Browsing by Issue Date, starting with "2019-11-25"
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- Safety assessment of a new bioactive compound, extracted from Lupinus albus seeds, through the analysis of its cytotoxic and genotoxic propertiesPublication . Bischoff, Nicolaj; Schwerdtle, Tanja; Silva, Maria JoãoColorectal cancer (CRC) is the third most common type of cancer worldwide, being highly metastatic and mostly resistant to anticancer treatment. Especially due to its high incidence in young people and a lack of adequate treatment CRC is imposing an increasing health risk for future generations. Patients who develop CRC often suffer from Inflammatory Bowel Diseases (IBD), such as Chron’s Disease (CD) or Ulcerative Colitis (UC), before the on-set of tumorgenesis. One of the most feared complications in CRC is the cancer metastasis from the colon into secondary tissues, which is primarily responsible for the high lethality of CRC. The induction of inflammation, as well as the metastasis formation, are strongly associated with an over-expression of a subgroup of matrixmetalloproteinases (MMP), the so-called gelatinases (MMP-2/MMP-9). Deflamin is a novel matrixmetallopoteinase-inhibitor (MMPI), extracted from the seeds of Lupinus albus (tremoço), which has shown anti-inflammatory properties in the gastrointestinal tract, and has been pointed as a promising antiinflammatory and cancer preventive agent. The mechanism of action from this plantbased protein shows a high inhibitory activity against MMP-9 and/or MMP-2. This makes deflamin a great candidate to become a valuable anti-inflammatory nutraceutical agent, as well as a powerful asset for the treatment and prevention of IBD and CRC. However, potential secondary adverse effects must be avoided and an early stage safety evaluation of its potential toxic effects on human colon cancer cells is needed. This work is aimed at contributing to the safety assessment of deflamin through the analysis of cytotoxic and genotoxic properties of the purified deflamin and a Lupinus extract in Caco-2 colon cancer cells. Furthermore, its bioavailability and transport via a polarized and differentiated cell monolayer was assessed, to get further information of possible uptake mechanisms. The cytotoxic effects were analyzed by the MTT assay, Neutral Red Uptake (NRU) assay, and monolayer integrity (TEER) following an incubation time of 24, 48 and 72 hours at a concentration range from 10-640 μg/ml. The cytotoxic assessment showed a dose- and time-dependent decrease of cell viability in Caco-2 cells that did not reach statistical significance. Genotoxicity was assessed by using the alkaline comet assay in combination with the cytokinesis-block micronucleus (CBMN) assay at the three non-cytotoxic concentrations of 20, 80 and 320 μg/ml. The evaluation of DNA damage with the alkaline comet assay did not show a dose- or time-dependent increase in the level of DNA damage. The exposure of Caco-2 cells to extract did not increase the frequency of micronuclei after 24 hours exposure to the extract. Preliminary data obtained from the CBMN assay, for the purified deflamin did show a dose-dependent increase in the number of micronuclei but further investigation to confirm these results is require.
- Cellular and molecular mechanisms of toxicity of ingested nanomaterialsPublication . Gramacho, Ana Catarina; Rolo, Dora; Martins, Carla; Assunção, Ricardo; Gonçalves, Lídia M.; Bettencourt, Ana; Alvito, Paula; Pereira, Joana; Jordan, Peter; Silva, Maria João; Louro, HenriquetaThe 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. Many of such products, already available, have NMs such as titanium dioxide nanomaterials (TiO2) and the 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 (GIT) 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 adverse outcomes (AO) such as genotoxic effects 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. INSA has previously shown that NMs with the same chemistry, but differing in primary properties may yield different biological effects. Conversely, the NMs properties are context-dependent, i.e. can be affected by the surrounding matrix. These secondary features may be potentially more relevant for determining toxicological outcomes. In particular, processes like digestion may modify the NMs characteristics leading to unexpected toxicity in intestine cells. INGESTnano project aims to investigate the nano-bio interactions of NMs, at cellular and molecular level, in the context of intestinal tract and digestion processes, to better understand their potential negative impacts on human health with special reference to organ-specific cells. TiO2 has been selected as case-study to setup a workflow for addressing nanosafety concerns that may be in the future applied to other NMs to which GIT may be exposed. It is expected that this project will contribute to the safety evaluation of the TiO2 ingested, by elucidating key events (KE) elicited by these NMs and linking exposure to AO.
