Browsing by Author "Silva, M.J."
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- Applicability of the harmonized in vitro digestion method to titanium dioxide nanomaterialsPublication . Alvito, Paula; Assunção, Ricardo; Gramacho, A.; Silva, M.J.; Louro, H.; Martins, C.Titanium dioxide nanomaterials (TiO2 NMs) have a high potential for ingestion by human populations, due to their increasing use as food additives, inclusion in dietary supplements and food packaging materials. Whether this oral exposure may lead to adverse local or systemic outcomes, has been the subject of research. In vitro studies have generated contradictory results, possibly due to differences in the physicochemical properties of the TiO2 NMs studied, which can be additionally affected by the surrounding matrix and interactions during digestion. INGESTnano is a national project aimed at investigating the nano-bio interactions of nanomaterials on intestinal cells, at the cellular and molecular levels, after the digestion process, to better understand their potential impacts on human health. Three TiO2 NMs were selected as case-studies to setup a workflow for addressing nanosafety concerns of ingested NMs, while considering the nano-bio interactions under physiological conditions. As an alternative to in vivo testing, this project is focused on the use of the harmonized in vitro digestion method for simulating the human digestion of NMs. This digestion comprises three compartments: a) mouth (pH 7.0, alpha-amylase, salts); b) stomach (pH 3, HCl, pepsin, salts) and c) small intestine (pH 7.5, bile salts, pancreatin, salts). The final product of digestion is tested in bioassays using intestinal cells, to ascertain its toxicity. A high level of toxicity of the final digestion product challenged the applicability of the digestion product in the biological assays. The results revealed that digestion products without the NM showed cytotoxic effects above the concentration of 10% in cell culture medium. Several modifications to the initial protocol were thus performed to overcome this issue. The results suggested that the addition of bile salts accounted for most of the toxicity observed. The applicability of the harmonized in vitro digestion method is discussed in view of its potential use as a tool for addressing the toxicity of ingested NMs or other food contaminants, mimicking the physiological processes, in alternative to animal models.
- Assessment of ecologic and human health risks from a contaminated estuarine environmentPublication . Silva, M.J.; Pinto, M.; Caeiro, S.Due to their particular geographic characteristics, estuaries are often used for diverse human and industrial activities that may generate and release potentially harmful chemical contaminants. Thus, estuaries may be reservoirs of a wide variety of chemicals, including mutagenic and carcinogenic ones (e.g., metals, polycyclic aromatic hydrocarbons and pesticides), particularly when considering the potential for aquatic sediments to accumulate and continuously re-suspend contaminants into the water column. Importantly, besides being available to the local biota, the proximity/use of these contaminated sites might also be unsafe to the local population, provided that they are directly exposed (e.g. fishing, agricultural or recreational activities) or indirectly, through their diet. This work was focused on the river Sado Estuary, where the northern banks include urban areas and a heavy-industry park while the southern banks are mainly impacted by extensive agriculture activities. Sizable amounts of hazardous contaminants (e.g., metals, pesticides and polycyclic aromatic hydrocarbons) were identified in the estuarine sediments raising concern to the local populations because estuarine organisms and locally produced vegetables constitute the basis of their diet. The study intended to assess the adverse effects of estuary contaminants on human and ecosystem health through an integrative methodology based on several lines of evidence. An epidemiological survey was conducted to assess the exposure of a local population to the estuary environment as well as their health status. Furthermore, analyses of sediments, soil and wells water contamination were performed. Biological effects were assessed in major estuarine species with commercial value (clams, sole and cuttlefish); the cytotoxic and genotoxic potential of sediments were characterized in a human cell line. The results showed that the sediments’ contaminants were capable of inducing concentration-dependent cytotoxic and genotoxic damage in vitro and also allowed the distinction between two ecogeographical areas (urban/industrial and rural) with respect to type and intensity of the genetic damage, likely reflecting different patterns of toxicants mixtures. The integration of data from all study components indicated that the estuary is moderately impacted by complex mixtures of toxicants, affecting the aquatic biota and potentially able of inducing adverse effects on human cells. Although no clear evidence of contaminant-related adverse health effects in the studied population was found, the dose-related genotoxicity observed in the in vitro approach together with the evidences of direct and indirect human exposure indicates that those toxicants might constitute a risk factor to the development of chronic-degenerative diseases.
- Avaliação toxicológica de contaminantes químicos em alimentosPublication . Silva, M.J.
