DGH - Apresentações orais em encontros internacionais
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- 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.
- Investigating ingested nanomaterials’ safety – the case of TiO2 and innovative nanocelluloses.Publication . Vital, Nádia; Silva, Maria João; Louro, HenriquetaIntroduction: The development of nanomaterials(NMs)-based technologies led to their increased use in key sectors and products related to food, food contact materials and feed. Many available products have NMs, as intentional constituents or contaminants from process ou food packaging release, such as silicon or titanium dioxide(TiO2) NMs. Others are being developed, like nanocelluloses(CNMs; doi:10.3390/nano12193375). However, it is recognised that the NMs’ specific physicochemical properties, conferring them unique benefi cial characteristics, can also elicit nano-bio interactions leading to toxic potential. Also, their dynamic behaviour in the surrounding matrix, may lead to secondary features determining the toxicological outcomes. Recognizing that processes like intake or digestion may modify the NMs’ characteristics leading to unexpected toxicity in human cells, EFSA included the use of in vitro digestion models in their specific guidelines concerning risk assessment of nanomaterials for food and feed(DOI:10.2903/j.efsa.2021.6768). Methodology: With the aim to contribute to the safety assessment of NMs, intestinal cell models (Caco-2 and HT29-MTX-E12 cells) were exposed to TiO2 NMs or innovative CNMs. Additionally, samples submitted previously to in vitro simulation of human digestion were used, and the genotoxicity(comet and micronucleus assays) was investigated with and without the digestion process. Results: After TiO2 NMs’ exposure, the micronucleus assay, an indicator of cancer risk, suggested eff ects on the chromosomal integrity in the HT29-MTX-E12 cells, for all the tested TiO2 NMs, especially after the in vitro digestion. Upon exposure to the two CNMs, no chromosomal damage was observed in the micronucleus assay, but the comet assay revealed DNA damage in the same cells, after 3h and 24h exposure, an effect slightly more relevant after the digestion of the cellulose nanofibril. Conclusion: Overall, the results show diff erent outcomes when using different NMs, and with/without digestion. Thus, it is important to consider the primary and secondary NMs’ characteristics determining the adverse eff ects, taking into account the human digestion for nanosafety assessment.
- The establishing of the program the human biomonitoring in the European Union - the portuguese perspetivePublication . Louro, HenriquetaThe portuguese perspetive about the establishing of the program the human biomonitoring in the European Union - the portuguese perspetive
- Epigenomics as a novel approach to explore the toxic effects of nanomaterialsPublication . Ventura, Célia; Vieira, Luís; Valente, Ana; Fernandes, Camila; Silva, Catarina; Louro, Henriqueta; Ferreira, Paulo J.T.; Silva, Maria JoãoIn recent years, there has been a huge development of innovative engineered nanomaterials with potential use in industrial and biomedical applications. This increased widespread use raised concerns that nanomaterials may elicit human adverse health effects through occupational, environmental or consumer exposure. Many toxicity studies, mainly in vitro, have showed that some nanomaterials, such as carbon nanotubes or titanium dioxide nanoparticles (TiO 2 NP), may cause genotoxicity, inflammation, and associated adverse health effects. Nevertheless, few studies have focused on the nanomaterials effect s on the epigenome, namely, modifications of histone tails, microRNA expression or DNA methylation. Here we wil l present two “omics” studies based on next generation sequencing , one focusing on the effect of three nanocellulose s derived from Eucaliptus globulus kraft pulp on the microRNA expression of BEAS 2B, and an other one focusing on the effect of three types of TiO 2 NP on the DNA methylation of Caco 2 cells. Regarding the former 24h exposure to fibrillar micro/nanocellulose s did not induced significant (FDR ≤ 0.05) differentially expressed microRNAs, as compared to non exposed cells. By contrast, the crystalline nanocelul l ose induced the over and under expression of 22 and 30 microRNAs, respectively. These microRNAs can be f urther explored as potential biomarkers for human biomonitoring and co ntribute to elucidate the mechanisms of action of crystalline nanocellulose. As to the genome wide methylation study Reduced Representative Bisulfite Sequencing allowed the identification of significant ( p ≤ 0.05) differential methylation of 92, 70, and 88 gene sequences for the anatase, rutile and brookite phase TiO 2 NP exposures, respectively. Functional pathway analysis of these methylation changes showed that all TiO 2 NP may affect cell proliferation, differentiation, and survival, and suggested different molecular mechanisms of action for each type of TiO 2 NP. In conclusion, epigenomics revealed to be a powerful tool to understand the key molecular events underlying nanomaterials effects.