- Topical delivery of nanostructured lipid carriers loaded with lipophilic active compounds on a 3D reconstructed human epidermis modelPublication . Pinto, Fátima; Fonseca, Luis; Souza, Sofia; Oliva, Abel; Barros, DraganaLipid nanocarriers refer to a wide group of drug delivery systems that are well-known as effective carriers for lipophilic and hydrophilic active compounds and that can be easily integrated into dermal formulations. Nanostructured lipid carriers (NLCs) belong to the wide group of lipid nanocarriers, representing an alternative – e.g. to liposomes, emulsions and polymeric nanoparticles. Usually, NLCs present a spherical shape with mean diameters ranging between 50 and 500 nm and are composed by an unstructured solid lipid matrix consisting on a mixture of liquid and solid lipids, stabilized by a surfactant or a mixture of surfactants dispersed in an aqueous phase. New optimized NLCs formulations loaded with retinyl palmitate (RP) and α-tocopherol (TOC) were evaluated regarding their topical distribution and efficacy on a 3D model of in vitro reconstructed human epidermis (RHE). NLCs were produced using sunflower oil and myristic acid as liquid and solid lipids, respectively and RP and TOC as lipophilic model compounds. Also, the fluorescent dye DiO was incorporated along with TOC, resulting in a TOC-DiO-NLCs formulation to enable a qualitative characterization on in vitro absorption studies. Physicochemical properties of empty NLCs, RP-NLCs and TOC-DiO-NLCs were characterized, as well as their surface morphology and internal structure. In vitro absorption studies were performed on the RHE model in customized Franz diffusion cells and were quantitatively and qualitatively characterized. The cytotoxicity of optimized NLCs formulations was evaluated through the in vitro skin irritation test on the RHE model. All characterized NLCs presented appropriate physicochemical properties for dermal formulations and an efficient distribution and release profile of active compounds across the reconstructed skin membrane. In vitro skin irritation tests demonstrated that the optimized NLCs formulations were no cytotoxic.
- Using confocal microscopy for monitoring the subcellular impact of nanomaterialsPublication . Matos, PauloThe physicochemical properties of nanomaterials, such as their small size and high surface area ratio, make them ideal for many applications in industry and biomedicine. However, those same properties increase their ability to interact with cells and tissues, allowing their permeation through several biological barriers. While these abilities have been exploited in the development of novel drug-delivery systems, the widespread use of nanomaterials makes the evaluation of the potential cytotoxicity of their raw materials an important public health issue. In vivo studies are the usual gold standard when assessing compound toxicity, however, in vitro studies have also provided a lot of information regarding the toxicity and MoA of many compounds, and have proved crucial to clarify how the intrinsic and extrinsic properties of certain nanomaterials contribute to their interaction with cells an tissues. In this talk we will describe how confocal microscopy can be used in in vitro cell cultures to evaluate the subcellular impact of nanomaterials. We will point out the advantages and limitations of using confocal fluorescent microscopy in investigating how cells interact and react to the presence of different types of nanomaterial and how these can affect basic cellular functions.