- Challenges in risk assessment of multiple mycotoxins in foodPublication . Assunção, Ricardo; Silva, M.J.; Alvito, PaulaMost fungi are able to produce several mycotoxins simultaneously and, consequently, to contaminate a wide variety of foodstuffs. Therefore, the risk of human co-exposure to multiple mycotoxins is real, raising a growing concern about their potential impact on human health. Besides, government and industry regulations are usually based on individual toxicities, and do not take into account the complex dynamics associated with interactions between co-occurring groups of mycotoxins. The present work assembles, for the first time, the challenges posed by the likelihood of human co-exposure to these toxins and the possibility of interactive effects occurring after absorption, towards knowledge generation to support a more accurate human risk assessment. Regarding hazard assessment, a physiologically-based framework is proposed in order to infer the health effects from exposure to multiple mycotoxins in food, including knowledge on the bioaccessibility, toxicokinetics and toxicodynamics of single and combined toxins. The prioritisation of the most relevant mixtures to be tested under experimental conditions that attempt to mimic human exposure and the use of adequate mathematical approaches to evaluate interactions, particularly concerning the combined genotoxicity, were identified as the main challenges for hazard assessment. Regarding exposure assessment, the need of harmonised food consumption data, availability of multianalyte methods for mycotoxin quantification, management of left-censored data, use of probabilistic models and multibiomarker approaches are highlighted, in order to develop a more precise and realistic exposure assessment. To conclude, further studies on hazard and exposure assessment of multiple mycotoxins, using harmonised methodologies, are crucial towards an improvement of data quality and a more reliable and robust risk characterisation, which is central for risk management and, consequently, to prevent mycotoxins-associated adverse effects. A deep understanding of the nature of interactions between multiple mycotoxins will contribute to draw real conclusions on the health impact of human exposure to mycotoxin mixtures.
- Combined exposure to multiple mycotoxins in food: are we ready for risk assessment and risk management?Publication . Alvito, Paula; Assunção, Ricardo; Silva, M.J.It is nowadays recognized that the risk of human co-exposure to multiple mycotoxins is real. In the last years, a number of studies have approached the issue of co-exposure and the best way to develop a more precise and realistic assessment. Likewise, the growing concern about the combined effects of mycotoxins and their potential impact on human health has been reflected by the increasing number of toxicological studies on the combined toxicity of these compounds. Nevertheless, risk assessment of these toxins, still follows the conventional paradigm of single exposure and single effects, incorporating only the possibility of additivity but not taking into account the complex dynamics associated to interactions between different mycotoxins or between mycotoxins and other food contaminants. Considering that risk assessment is intimately related to the establishment of regulatory guidelines, once the risk assessment is completed, an effort to reduce or manage the risk should be followed to protect public health. Risk assessment of combined human exposure to multiple mycotoxins thus poses several challenges to scientists, risk assessors and risk managers and opens new avenues for research. This presentation aims to give an overview of the different challenges posed by the likelihood of human co-exposure to mycotoxins and the possibility of interactive effects occurring after absorption, towards knowledge generation to support a more accurate human risk assessment and risk management. For this purpose, a physiologically-based framework that includes knowledge on the bioaccessibility, toxicokinetics and toxicodynamics of multiple toxins is proposed. Regarding exposure assessment, the need of harmonized food consumption data, availability of multianalyte methods for mycotoxin quantification, management of left-censored data and use of probabilistic models will be highlight, in order to develop a more precise and realistic exposure assessment. On the other hand, the application of predictive mathematical models to estimate mycotoxins’ combined effects from in vitro toxicity studies will be also discussed. Results from a recent Portuguese project aimed at exploring the toxic effects of mixtures of mycotoxins in infant foods and their potential health impact will be presented as a case study, illustrating the different aspects of risk assessment highlighted in this presentation. Further studies on hazard and exposure assessment of multiple mycotoxins, using harmonized approaches and methodologies, will be crucial towards an improvement in data quality and contributing to holistic risk assessment and risk management strategies for multiple mycotoxins in foodstuffs.
