might take. This could be in terms of exploring exposure to new emerging risks or understanding exposure to known chemical risks, such as heavy metals. In addition, it explored interest in participating in a new initiative and openness towards sharing of best practice. This included questions regarding the possible role that the EPAs and NRCs might play in such an initiative. The invitation was sent out to 29 EPA countries and 15 NRCs, from which 19 answers were obtained in total with 4 EPAs answered and 13 NRCs. One of the EPAs is also a HBM4EU partner, whereas from the 15 NRCs, 13 of those provided their countries/institution from which 7 are HBM4EU partners. The survey produced quite interesting results, where it was pointed out that HBM is the only instrument that can assess human exposure in an integrated and reliable way. From the institutions that are not part of HBM4EU, most of them were aware of the project. The ones that were already part of it would like to carry on as such if a future initiative is to take place. Despite the fact that a slightly higher percentage of institutions do not use HBM at the moment in their work (53 % vs 47 %), all of the institutions recognised the value of HBM and plan on using it in the future. All the NRCs and 75 % of the EPAs are interested in being part of a future initiative with a possible role in: positioning Human Biomonitoring in the strategic agenda of implementation of environmental policy and state of the environment in Europe, supporting the activities of the HBM initiative through an existing interest group under the NRC Network, creating joint working initiatives with other relevant networks. It was clear from the answers given that HBM has triggered an interest and there is a willingness to be part of the future initiative.
- Occupational exposure to emerging cellulose nanomaterials and potential respiratory effects: uncovering exposure-effect relationships using in vitro systemsPublication . Ventura, Célia; Pinto, Fátima; Vieira, Luís; Louro, Henriqueta; Silva, Maria JoãoThe application of innovative cellulose nanomaterials (CNMs), particularly, cellulose micro- and nanofibrils (CMF, CNF) and nanocrystals (CNC), in technology and biomedicine has expanded in recent years. However, there is still a lack of reliable indicators to assess workers’ exposure, mainly by inhalation, and the associated health outcomes. This study aimed at identifying genotoxic, oxidative stress, or epigenetic endpoints that could be used as promising effect biomarkers to predict health outcomes from CNMs exposure, exploitable for human biomonitoring. Three well-characterized CNMs were used, at a low concentration range, to expose cell systems representative of the respiratory tract, namely, bronchial epithelial (BEAS-2B) cells or co-cultures of lung (A549) and macrophage-like (THP-1) cells. No induction of chromosomal instability was detected in co-cultures for all types of CNMs analysed through the cytokinesis-block micronucleus assay. Likewise, neither significant toxic effects nor oxidative stress was found. Interestingly, preliminary data showed differentially expressed microRNAs in BEAS-2B cells after exposure to CNC, but not to CMF or CNF, compared to untreated cells. Moreover, a profile of microRNAs associated with CNC exposure was identified. Overall, no major concern regarding CNMs genotoxicity and oxidative stress was raised. Epigenetic effects were uncovered for CNC and the identified miRNA profile has potential to be used as an effect biomarker for occupational biomonitoring. However, further investigation with longer exposures and validation of that profile using in vivo models must be conducted, to prove its value to assess early effects from CNMs exposure.