- A predictive toxicology approach to characterize potential respiratory effects of functionalized nanocellulose fibres in a co-culture systemPublication . Ventura, Célia; Teixeira, Sara; Louro, Henriqueta; Lourenço, Ana Filipa; Ferreira, Paulo J.T.; Silva, Maria JoãoCellulose nanofibrils (CNF) have an enormous potential for industrial and biomedical applications, assuming a great economic value. Because other nanofibres, e.g. carbon nanotubes (CNT), have revealed toxicity 1,2, there is a need to comprehensively evaluate the toxic potential of CNF along the value chain, before they enter the market. This project is aimed at a safety evaluation of several CNF comparatively to CNT, in a co-culture of human-derived alveolar epithelial cells and macrophages3. A predictive toxicology approach is used, i.e., the toxicity will be characterized alongside the specific fibre-associated mode of action, including immunotoxicity, genomic and epigenetic effects. The data obtained for two CNF synthesized from Eucalyptus gobulus but using different pre-treatments will be presented. Future work includes the use of omics-based tools adapted to the toxicity assessment of CNF and other NMs that will give some insights on cellular and molecular mechanisms underlying CNF toxicity. The overall results will be used to ensure the safety of these CNF or to allow the modification of toxic CNF in order to reduce the adverse outcomes, thereby complying with the safer-by-design approach.
- New “omics” approaches as a tool to explore mechanistic nanotoxicologyPublication . Ventura, Célia; Vieira, Luís; Silva, Catarina; Silva, Maria JoãoIn the last years, genomic approaches have been applied to study the toxicity of nanomaterials with the aim of obtaining insightful information on their effects on gene expression and consequent cellular changes. Toxicogenomics expects to find unique transcriptional profiles that, besides providing evidence of the mechanistic mode of action of nanomaterials, may also be used as biomarkers for biomonitoring purposes. Moreover, several nanomaterials have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by DNA methylation, histone tail alterations and differential microRNA (miRNA) expression. DNA methylation is frequently studied when analysing the epigenetic regulation of gene expression and the role of miRNAs is being increasingly understood, either promoting or supressing biological pathways. Consequently, the identification of the differently expressed miRNAs in cells or tissues after exposure to a toxic can allow the recognition of its possible mechanisms of action. An example of an epigenomic study will be presented, focusing on the exposure of epithelial alveolar cells to a multi-walled carbon nanotube (MWCNT) and asbestos (crocidolite). MWCNTs are one of the most promising products of nanotechnology with an extensive variety of applications in industry and biomedicine. Several toxicological studies have demonstrated that exposure to some MWCNTs can induce immunotoxic, cytotoxic and genotoxic effects, and nowadays they are considered as an occupational hazard. Particularly, those with a fiber-like shape similar to asbestos have raised concern about their carcinogenicity, and one (MWCNT-7) was classified in Group 2B (IARC) as a possibly human carcinogenic. By elucidating the molecular pathways that are involved in key events of nanomaterials toxicity, the new “omics” studies are expected contribute to exclude or reduce the handling of hazardous nanomaterials in the workplace and support the implementation of regulation to protect human health.
- Tackling the molecular basis of lipid metabolism: from candidate genes testing in a disease cohort to multi-omics approaches in unselected populationsPublication . Rossi, Niccolò; Bourbon, Mafalda; Falchi, MárioDyslipidemia, broadly defined as an unhealthy deviation of plasma lipid levels, is a well-known heritable risk factor for c ardiovascular diseases (CVD), the first cause of death worldwide. Uncovering the genetic basis of p lasma lipids is, therefore, fundamental for CVD prevention and treatment. In this work, I tackled dyslipidemia from two different perspectives, namely genetics of severe dyslipidemia in a disease cohort and multi-omics of plasma lipids variation in unselected populations. First, I aimed to investigate if mutations in genes involved in miscellaneous monogenic dyslipidemia can mimic a Familial Hypercholesterolemia-like phenotype. By knocking-down