- Contribute of the MYCOMIX project and future perspectives for Human Biomonitoring and health risk assessment in PortugalPublication . Martins, Carla; Assunção, Ricardo; Silva, M.J.; Louro, H.; Pinhão, M.; Nunes, Baltazar; Vasco, Elsa; Alvito, PaulaThere is a growing concern within public health about mycotoxin involvement in human diseases, namely those related to children. The MycoMix project (2012-2015), funded by the Portuguese Foundation for Science and Technology, gathered a multidisciplinary team aiming at answering several questions: 1) Are Portuguese children exposed daily to one or several mycotoxins through food? 2) Can this co-exposure affect children´s health? and 3) Are there interaction effect between mycotoxins? Mycomix results revealed that Portuguese children (< 3 years old, n=103) are exposed to multiple mycotoxins through food consumption. Cumulative risk assessment results revealed a potential health concern for the high percentiles of intake, specially for aflatoxins which are carcinogenic compounds. This fact assumes particular importance considering the interactive effects found in in vitro bioassays. These results highlight the need for a more accurate approach to assess the human exposure to mycotoxins6. Within the Mycomix project the assessment of mycotoxin exposure was based on calculations combining mycotoxin data in food with population data on food consumption. This approach does not consider some aspects as the inter-individual metabolism variation, the exposure through sources other than food and the heterogeneous distribution of mycotoxins in food. Exposure assessment of mycotoxins in Portuguese population through biomarkers is still missing and further studies are urgent to be developed. The European Human Biomonitoring Initiative (EHBMI), a proposal within the European Joint Programme, aims to advance the understanding of the extent of exposure to environmental chemicals across Europe and the impact on human health, by gathering national expertise in human biomonitoring domain. At national level Mycomix project uncovered the potential health risk of exposure of Portuguese children to multiple mycotoxins. The risk assessment expertise acquired within Mycomix, namely in analysis and toxicology of chemical mixtures, will be brought together as a contribute to EHBMI objectives.
- Contribution to macrophage activation to the genotoxic effect of nanofibers in lung epitheliumPublication . Ventura, C.; Uva, A.S.; Silva, M.J.Nanofibers are nano-objects with two similar dimensions in the nanoscale (size range from approximately 1nm to 100nm) and the third dimension significantly larger. The most widespread group of nanofibers is carbon nanotubes (CNTs) that consist of graphite sheets with a cylindrical arrangement of various lengths, and diameter at the nanoscale. CNTs may have a single, double or multiple walls arranged in concentric layers, and embedded metals. In recent years, several studies performed in rodents exposed to liquid suspensions of CNTs by intratracheal instillation or pharyngeal aspiration showed the development of acute or persistent pulmonary inflammation and persistent interstitial fibrosis with granuloma formation, and bronchiolar or bronchioloalveolar hyperplasia. High thin and crystalline CNTs may also have a carcinogenic effect similar to asbestos. Macrophages play a key role in the response to poorly soluble nanomaterials as nanofibers. Activated macrophages degrade SWCNTs via NADPH oxidase pathway facilitating lung clearance, and if oxidative species formation is exaggerated, injury to the neighbour cells can occur. After macrophage activation, nanofibers phagocytosis also leads to the release of cytokines (TNF-α, IL-6, etc.) and transcription factors associated to inflammation (NF-κB and AP-1). In addition, when nanofiber length exceeds the pleural macrophages length, it triggers an inflammatory response in the pleural cavity due to "frustrated phagocytosis", which in turn stimulates a cytokine proinflammatory response by adjacent mesothelial cells. Thin and highly crystalline CNTs may also have a piercing effect in the mesothelial cell membrane causing in vitro cytotoxicity, and in vivo inflammatory and carcinogenic effects. The aim of this work is to elucidate the role of macrophage activation in the genotoxic effects of a thick and high aspect ratio multiwalled CNT in human lung epithelium.
- Development and Characterization of Engineered Acrylic Particulate SystemsPublication . Graça, Diogo; Ferreira, I.S.F.; Matos, A.; Gonçalves, L.M.D.; Silva, M.J.; Almeida, A.J.; Bettencourt, A.Polymethylmethacrylate (PMMA) is one of the most widely explored biomaterials because of its biocompatibility. Recent publications have shown an increasing interest in its application as a carrier system in drug delivery[1]. A major drawback in its use refers to the incomplete drug release from PMMA particles. To improve release profiles, recent strategies are focusing on formulating PMMA composites with permeable polymers. The main aim of the study was to evaluate the effect of the incorporation of a specific permeable polymer - an Eudragit polymer (EUD) - to PMMA particles. Both PMMA and PMMA-EUD particles were obtained through simple emulsion solvent evaporation methodology (SESE), previously optimized in our group[1]. Comparison between particles was mainly focused on physicochemical properties with impact on drug release, including particle size, surface charge, hydrophobicity and chemical composition, which were conducted using suitable methods such as laser diffraction, zeta potential, hydrophobic interaction chromatography (HIC), Fourier Transform Infrared (FT-IR) spectroscopy and transmission electron microscopy (TEM). SESE methodology allowed the production of particles with spherical and smooth surfaces within the micrometer range (PMMA = 572.7 ± 20 nm; PMMA-EUD = 508.9 ± 8 nm) with unimodal and narrow size distribution. Surface charge modification, as measured through zeta potential, was the main feature observed after inclusion of EUD in the formulation. Initial PMMA particles were strongly negative (-32.9±1.4 mV) whereas PMMA-EUD particles were strongly positive (+33.7±4.4 mV). The formulated PMMA-EUD particles also have shown slightly higher levels of hidrophobicity than the PMMA particles. Protein adsorption was also studied using a model protein (bovine serum albumin) through UV-Vis spectroscopy with both types of particles showing no considerable adsorption. The next step will be the evaluation of the effect of PMMA particles composition change in the release of a model drug. References: [1] Bettencourt, A., Almeida, A.J., J Microencapsul., 2012, 29(4):353-67.
