Browsing by Author "Louro, Henriqueta"
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- Abordagens experimentais à avaliação do efeito genotóxico de agentes químicos e físicosPublication . Silva, Maria João; Louro, HenriquetaAula 2: Abordagens experimentais à avaliação do efeito genotóxico de agentes químicos e físicos: Ensaios de longo-termo em roedores; Ensaios de curto-termo para avaliação de efeitos genotóxicos in vitro e in vivo; Linhas orientadoras para produtos farmacêuticos (ICH guidelines)
- 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.
- 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.
- Adverse Outcome Pathways Associated with the Ingestion of Titanium Dioxide Nanoparticles - A Systematic ReviewPublication . Rolo, Dora; Assunção, Ricardo; Ventura, Célia; Alvito, Paula; Gonçalves, Lídia; Martins, Carla; Bettencourt, Ana; Jordan, Peter; Vital, Nádia; Pereira, Joana; Pinto, Fátima; Matos, Paulo; Silva, Maria João; Louro, HenriquetaTitanium dioxide nanoparticles (TiO2-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO2-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO2-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO2-NPs in food, announced by the European Food Safety Authority (EFSA).
- Aflatoxins and ochratoxin A in baby foods and analysis of interactive cyto- and genotoxic effects in a human intestinal cell linePublication . Tavares, Ana; Alvito, Paula; Loureiro, Susana; Louro, Henriqueta; Silva, Maria JoãoMycotoxins are natural fungal metabolites and food contaminants with potential to cause severe acute and chronic conditions. Food contamination with mycotoxins such as aflatoxins (AF) and ochratoxin A (OTA) have been causing great concern, especially due to their potential mutagenic and carcinogenic effects. Children are especially vulnerable to the deleterious effects of these mycotoxins due to their physiological immaturity and high metabolic rate. Previous studies showed the co-occurrence of low concentrations of aflatoxins and OTA in baby foods. However, studies addressing potential interactive cyto- and genotoxic effects between these toxins are still scarce. In the present study we aimed to develop and validate a method for detection and quantification of total aflatoxins (B1, B2, G1, G2), AFM1 and OTA, and to evaluate the cytotoxic and genotoxic effects of mixtures of AFM1 and OTA, comparatively to their individual effects, in a human-derived intestinal cell line. A method based on immunoaffinity column cleanup and High Performance Liquid Chromatography with fluorescence detection (HPLC-FD), was applied and validated for total aflatoxins, AFM1 and OTA. The method was adequate for the analysis of these mycotoxins in baby foods and met the requirements of validation and quality control. The application of the method to a small set of baby foods marketed in Portugal showed an absence of quantifiable amounts of these mycotoxins. The individual and combined cytotoxic and genotoxic effects of AFM1 and OTA were characterized in Caco-2 cells using the Neutral Red and the Comet assays, respectively. A dose-dependent cytotoxicity was observed after individual exposure to OTA and AFM1, and the IC50 values were determined. The cytotoxic effect observed for several AFM1 and OTA mixtures was compared to the expected effect predicted by concentration addition (CA) and independent action (IA) conceptual models, using the MIXTOX model. A preliminary approach regarding the total data pool and considering the CA model as the most conservative model, pointed to an antagonistic cytotoxic effect caused by the mixture of both mycotoxins. However, a dose level deviation was observed after IA modelling, reflecting antagonism at low dose levels and synergism at higher dose levels. To better support data modelling, further cytotoxicity results from mixtures will be obtained and analyzed. To which respects the genotoxic effects, no induction of DNA damage was observed for the tested low doses, neither for individual toxins nor for their mixtures. The present study reinforces the relevance of exploring possible interactive adverse effects of mycotoxins that can contaminate foodstuff and thus having impact in human health. Future studies will face the challenge of understanding the mode of action of such mycotoxins when in mixture, in order to try predicting their effects.
- An integrative assessment to determine the genotoxic hazard of estuarine sediments: combining cell and whole-organism responsesPublication . Costa, Pedro Manuel; Pinto, Miguel; Vicente, Ana M.; Gonçalves, Cátia; Rodrigo, Ana P.; Louro, Henriqueta; Costa, Maria Helena; Caeiro, Sandra; Silva, Maria JoãoThe application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors,e.g.,when dealing with estuarine sediments,that hampers the drawing of cause-effect relationships. Although the in vitro The application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors, e.g., when dealing with estuarine sediments, that hampers the drawing of cause-effect relationships. Although the in vitro Comet assay may circumvent confounding factors, its application in environmental risk assessment (ERA) still needs validation. As such, the present work aims at integrating genotoxicity and oxidative DNA damage induced by sediment-bound toxicants in HepG2 cells with oxidative stress-related effects observed in three species collected from an impacted estuary. Distinct patterns were observed in cells exposed to crude mixtures of sediment contaminants from the urban/industrial area comparatively to the ones from the rural/riverine area of the estuary, with respect to oxidative DNA damage and oxidative DNA damage. The extracts obtained with the most polar solvent and the crude extracts caused the most significant oxidative DNA damage in HepG2 cells, as measured by the formamidopyrimidine-DNA glycosylase (FPG)-modified Comet assay. This observation suggests that metals and unknown toxicants more hydrophilic than polycyclic aromatic hydrocarbons may be important causative agents, especially in samples from the rural part of the estuary, where oxidative DNA damage was the most significant. Clams, sole, and cuttlefish responded differentially to environmental agents triggering oxidative stress, albeit yielding results accordant with the oxidative DNA damage observed in HepG2 cells. Overall, the integration of in vivo biomarker responses and Comet assay data in HepG2 cells yielded a comparable pattern, indicating that the in vitro FPG-modified Comet assay may be an effective and complementary line-of-evidence in ERA even in particularly challenging, natural, scenarios such as estuarine environments.