dyslipidemia genes in cultured cells, I observed that sitosterolemia and hypertriglyceridemia causing genes are negative regulators o f LDL-uptake in vitro. Targeted sequencing of 1 85 FH mutation-negative individuals from the Portuguese FH study and subsequent cascade family screening for c andidate pathogenic variants, highlighted nine variants in ABCG5, ABCG8 and GPD1 co-segregating with the FH-phenotype. Mutations in these three genes, in heterozygosity, were associated with increased plasma LDL-cholesterol (LDL-C) as compared to the normal population (β = +71.38±9.57, +76.11±10.14 and +65.96±8.77 mg/dL, respectively). Rare genetic variants underlying extreme dyslipidemia tend to be conserved across ethnic groups. Instead, the study of rare variations underlying non-monogenic dyslipidemia in multi-ethnic populations remains challenging. Here, I looked for rare single nucleotide variants, individually or in aggregate, associated with plasma LDL-C from whole-genome sequencing data in 1,751 participants f rom the TwinsUK c ohort and replicated my findings in 2,587 individuals from the Qatar Genome Programme. I identified a conserved locus located upstream the KCNJ2 gene associated with LDL-C levels, at both single and aggregate variants levels in the two cohorts, and with myocardial infarction risk in TwinsUK. Loci identified by association studies have the potential to reveal novel genes and pathways involved in dyslipidemia biology. However, individual genes do not work alone, but rather interact with one another and jointly affect human health. I constructed gene co-expression networks based on RNA sequencing data generated from subcutaneous adipose and skin tissues, and lymphoblastoid cell lines from 856 subjects from the TwinsUK cohort. First, by testing the enrichment of co-expression modules for l ipid-related gene ontologies and GWAS hits, I defined a lipid functional gene module. Within this module, the expression level of the long non-coding RNA LINC00263 and transcription factor Srebf1, a key player in adipogenesis, were found to be highly correlated (Pearson’s ρ = 0.62; P = 3.71x10 -81) . In addition, I observed that LINC00263 predicted interactors are specifically expressed in adipocytes and are enriched for lipid-related pathways. Thus, I propose LINC00263 as a novel candidate lipid regulator in subcutaneous adipose tissue. Together, the results presented in this thesis provide new insights into dyslipidemia complex aetiology both at the genomic and transcriptomic level, and improve CVD risk assessment and prevention.
- Workshop 'Nanotoxicology in the context of the safety assessment of nanomaterials': Abstract BookPublication . Louro, Henriqueta; Silva, Maria JoãoRemuno dos trabalhos apresentados no Workshop 'Nanotoxicology in the context of the safety assessment of nanomaterials'. Enquadramento: Tem vindo a aumentar a preocupação com os potenciais riscos para a saúde humana e para o ambiente decorrentes da exposição a nanomateriais em contexto ocupacional, ambiental ou através de produtos de consumo, representando um desafio para as autoridades de saúde e ambiente, à escala mundial. Em particular, o impacto dos nanomateriais sobre o genoma humano (efeitos genotóxicos e epigenéticos) pode implicar riscos acrescidos de doenças crónicas, incluindo doenças oncológicas que se poderão manifestar apenas a longo prazo e cujo impacto ainda se desconhece. Nesta perspetiva, mostra-se necessário que investigadores, trabalhadores, profissionais da Saúde e cidadãos em geral, tomem consciência dos potenciais riscos para a saúde decorrentes da exposição a nanomateriais, bem como formas de os prevenir ou mitigar, no sentido de assegurar a Saúde Pública. Neste workshop, pretende-se estimular sinergias entre projetos nacionais sobre a segurança de nanomateriais e as nanotecnologias. Os aspetos a abordar incluem a síntese e aplicação de nanomateriais, a sua potencial toxicidade, particularmente, ao nível do genoma humano, a avaliação de risco dos nanomateriais e sua aplicação em termos de avaliação e de gestão do risco. Como destinatários, os investigadores das áreas da síntese e aplicação de nanomateriais, da nanotoxicologia, das áreas da saúde e ambiente, bem como os profissionais dos setores industrial e regulamentar e cidadãos com interesse em nanotecnologias, nanotoxicologia, saúde ambiental e ocupacional. A iniciativa enquadra-se nos projetos ToxApp4NanoCELFI e INGESTnano, financiados pela Fundação para a Ciência e Tecnologia.