- Effect biomarkers in e-waste management workersPublication . Silva, Maria João; Aimonen, K.; Louro, Henriqueta; Tavares, A.; Moreira, R.; Catalan, J.; Duca, R.C.; Godderis, L.; Mahiout, S.; Martins, C.; Martinsone, I.; Matisane, L.; Namorado, S.; Van Nieuwenhuyse, A.; Pinhal, H.; Porras, S.; Remes, J.; Scheepers, P.; Verdonck, J.; Viegas, S.; Santonen, T.; HBM4EU E-waste study teamDuring e-waste handling/processing, a broad range of toxic chemicals (metals and persistent organic compounds), are released and may affect workers’ health. This work intended to identify genotoxic effects in workers from European e-waste management companies. Micronuclei were analysed in peripheral blood lymphocytes (MNPBL) from 95 workers and 50 controls and in reticulocytes (MNRET) from 82 workers and 41 controls. No statistically significant differences were detected between the total exposed and control groups, for both MNPBL and MNRET frequencies. Stratification of workers in subgroups according to the main activities performed revealed that the subgroup involved in batteries recycling (n=23) presented a frequency of MNPBL significantly higher than that of controls. Significant differences in MNPBL frequencies were also found between battery workers and the subgroups handling/processing white goods, metals and plastics, and miscellaneous E-waste; no differences in MNRET frequencies among subgroups were detected. Worth to note, the subgroup dealing with brown goods (n=12) displayed the highest MNPBL and MNRET frequencies, although statistical significances were not observed when comparing with the other subgroups or controls. These preliminary results highlight the value of adding effect biomarkers to biomonitoring campaigns, to uncover groups of workers at enhanced risk and to prioritize risk management measures’ implementation.
- HBM4EU diisocyanates study – results from a collaborative European human biological monitoring study on occupational exposurePublication . Jones, Kate; Galea, K.S.; Scholten, B.; Loikala, M.; Porras, S.P.; Bousoumah, R.; Ndaw, S.; Leese, E.; Louro, Henriqueta; Silva, Maria João; Viegas, S.; Godderis, L.; Verdonck, J.; Poels, K.; Gӧen, T.; Duca, R.C.; Santonen, T.; HBM4EU diisocyanates study teamDiisocyanates have long been a leading cause of occupational asthma in Europe, and they are now restricted under the REACH regulation. As part of the European Human Biomonitoring project (HBM4EU), we conducted an occupational exposure survey on diisocyanates in five European countries. 116 workers were recruited across four job categories: (1) Use of diisocyanates-based glues, adhesives or sealants; (2) Polyurethane coating of large surfaces; (3) Spray application of urethane foam; (4) Spray coating of vehicles. Fifty controls (within the same companies) were also recruited. The study collected urine samples (analysed for diisocyanate-derived diamines and lysine conjugates), blood samples (analysed for diisocyanate-specific antibodies, inflammatory markers, and diisocyanate-specific haemoglobin adducts), buccal cells (micronucleus analysis) and measured fractional exhaled nitric oxide (FeNO). In addition, occupational hygiene measurements (air monitoring and skin wipe samples) and questionnaires were collected. Initial results showed significant airborne exposures for some tasks (spraying polyurethane foam insulation), with elevated urinary diamine levels compared to controls (p<0.001), and detection of the specific MDI-lysine conjugate. Most workers did not show increased FeNO, but some individuals had elevated results (controls max 30 ppb (n=49), workers max 161 ppb (n=108). Further ongoing data analysis will be presented.
- The HBM4EU e-waste study: exploratory survey of worker’s exposure to toxic contaminantsPublication . Scheepers, Paul; Viegas, S.; Duca, R.C.; Cseresznye, A; Cleys, P.; Covaci, A.; Goën, T.; Galea, K.S.; Godderis, L.; Hardy, E.; Leese, E.; Louro, Henriqueta; Mahiout, S.; Ndaw, S; Poels, K.; Silva, Maria João; Verdonck, J.; Porras, S.; Santonen, T.; HBM4EU E-waste Study TeamSo far, human biomonitoring (HBM) has not been much used to study exposure of workers involved in the processing of e-waste in the EU. In this study we aimed to explore exposures of workers to chemical contaminants, contribute to raise awareness of potential hazards and to further improve work practices. The study was conducted in eight European counties in a target population of 195 exposed and 73 controls. Biomarkers of exposure were used for selected metals and organic contaminants. Occupational hygiene sampling methods and contextual information were collected to facilitate the interpretation of the biomarker results. We found somewhat elevated exposures in workers for cadmium and mercury in blood and urine compared to controls. Blood analysis indicated high lead levels in post-shift compared to pre-shift in battery workers. Some urinary phthalate metabolite levels indicated a contribution from work-related exposures and were more pronounced in battery workers. Only small differences were observed in urinary excretion before and after the shift for organophosphorus flame retardants. Brominated flame retardant and PCB serum levels were in the range of general population background. From this exploratory study we conclude that more studies are needed to better understand chemical exposure in the processing of e-waste.
