Browsing by Author "Alverca, Elsa"
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- Are standard genotoxicity tests useful for the safety evaluation of nanomaterials?Publication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Antunes, Susana; Costa, Pedro; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNanomaterials (NMs) are widely used in a diversity of consumer products, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. One of the major concerns is the potential to induce cancer. To analyze in a short term the carcinogenic properties of a compound, genotoxicity assays in mammalian cell lines or animal models are frequently used. In the context of an EU Joint Action, in the present work we have used standard genotoxicity assays (comet, micronucleus and mutation assays) to investigate the effects associated with the exposure to titanium dioxide nanomaterials (TiO2), following standardized dispersion and assay procedures, in three types of human cells and in a mouse model. The results showed slight but significant increases in the frequencies of micronuclei after exposure to some of the NMs, as compared to controls. No clear dose-response relationships could be disclosed. One of the tested TiO2 yielded equivocal results in vitro micronucleus assay and was positive in the comet assay in pulmonary cells. In view of the inconclusive results,it was further analyzed in vivo, using the lacZ transgenic mouse model. It did not induce genotoxic effects in mice, 28 days after injection, despite the accumulation of the NM observed in the mouse liver. Regarding safety assessment, the different genotoxicity observed for closely related NMs, but that differ in some physicochemical characteristics, highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs. The equivocal genotoxicity of the nanosized TiO2 in human cells in vitro was not confirmed in vivo, and therefore the predictive value of these in vitro tests for identifying genotoxic (and potentially carcinogenic) NMs in vivo should be clarified, before extrapolating the conclusions for human health.
- Avaliação integrada dos efeitos genotóxicos de nanomateriais manufaturados no ratinho transgénico LacZPublication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Costa, Pedro M.; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNum estudo recente, demonstrámos que um nanomaterial de dióxido de titânio na forma cristalina designada anatase, o NM-102 (do repositório do Joint Research Center; Ispra, Itália), induziu um aumento significativo de quebras cromossómicas - detetáveis na forma de micronúcleos - em linfócitos humanos expostos ex vivo(3) não se tendo, porém, observado um efeito dose-resposta. No sentido de prosseguir e aprofundar a avaliação da genotoxicidade do NM-102, o presente estudo teve por objetivo caracterizar os seus efeitos genotóxicos, num modelo animal, utilizando uma abordagem integrada, a qual abrangeu a análise de vários parâmetros de genotoxicidade no mesmo animal.
- Coleção de culturas de algas Estela Sousa e SilvaPublication . Menezes, Carina; Churro, Catarina; Paulino, Sérgio; Sam-Bento, Filomena; Alverca, Elsa; Dias, Elsa; Pereira, PauloA coleção de culturas de algas Estela Sousa e Silva (ESSACC) foi criada em 1956 e reside atualmente no Laboratório de Biologia e Ecotoxicologia no Instituto Nacional de Saúde Dr. Ricardo Jorge. A ESSACC foi implementada em resposta à necessidade de um repositório de material biológico para investigação na área do fitoplâncton. Este importante recurso biológico contém culturas monoclonais de algas eucarióticas e cianobactérias provenientes de águas costeiras e albufeiras portuguesas. Atualmente, a coleção mantém acima de 176 isolados vivos, dos quais 151 são cianobactérias de água doce e 25 são dinoflagelados marinhos. Adicionalmente são também mantidos alguns isolados pertencentes a outros grupos de fitoflagelados. Esta coleção permitiu até agora a identificação e caracterização de espécies assim como a produção e purificação de toxinas para aplicação em estudos toxicológicos entre outras diversas áreas de investigação. Deste modo, a ESSACC constitui uma ferramenta importante no fornecimento de culturas de algas a investigadores na área do fitoplâncton, particularmente no estudo de espécies nocivas.