- Exploring Research Priorities of Parents Who Have Children With Down Syndrome, Cleft Lip With or Without Cleft Palate, Congenital Heart Defects, or Spina Bifida Using ConnectEpeople: A Social Media Coproduction Research StudyPublication . Sinclair, Marlene; McCullough, Julie E.M.; Elliott, David; Latos-Bielenska, Anna; Braz, Paula; Cavero-Carbonell, Clara; Jamry-Dziurla, Anna; João Santos, Ana; Páramo-Rodríguez, LucíaBackground: Using social media for research purposes is novel and challenging in terms of recruitment, participant knowledge about the research process, and ethical issues. This paper provides insight into the recruitment of European parents of children with specific congenital anomalies to engage in coproduction research by using social media. Secret Facebook groups, providing optimal security, were set up for newly recruited research-aware parents (RAPs) to communicate privately and confidentially with each other and for the research team to generate questions and to interpret findings. Objective: This study aimed to use social media for the recruitment and engagement of parents in research and to determine the research priorities of parents who have children with Down syndrome, cleft lip with or without cleft palate, congenital heart defects, and spina bifida. Methods: The design was exploratory and descriptive with 3 phases. Phase 1 included the recruitment of RAPs and generation of research questions important to them; phase 2 was a Web-based survey, designed using Qualtrics software, and phase 3 included analysis and ranking of the top 10 research questions using an adapted James Lind Alliance approach. Simple descriptive statistics were used for analysis, and ethical approval was obtained from the Ethics Filter Committee of the Institute of Nursing and Health Research, Ulster University. Results: The recruitment of 32 RAPs was a sensitive process, varying in the time taken to consent (mean 51 days). However, parents valued the screening approach using the State-Trait Anxiety Inventory as a measure to ensure their well-being (mean 32.5). In phase 1, RAPs generated 98 research questions. In phase 2, 251 respondents accessed the Web-based survey, 248 consented, and 80 completed the survey, giving a completeness rate of 32.3% (80/248). Most parents used social media (74/80, 92%). Social media, online forums, and meeting in person were ranked the most preferable methods for communication with support groups networks and charities. Most respondents stated that they had a good understanding of research reports (71/80, 89%) and statistics (68/80, 85%) and could differentiate among the different types of research methodologies (62/80, 78%). Phase 3 demonstrated consensus among RAPs and survey respondents, with a need to know the facts about their child's condition, future health, and psychosocial and educational outcomes for children with similar issues. Conclusions: Social media is a valuable facilitator in the coproduction of research between parents and researchers. From a theoretical perspective, ocularcentrism can be an applicable frame of reference for understanding how people favor visual contact.
- Adverse Outcome Pathways (AOPs) development, a tool for predictive nanotoxicologyPublication . Rolo, Dora; Louro, HenriquetaNanomaterials (NMs) have the potential to improve novel and useful wide applications in electronics, chemicals, environmental protection, biological medicine, food and others. Therefore, NMs rapid proliferation presents a dilemma to regulators regarding hazard identification, with increased concerns for public health. Predictive nanotoxicology describes a multidisciplinary approach to NMs evaluation that uses a set of in vitro and in silico methods to forecast the effects on biological systems. This approach offers advantages to traditional hazard assessment methods, such as reducing the reliance on animal studies, associated costs and ethical issues. It may be used with several applications in environmental and human health risk assessment and NMs hazard identification, as well as for regulation. The Adverse Outcome Pathways (AOPs) are the central element of a toxicological knowledge framework, promoted by member countries through OECD, built to support chemical risk assessment based on mechanistic reasoning. AOPs describes a logical sequence of causally linked events at different levels of biological organisation, which follows exposure and leads to an adverse health effect in humans or wildlife. The integrative analysis of the cellular and molecular mechanisms of nanotoxicity towards a definition of key events, may lead to adverse outcomes, driving a sequential line and defining an AOP landscape. Each defined AOP is available for crossing data, linking known and unknown landscapes. Since the biological effects that relate to possible genotoxicity and increased risk of cancer due to NMs exposure are under analysis, the development and assessment of AOPs are important novel strategic tools for predictive nanotoxicology.