- Analysis of the Characteristics and Cytotoxicity of Titanium Dioxide Nanomaterials Following Simulated In Vitro DigestionPublication . Bettencourt, Ana; Gonçalves, L.; Gramacho, A.; Vieira, A; Rolo, D.; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Silva, Maria João; Louro, HenriquetaSeveral metallic nanomaterials (NMs), such as titanium dioxide nanomaterials (TiO2), present beneficial properties with a broad range of innovative applications. The human population is exposed to TiO2, particularly by ingestion, due to its increasing use as a food additive and 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, but studies have generated contradictory results, reflecting differences in the physicochemical properties of the TiO2 studied, effects of the surrounding matrix, and modifications during digestion. This work aimed to investigate the toxic effects of three different TiO2 NMs (NM-103, NM-103 and NM-105) on the gastrointestinal tract cells, Caco-2 and HT29-MTX-E12, after the use of the standardized static INFOGEST 2.0 in vitro digestion method to mimic human digestion of TiO2, contributing to hazard assessment. The results show that, for one of the digested TiO2 NMs studied (NM-105), a more pronounced toxicity occurs after exposure of HT29-MTX-E12 intestinal cells, as compared to undigested NM, concomitantly with subtle changes in characteristics of the NM. Thus, the inclusion of the digestion simulation in the safety evaluation of ingested NMs through in vitro bioassays can better integrate the modifications that NMs suffer in the organism. It is expected that such an approach will reduce uncertainties in the hazard assessment of ingested NMs for human health.
- Analysis Of The Cytotoxicity And Genotoxicity of Digested Titanium Dioxide Nanomaterials (TiO2) In Intestinal CellsPublication . Louro, Henriqueta; Vieira, Adriana; Gramacho, Ana Catarina; Rolo, Dora; Vital, Nádia; Martins, Carla; Assunção, Ricardo; Alvito, Paula; Gonçalves, Lídia; Bettencourt, Ana Francisca; Silva, Maria JoãoTitanium dioxide nanomaterials (TiO2) have been frequently applied as food additives, in pharmaceuticals and in personal care products, such as toothpastes. Despite some regulators like EFSA concluded that the absorption of orally administered TiO2 is low, and that the use of TiO2 as a food additive does not raise a genotoxic concern, the presence of TiO2 in human organs was recently reported. This exposure may lead to adverse outcomes and has been poorly investigated. Furthermore, many of the biological effects of TiO2 described in the literature often overlook adequate physicochemical properties and their modification due to NMs interaction with the surrounding physiological matrices happening, e.g, during digestion. This work aimed to investigate in intestinal cells, the cyto- and genotoxic effects of TiO2 after the simulation of the human digestive process using the standardized INFOGEST in vitro digestion method, to better understand their potential negative impacts on the gastrointestinal tract. The TiO2 were characterized before and after digestion using DLS, zeta potential and TEM-EDS. The digestion product was used for cytotoxicity (MTT) and genotoxicity (comet, micronucleus) assays in two types of intestinal cells (Caco-2 and mucus secreting HT29-MTX cells). The results of the cytotoxicity and genotoxicity assays are discussed in view of the TiO2 secondary characteristics, to further understand the potential adverse intestinal outcomes in light of the transformation they suffer during digestion.
- Analysis of the in vitro cytotoxicity and genotoxicity of cellulose nanomaterials in intestinal cellsPublication . Vital, Nádia; Pinto, Fátima; Kranendonk, Michel; Silva, Maria João; Louro, HenriquetaCellulose nanomaterials(CNMs) are recognized as promising bionanomaterials due to their natural and renewable source and attractive properties, with potential applications in multiple food-related products, as zero-calorie filler/thickener/stabilizer, or as substitutes of petroleum-based food packaging materials. These applications in food lead to human exposure through ingestion and their biopersistence raise concerns about their potential impact on the gastrointestinal tract. The present study aimed to investigate the in vitro cytotoxicity and genotoxicity of two types of micro/nanofibrillated celluloses(CMF/CNFs) using a human intestinal cell line. CMF and CNF were synthetized from industrial bleached Eucalyptus globulus kraft and their physicochemical properties were previously characterized. Following Caco-2 cells exposure to a concentration-range of CMF or CNF, the cytotoxicity was assessed by the MTT and clonogenic assays, while genotoxicity was assessed by the cytokinesis block micronucleus assay(CBMN). Outcomes indicated no cytotoxic or genotoxic effects upon exposure of Caco-2 cells to these CNMs for the tested concentration-range, suggesting their biocompatibility. This is the first study using the CBMN assay after Caco-2 cells exposure to CNMs. The results of ongoing studies using in vitro simulation of human digestion will allow a more comprehensive assessment of CNMs safety. By incrementing the knowledge on the cellular effects of novel CNMs in the human intestine, we expect to contribute to their safety assessment at an early-stage of their technological development towards the sustainable innovation in food technology.
- 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.