- Cytotoxic and morphological effects of microcystin-LR in in vitro models - a comparision between HepG2, Vero-E6, MDCK and CaCo-2 cell linesPublication . Menezes, Carina; Alverca, Elsa; Dias, Elsa; Sam-Bento, Filomena; Paulino, Sérgio; Pereira, PauloMicrocystin-LR (MCLR), a potent hepatotoxin, is transported selectively into the cells throught specific membrane polypeptides mostly present in the liver. These transporters are also expressed in the brain, kidneys and intestine, although the toxicity of MCLR in these cell types is still poorly understood. In this study, morphological, ultrastructural and biochemical analyses were performed in hepatic, renal and intestinal cell lines in order to evaluate the toxicity of MCLR obtained from semi-purified cyanobacterial extracts. Our results show that after 24h of exposure, MCLR induces the viability decrease of all cell lines, in a concentration-dependent manner. HepG2 cells are the most susceptible, followed by Vero, MDCK and CaCo-2. Ultrastructural analyses show that subcytotoxic concentrations of MCLR induce the formation of large cytoplasmic vacuoles, particularly in the HepG2 cell line. Immunofluorescence microscopy reveals that these vacuoles are enriched in LC3B protein, suggesting autophagy as an early cellular response of HepG2 and Vero cells to MCLR. At cytotoxic MCLR concentrations, lysossomal dysfunction in both cell lines occurs prior to mitochondrial disruption, as demonstrated by the specific labeling with Acridine Orange and Rhodamine-123. This suggests that besides mitochondria, lysossomes may also be an MCLR-early target. Immunolocalization and western blot analysis of the endoplasmic reticulum anti-apoptotic protein GRP94 show distinct MCLR-induced effects in Vero and HepG2 cells: re-localization of GRP94 within Vero cells and decrease of GRP94 expression in the HepG2 cell line. These results demonstrate that all the studied cell lines are susceptible to MCLR although with cell type specificity and differential organelle targeting.
- Efeitos de microcistina-LR em células HepG2, Vero, MDCK e CaCo2Publication . Menezes, Carina; Alverca, Elsa; Dias, Elsa; Sam-Bento, Filomena; Pereira, PauloA microcistina-LR (MCLR) é amplamente reconhecida pela sua hepatotoxicidade. No entanto sabe-se que também afecta outros orgãos tais como o cérebro, rins e intestinos. Este trabalho tem como objectivo comparar os efeitos tóxicos da exposição a uma gama de concentrações de MCLR pura e de extractos de cianobactérias em linhas celulares hepáticas, renais e de intestino, representativas dos orgãos de acumulação da MCLR, ao nível da viabilidade celular e ultrastrutura. As linhas celulares HepG2 (hepatoma), Vero-E6 e MDCK (renais) e CaCo2 (adenocarcinoma do cólon) foram expostas a 1-100 ìM de MCLR durante 24h e a viabilidade celular foi determinada através do teste do Neutral Red. Observou-se em todas as linhas celulares um decréscimo da viabilidade dependente da concentração de MCLR. Contudo, as células HepG2 mostraram uma maior sensibilidade, seguidas das células Vero e das células MDCK e CaCo2. A observação da ultrastrutura celular a concentrações citotóxicas de MCLR revelou a presença abundante de células apoptóticas nas quatro linhas celulares. A baixas concentrações de MCLR, as células HepG2 e Vero apresentaram numerosos vacúolos citoplasmáticos, com conteúdo electrodenso, indicativo de autofagia. Nas células Vero foram ainda visíveis alterações no retículo endoplasmático, o que sugere que ambos os organelos estão envolvidos num mecanismo de resposta celular a concentrações sub-citotóxicas de toxina. Em células MDCK os alvos intracelulares primários parecem ser o complexo de Golgi e as mitocôndrias, tal como em células CaCo2, ainda que neste caso os efeitos tóxicos sejam observáveis apenas a concentrações de MCLR mais elevadas. Os resultados deste estudo in vitro mostram que a MCLR induz efeitos mais pronunciados nas células de fígado tal como indicado pelos estudos in vivo. Para todas as linhas celulares estudadas a perda de viabilidade é dependente da concentração de MCLR apesar de o tipo celular parecer interferir na sensibilidade e alvos intracelulares da MCLR.