- Unlocking the Potential of Environmental and Health Research with FAIREHRPublication . Zare Jeddi, Maryam; Hopf, N.; Louro, Henriqueta; Silva, Maria João; Costa, Carla; Viegas, S.; Scheepers, P.; Cubadda, F.; Ghosh, M.; Ali, I.; Santonen, T.; von Goetz, N.; Bessems, J.; Galea, K.S.Current challenges in data comparability, integration, and management, hinder effective utilization of the large amount of data generated in environment and health studies. The European chapter of the International Society of Exposure Science (ISES Europe) Human Biomonitoring (HBM) Working Group is developing a global preregistration platform “FAIR Environment and Health Registry (FAIREHR)” to address these challenges. The focus is initially on the HBM domain, towards the implementation of FAIR (findable, accessible, interoperable, reusable) principles throughout the data lifecycle. Preregistration of HBM studies in a peer review-based registry like FAIREHR would stimulate communication and interaction among HBM communities leading to improved HBM study designs as well as generating comparable results worldwide. Using common standards and ontologies will make data better interoperable and functional for machine discovery. FAIREHR will also provide information on data licenses and request procedures necessary to access datasets of interest. Overall, the FAIREHR platform gathers many stakeholders (scientists, regulators, policy makers, life science companies, publishers, and funding bodies) interested in tracking and identifying planned, ongoing, and completed studies. FAIREHR is expected to benefit research, innovation and environment and public health policies by providing FAIR data that can be readily utilized for protecting human health.
- How can multi-national occupational studies support policy making in Europe? Experiences from HBM4EU and PARC occupational studiesPublication . Santonen, Tiina; Louro, Henriqueta; Bocca, Beatrice; Bousoumah, Radia; Corneliu Duca, Radu; Fucic, Aleksandra; Galea, Karen S.; Godderis, Lode; Göen10, Thomas; Iavicoli, Ivo; Janasik, Beata; Jones, Kate; Leese, Elizabeth; Leso, Veruscka; Ndaw, Sophie; Poels, Katrien; Porras, Simo P.; Ruggieri, Flavia; Silva, Maria João; Van Nieuwenhuyse, An; Verdonck, Jelle; Wasowicz, Wojciech; Tavares, Ana; Sepai, Ovnair; Scheepers, Paul T.J.; Viegas, SusanaProviding policy relevant data on chemical exposures was a major aim of the EU biomonitor-ing initiative, HBM4EU. Similarly, the recently launched “Partnership for the risk assessment of chemicals” (PARC) emphasizes the relevance of the research findings to regulatory deci-sion making. Occupational biomonitoring studies performed under HBM4EU were focused on HBM4EU priority substances with specific regulatory relevance. Chromates and diisocya-nate studies were developed since these substances are of high concern for workers and to support regulatory measures under both EU chemicals regulation (REACH) and occupational safety and health (OSH) legislation. E-waste study was targeted to support European circular economy strategies and identify issues e.g. under OSH legislation. Based on the results of the chromates study, we drew several policy relevant conclusions which support the future up-dating of occupational exposure limits and the development of monitoring and risk man-agement practices in industry. A survey to policy makers on the usefulness of the study re-sults gave us confidence. We emphasise the importance of early and continued engagement of policy makers to ensure the usability of the results. Early communication with regulators of the anticipated benefits and formatting outputs appropriately are important and have been considered and improved within the EU PARC project.
- HBM4EU chromates study: untargeted metabolomics study of workers exposed to hexavalent chromiumPublication . Kozłowska, Lucyna; Santonen, T.; Duca, R.C.; Godderis, L.; Jagiello, K.; Janasik, B.; Van Nieuwenhuyse, A.; Poels, K.; Puzyn, T.; Scheepers, P.; Sijko, M.; Silva, Maria João; Sosnowska, A.; Viegas, S.; Verdonck, J.; Wąsowicz, W.; HBM4EU Chromates Study Team and Statistical TeamDue to the wide use of Cr(VI), a large number of workers in a variety of industries are ex-posed to the potential harmful effects of this compound. The aim of this study was to inves-tigate changes in metabolic pathways in response to Cr(VI) exposure and to search for new urinary biomarkers. The study included 220 male workers who were exposed to Cr(VI) (through welding, chromium plating, surface treatment) and 102 male controls from Bel-gium, Finland, Poland, Portugal, and the Netherlands. Urinary metabolite profiles were de-termined using liquid chromatography mass spectrometry. The changes in the abundancy of excreted metabolites observed in post-shift workers vs. controls reflect fatty acid and mon-oamine neurotransmitter metabolism, oxidative modifications of amino acid residues, the excessive formation of abnormal amino acid metabolites and changes mainly in steroid hormones. We observed that: argininosuccinic acid, ubiquinone-1, indole-3-propionicacid, 6-hydroxyphenylpropionylglycine, 20-oxo-leukotriene E4, 3,4-dihydroxybenzylamine, 3,4-dimethoxyphenylethylamine and succinylacetone are potential biomarkers for Cr(VI) exposure (area under the curve > 0.9). The results of our study could form the basis for vali-dating early biomarkers of Cr(VI) exposure and their application in screening tests, and also serve as a starting point for further targeted metabolomics studies to better understand the disorders associated with Cr(VI) exposure.