- Drinking water contaminants: toxicity of halogenated polycyclic aromatic hydrocarbonsPublication . José, S.S.; Pinto, M.; Antunes, A.M.M.; Louro, H.; Jordão, Luísa; Silva, M.J.; Cardoso, A.S.Food may be contaminated with polycyclic aromatic hydrocarbons (PAHs) in the process of smoking or heating. These contaminants or their derivatives can also be present in drinking water when raw water contacts with discharges of untreated industrial/waste water effluents, forest fires or by solubilisation of organic material from contaminated soils. A few studies have shown that water disinfection can lead to halogenated derivatives of PAHs (HPAHs) as chlorinated and brominated derivatives, and there are evidences that these compounds may have greater mutagenicity than the parent PAHs. In this study the cytotoxic and genotoxic effects of chlorinated/brominated derivatives of pyrene (Pyr) and benzo[a]anthracene (BaA), 1-ClPyr, 1-BrPyr and 7-ClBaA, which can be formed as water disinfection by-products, were studied in HepG2 cells to assess their potential hazard to human health. The formation of 1-ClPyr, 1-BrPyr and 7-ClBaA under aqueous disinfection conditions in waters contaminated with Pyr and BaA, was confirmed with an optimized gas chromatography method. Cells exposed (24h) to several concentrations of BaA and 7-ClBaA (1 to 200μM) displayed a dose-related and significant increase of cytotoxicity (neutral red assay) with IC50 values of 3.37 and 12.63µM respectively. For Pyr, 1-ClPyr and 1-BrPyr (10 to 200μM), a lower but significant dose-related cytotoxicity was observed. At non-cytotoxic concentrations (10 and 15µM), 7-ClBaA was able to induce a significantly higher level of oxidative DNA damage in HepG2 cells than its parent compound, as assessed by the FPG-modified comet assay. Under these conditions neither Pyr nor its derivatives were genotoxic. In conclusion, the disinfection process may give rise to genotoxic HPAHs with potential impact on human health and it should be performed in raw waters with minimal content of total organic carbon. In real conditions, humans may be exposed to a mixture of these organic compounds and thus their combined toxic effects should be further evaluated.
- earlyMYCO project - Early-life exposure to mycotoxins: a neglected issue?Publication . Alvito, Paula; Amador, P.; Broeiro, Paula; Caldeira, T; De Boevre, Marthe; De Saeger, Sarah; Duarte, E.; Ferreira, M.; Lamy, Elisabete; Mexia, R.; Namorado, S.; Nunes, B.; Nunes, C.; Pires, S.; Silva, M.J.; Silva, Susana; Vidal, A.; Martins, C.; Assunção, R.Recent studies under MYCOMIX project reported that Portuguese children until 3 years old are exposed to multiple mycotoxins through food consumption, constituting a potential health threat. Aflatoxins (carcinogenic toxins) represented the main risk contributors and deoxynivalenol (a non-carcinogenic toxin associated with immunological and gastrointestinal toxic effects) showed the highest daily intake of the studied mycotoxins. These results opened new research perspectives and emphasized the need to accurately assess the prenatal and lactational exposure to mycotoxins in a critical and vulnerable period of life. Early-life exposure of children occurs during gestation through transfer of toxic substances present in the maternal diet to the fetus and later on, during lactation, through the breast milk. Considering this, the national project earlyMYCO – Early-life exposure to MYCOtoxins and its impact on health aims at assessing the risk of early-life exposure to mycotoxins. earlyMYCO proposes to answer several key questions including what extent are pregnant women and infants until six months exposed to mycotoxins in Portugal? Is this exposure a health threat? With this purpose, earlyMYCO gathered a multidisciplinar team with expertise on medical sciences, public health and toxicology to perform i) an epidemiological study, including the recruitment of pregnant women and infants, food survey and biological sample collection and ii) mycotoxin exposure assessment in pregnant women and infants using biomarkers of exposure. The epidemiological study was approved by INSA’s Ethical Committee and will be conducted in the Primary Health Care of Central Lisboa. The biomonitoring study will use advanced analytical methodologies and will provide data to perform the exposure assessment. Due to the increasing prevalence in food commodities, mycotoxins appear to be important, but often neglected contaminants in terms of health impact on human population especially in vulnerable groups as children. It is expected that results obtained within earlyMYCO will contribute to understand the impact of mycotoxin early-life exposure.