- Effects of bacillamide and newly synthesized derivatives on the growth of cyanobacteria and microalgae culturesPublication . Churro, Catarina; Alverca, Elsa; Sam-Bento, Filomena; Paulino, Sérgio; Figueira, Valdemar; Bento, Artur; PrabhaKar, Sundaresan; Lobo, Ana; Calado, António; Pereira, PauloThe antialgal activity of newly synthesized bacillamides against several cyanobacteria and microalgae isolates was screened using a rapid 96-well microplate bioassay. Cultures were exposed to serial dilutions of each bacillamide derivative (0–160 μg mL−1) in the microplate wells and daily optical measurements were used to estimate growth over a 216 h period. Inhibition values (%) were calculated from the estimated growth curves and inhibitory concentrations (IC50-216 h) were obtained from the sigmoidal inhibition curves fitted by regression analysis. The effects of bacillamides on cell morphology and ultrastructure were also analysed by light and transmission electron microscopy. In general, the toxic cyanobacteria Microcystis aeruginosa, Aphanizomenon gracile, Anabaena circinalis and Anabaenopsis circularis were much more sensitive to bacillamides then the chlorophytes Ankistrodesmus falcatus and Scenedesmus obliquus. However, clear signs of morphological and ultrastructural changes induced by bacillamide were observed on both cyanobacteria and chlorophytes. Other cyanobacteria, namely the nostocalean Nodularia spumigena and the oscillatorialeans Leptolyngbya sp. and Planktothrix rubescens, exhibit higher tolerances to bacillamides, similar to that shown by different eukaryotic microalgae. Diatoms, on the other hand, proved to be quite as sensitive to most bacillamides as the most affected cyanobacteria. The properties of 5-iodo-Bacillamide (algicide or algistatic) were further investigated. This compound acted as an algistactic agent against eukaryotic algae and, depending on its concentration, acted as either an algicide or algistactic agent against most of the cyanobacteria tested. Although bacillamides cannot be considered as broad spectrum cyanobacterial algicides, different bacillamides might be of use in selectively controlling the growth of particular species of cyanobacteria.
- Estudo comparativo da citotoxicidade e alterações citopatológicas induzidas pela microcistina-LR em linhas celulares renais e hepáticas (Vero e HepG2).Publication . Menezes, Carina; Alverca, Elsa; Dias, Elsa; Paulino, Sérgio; Sam-Bento, Filomena; Pereira, PauloMicrocystin-LR (MCLR) is a potent hepatotoxin produced by freshwater cyanobacteria, being responsible for acute and chronic hazards to animal and human health. Despite the increasing recognition of its toxic effects, the cellular basis of MCLR-induced toxicity is still poorly understood. In this work, morphological, ultrastructural and biochemical (MTT and Neutral Red-NR) analyses were performed to evaluate the effects of a semi-purified MCLR-containing cyanobacterial extract on Vero and HepG2 cell lines. Our results showed that cell viability decayed markedly after 24h of exposure to toxin concentrations greater than 25μg.ml-1 in both cell lines. The ultrastructural analysis revealed that at subcytotoxic MCLR doses, cells presented large cytoplasmic vacuoles, which were more prominent in HepG2. These vacuoles showed to be enriched in the LC3 protein, suggesting that autophagy is an early cellular response to MCLR. At higher doses, the specific staining Acridine Orange (AO) and Rhodamine-123 (Rh-123), demonstrated that lysosome destabilization precedes mitochondrial dysfunction. Besides, MCLR seemed to induce a decrease in the expression of the anti-apoptotic endoplasmic reticulum protein Grp94, particularly evident in HepG2 cells. These results suggest that MCLR-induced cytotoxicity involves a considerable crosstalk among several organelles and that despite some cell-specific features, the general cellular basis underlying this toxicity is common to both Vero and HepG2 cells.
- Exploring a link between mycobacteria structure and virulencePublication . Silva, Carla; Perdigao, Joao; Alverca, Elsa; Matos, António Pedro Alves; Carvalho, Patrícia A; Portugal, Isabel; Jordão, LuísaTuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR)Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely-related mycobacteria known collectively as Lisboa family and Q1 cluster. The genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless, little is known about other factors involved in drug resistance development. Here we focused on the study of morphological and structural features of Mtb isolates, collected during 2008-2009 in Lisbon, in order to complement the genetic analysis. For this propose scanning and transmission electron microscopy techniques were used. Particular attention was given to Lisboa family and Q1 cluster isolates since together they account for the majority of reported MDR-TB cases. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates, which are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of Lisboa family isolates over other circulating Mtb isolates.
- Exploring the contribution of mycobacteria characteristics in the interaction with human macrophagesPublication . Silva, Carla; Perdigao, Joao; Alverca, Elsa; de Matos, António Pedro Alves; Carvalho, Patrícia A; Portugal, Isabel; Jordão, LuísaTuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely-related mycobacteria known collectively as Lisboa family and Q1 cluster. The genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless little is known about other factors involved in drug resistance development. Here, for the first time, we complemented the genetic analysis with the study of morphological and structural features of Lisboa family and Q1 cluster isolates by using scanning and transmission electron microscopy. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates, which are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of Lisboa family isolates over other circulating Mtb isolates.
- Importância da monitorização de cianobactérias em albufeiras portuguesasPublication . Churro, Catarina; Dias, Elsa; Paulino, Sérgio; Alverca, Elsa; Pereira, Paulo
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