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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 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.
- 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.
- Aula 5: Efeitos Genéticos e Epigenéticos da Exposição a NanomateriaisPublication . Ventura, Célia; Louro, Henriqueta; Silva, Maria João1. Nanomateriais Manufaturados – NM 2. Possíveis vias de exposição a NM e efeitos na saúde 3. Interações NM - sistemas biológicos 4. A Nanotoxicologia 5. Genotoxicidade dos Nanomateriais 6. Estudos realizados In vitro 7. Estudos realizados In vivo 8. Estudos efectuados sobre exposição ocupacional a nanofibras 9. Toxicogenómica, toxicotranscriptómica e toxicoepigenómica 10. Conclusões dos estudos in vitro e in vivo
- 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.
- Early-stage nanosafety assessment as a critical tool for innovative nanomaterials developmentPublication . Louro, Henriqueta; Ventura, Célia; Rolo, Dora; Vital, Nádia; Pinto, Fátima; Silva, Maria JoãoThe 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.
- Assessing the Genotoxicity of Cellulose Nanomaterials in a Co-Culture of Human Lung Epithelial Cells and Monocyte-Derived MacrophagesPublication . Ventura, Célia; Pinto, Fátima; Lourenço, Ana Filipa; Pedrosa, Jorge F. S.; Fernandes, Susete N.; da Rosa, Rafaela R.; Godinho, Maria Helena; Ferreira, Paulo J. T.; Louro, Henriqueta; Silva, Maria JoãoCellulose micro/nanomaterials (CMNMs) are innovative materials with a wide spectrum of industrial and biomedical applications. Although cellulose has been recognized as a safe material, the unique properties of its nanosized forms have raised concerns about their safety for human health. Genotoxicity is an endpoint that must be assessed to ensure that no carcinogenic risks are associated with exposure to nanomaterials. In this study, we evaluated the genotoxicity of two types of cellulose micro/nanofibrils (CMF and CNF) and one sample of cellulose nanocrystals (CNC), obtained from industrial bleached Eucalyptus globulus kraft pulp. For that, we exposed co-cultures of human alveolar epithelial A549 cells and THP-1 monocyte-derived macrophages to a concentration range of each CMNM and used the micronucleus (MN) and comet assays. Our results showed that only the lowest concentrations of the CMF sample were able to induce DNA strand breaks (FPG-comet assay). However, none of the three CMNMs produced significant chromosomal alterations (MN assay). These findings, together with results from previous in vitro studies using monocultures of A549 cells, indicate that the tested CNF and CNC are not genotoxic under the conditions tested, while the CMF display a low genotoxic potential.
- Cyto-genotoxic effects of distinct micro/nanocelluloses in human osteoblastic and mice fibroblastic cellsPublication . Ventura, Célia; Marques, Catarina; Cadete, João; Vilar, Madalena; Pedrosa, J.F.S.; Pinto, P.; Rosa, R.R.; Fernandes, S.N.; Godinho, M.H.; Ferreira, P.J.T.; Louro, Henriqueta; Silva, Maria JoãoThe unique characteristics of micro/nanocelluloses (MNCs) have been boosting their use in a wide variety of industrial and biomedical applications. With the expected decrease of their production cost, it is likely to observe an even faster growing adoption of these materials in the coming years. With the increasing use of MNCs comes an increased likelihood of human exposure to these materials, raising concerns about potential human health effects. The number of studies on the potential adverse effects of MNCs is still scarce and sometimes inconclusive. The aim of the present study is to shed some light on the impact of human exposure to cellulose-based nanomaterials. For that, three MNCs were produced from bleached Eucalyptus kraft pulp using different pretreatments. A microfibrillated (CMF) and a nanofibrillated cellulose (CNF) were respectively obtained by enzymatic and TEMPO-mediated oxidation pretreatments followed by high-pressure homogenization. In addition, cellulose nanocrystals (CNC) were obtained by acid hydrolysis with diluted sulfuric acid. The potential cytotoxicity of the MNCs was evaluated in vitro in two mammalian cell lines (human MG-63 osteoblasts and mice V79 lung fibroblasts) by the MTT and the clonogenic assays, and the genotoxicity was assessed by the micronucleus assay. Cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNC, but not by the MTT assay. CNF induced micronuclei in both cell lines and nucleoplasmic bridges in MG-63 cells. CMF and CNC induced micronuclei and nucleoplasmic bridges in MG-63 cells, but not in V79 cells. From the results, it was concluded that under the tested conditions all MNCs present cytotoxicity and genotoxicity, although at different concentration levels, which may be related to their physicochemical differences, to the availability for cell uptake, and to differences in the DNA damage response of the cell model.
- Occupational exposure to Hexavalent chromium: the portuguese case within the collaborative european human biological monitoring studyPublication . Afonso, Catarina; Gomes, Bruno Costa; Louro, Henriqueta; Ladeira, Carina; Pinhal, Hermínia; Nogueira, Ana; Santos, Sílvia; Ventura, Célia; Ribeiro, Edna; Santonen, Tiina; Viegas, Susana; Silva, Maria JoãoBackground: The EU human biomonitoring initiative (HBM4EU) aims to coordinate and advance human biomonitoring across Europe. A multicenter study intending to characterize occupational exposure to hexavalent chromium [Cr(VI)], a human carcinogen, was setup in several industrial settings across Europe. In Portugal, the study was developed in one aircraft maintenance company since the substitution of Cr(VI) in this sector is not expected in the near future. Methods: The work processes that could imply exposure to Cr(VI) were characterized and exposure estimation was done. Following the company agreement and the volunteers informed consent, a survey was applied to each worker for detailed information on lifestyle, health status, etc.. Sampling for exposure and effect biomarkers analyses involved industrial hygiene samples (HIS) and biological specimens from 50 workers and 25 non-exposed individuals (controls). Biomarkers of early biological effects were studied in leukocytes from all individuals, involving the analysis of DNA damage (comet assay) and chromosome alterations (micronucleus assays); the results obtained for exposed and control individuals were statistically compared. Results and Discussion: Besides HIS results, Cr (VI) concentrations in biological samples and effect biomarkers results will be presented. The complementarity of the environmental and biological sampling, together with the sensitivity and specificity of the biomarkers used, is expected to allow exposure characterization and possible early genotoxic effects identification. Potential associations between exposure and effect biomarkers will be explored. Conclusions: An association between the levels of exposure to Cr(VI) and the early effects characterized in workers, if existent, is expected to predict potential health outcomes for Cr(VI)-exposed workers, particularly, on the long-term. All the information generated will help to support new priorities and investments regarding risk management measures to apply in this company and in the aircraft maintenance sector, in general.
- The Use of Human Biomonitoring to Assess Occupational Exposure to PAHs in Europe: A Comprehensive ReviewPublication . Louro, Henriqueta; Gomes, Bruno Costa; Saber, Anne Thoustrup; Iamiceli, Anna Laura; Göen, Thomas; Jones, Kate; Katsonouri, Andromachi; Neophytou, Christiana M.; Vogel, Ulla; Ventura, Célia; Oberemm, Axel; Duca, Radu Corneliu; Fernandez, Mariana F.; Olea, Nicolas; Santonen, Tiina; Viegas, Susana; Silva, Maria JoãoPolycyclic aromatic hydrocarbons (PAHs) are among the chemicals with proven impact on workers’ health. The use of human biomonitoring (HBM) to assess occupational exposure to PAHs has become more common in recent years, but the data generated need an overall view to make them more usable by regulators and policymakers. This comprehensive review, developed under the Human Biomonitoring for Europe (HBM4EU) Initiative, was based on the literature available from 2008–2022, aiming to present and discuss the information on occupational exposure to PAHs, in order to identify the strengths and limitations of exposure and effect biomarkers and the knowledge needs for regulation in the workplace. The most frequently used exposure biomarker is urinary 1-hydroxypyrene (1-OH-PYR), a metabolite of pyrene. As effect biomarkers, those based on the measurement of oxidative stress (urinary 8-oxo-dG adducts) and genotoxicity (blood DNA strand-breaks) are the most common. Overall, a need to advance new harmonized approaches both in data and sample collection and in the use of appropriate biomarkers in occupational studies to obtain reliable and comparable data on PAH exposure in different industrial sectors, was noted. Moreover, the use of effect biomarkers can assist to identify work environments or activities of high risk, thus enabling preventive risk mitigation and management measures.