DSA - Apresentações orais em encontros internacionais
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- 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.
- 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.
- Condições de iluminação em ambiente de escritório: Influência no conforto visual [comunicação oral]Publication . Pais, Aida; Melo, Rui BettencourtAs condições de iluminação condicionam o conforto visual, podendo provocar fadiga visual, irritabilidade ocular, dores de cabeça, dores musculares, dificuldade de concentração. Assim sendo, este estudo pretendeu identificar e avaliar a relação entre a iluminação de postos de trabalho administrativos e o conforto visual dos seus ocupantes. A amostra foi constituída no seio de 3 empresas do sector administrativo e integrou 124 indivíduos. Os instrumentos usados para recolha de dados foram um questionário e uma checklist, um luxímetro e uma máquina fotográfica. Para o tratamento dos dados recorremos ao SPSS (Statistical Package for the Social Sciences), versão 18, tendo-se efectuado uma análise estatística descritiva e inferencial, pela aplicação dos testes de Kruskal-Wallis e de comparações múltiplas. Para a medição e avaliação da iluminância e da uniformidade foram seguidas as normas europeias EN 12464-1 (2002), DIN 5035 (1990) e ISO 8995-1 (2002). Os resultados do estudo revelaram que a ocorrência de desconforto visual e da Síndrome de Visão de Computador estão relacionados com valores de iluminância abaixo dos recomendados, tanto na área da tarefa como na área da vizinhança, com a existência de brilhos e de reflexos no posto de trabalho, assim como com o tempo de trabalho em computador, sem pausas.
- Trihalomethanes in Lisbon Indoor Swimming Pools: Occurrence and Determining FactorsPublication . Silva, Z.; Rebelo, H.; Silva, M.M.; Alves, A.; Cabral, C.; Almeida, A.C.; Aguiar, F.; Oliveira, A.; Nogueira, A.; Pinhal, H.; Matos, A.; Ramos, C.D.; Pacheco, P.; Aguiar, P.; Cardoso, A.S.The presence of water disinfection by-products (DBPs) in swimming pools constitutes today a public health concern, particularly because swimming is an activity used by a high percentage of the population, namely elderly and young children. Moreover, several adverse short-term and long-term health effects have been associated with these compounds (Lakind et al., 2010; Zwiener et al., 2007). Water disinfection methods are used in all swimming pools, namely in public pools, to ensure an adequate and effective protection of users against microbiological pathogens. Chlorination is the most common disinfection method used worldwide, because it is low cost, easy to use, efficient against a broad spectrum of microorganisms, and enables the maintenance of a residual protection. The use of chlorine based treatment techniques has one strong drawback, which is the generation of several DBPs, potentially harmful products, that can be absorbed by ingestion, inhalation and absorption through the skin (Nieuwenhuijsen et al., 2009; Caro and Gallego, 2007). DBPs comprise several compounds that are formed through the reaction of chlorine with organic matter present in water. Amongst DBPs, the most relevant and better characterized are trihalomethanes (THMs): chloroform (CF), bromoform (BF), bromodichloromethane (BDCM), and chlorodibromomethane (DBCM). These compounds have been associated with health effects such as respiratory, ocular and cutaneous symptoms and also with some long-term health effects such as bladder cancer and adverse reproductive outcomes (Lee et al., 2009; Zwiener et al., 2007). Accurate exposure assessments to THMs in indoor pool environment is particularly difficult because their formation depends on many factors such as water and air temperature (Tw and Tair), humidity (Hu), pH, free residual chlorine (FrCl), total organic content and number of pool users (Lee et al., 2009). In Portugal a specific legislation to assess the quality of swimming pool waters is inexistent. The guideline value used in this study for total THMs (TTHMs) was the one established in the Portuguese Law 306/2007, for drinking water quality - 100 μg/L. Some other water parameters determined in swimming pools such as Tw turbidity (Turb), pH, FrCl, total residual chlorine, conductivity (Cond), permanganate index (COD) and isocyanuric acid were assessed by Portuguese Law 5/97, regarding technical and safety conditions of closed environments with water diversions. WHO guidelines for safe recreational water environments (2006) were also used to assess some water and air parameters in pools. Standard 62.1 (2006) from American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASRHAE) was also used to assess pool air parameters. The main goal of this investigation project was to study the occurrence, distribution and determinants of THMs in indoor swimming pools. This will enable an evaluation of swimming pool users’ exposure to THMs. In order to achieve this goal, the characterization of water quality of 30 Lisbon indoor swimming pools, using chorine based treatment techniques, was made during a six month period. Because of laboratory working restrictions, CF concentration in pool air was studied only in 6 swimming pools, at the same period. Several parameters such as TTHMs concentration, CF, BDCM, DBCM, BF concentration in water, FrCl, combined residual chlorine (CrCl), pH, Tw and Tair, Hu, COD, Cond, Turb and chloride (Cl) were determined in each pool, once a month. THMs water sampling was made in duplicates in two pool water spots, physicochemical parameters were determined in one of the THMs water sampling spots and THMs air sampling was made in two pool spots, considered to be representative of the swimming pool air. All sampling procedures were performed by specialized sampling technicians from Lisbon Health Centres Group (ACES). Statistical analysis was performed using the SPSS 17.0 software. Descriptive analysis was applied to all variables in order to determine mean, median, standard deviation, minimum and maximum. Student’s t-test and Mann-Whitney tests were used to compare numerical variables and Spearman’s rank correlation coefficient were used to examine correlation between all environmental parameters. TTHMs water level ranged from 10 to 160 μg/L, while CF water level typically ranged from 5 to 150 μg/L, with occasional higher levels detected. Regarding other THMs levels in pool water, BDCM ranged from 0.5 to 15 μg/L, DBCM from 0.4 to 10 μg/L and BF was obtained in concentrations less than 2 μg/L. Moreover, in some pools, DBCM and BF were not detected. From these results, it can be concluded that there was a large variation in TTHMs and CF water levels between the pools and that CF was the THM obtained in higher concentrations. These results are in agreement with studies presented by other authors (Lee et al., 2009; Chu and Nieuwenhuijsen, 2002). In air, CF level ranged from 40 to 200 μg/m3, with occasional higher levels detected. Tw presented less variation (27 to 31 ºC) than Tair (21 to 33 ºC), although sporadic higher values were found. FrCl and Cond presented higher values than the guidelines from DR 5/97 only in a small number of pools. Turb and COD values were within the guidelines from DR 5/97. pH varied from 7 to 8.5, with only a small number of pools having values higher than 7.8. As referred in WHO guidelines, pH should be maintained between 7.2 and 7.8 for chlorine disinfectants to ensure efficient disinfection. Using the statistical tests mentioned before, it was possible to obtain some interesting results. There was a clear positive linear correlation between CF water concentration (CFW) and TTHMs water concentration (R>0.98, p<0.01), good correlation other THMs, there was a clear positive linear correlation between BDCM and DBCM water concentration (R>0.78, p<0.01) and good correlation between TTHMs water concentration and Tw (R>0.45, p<0.05). These results are in agreement with Lee et al. (2009) which obtained positive linear correlations between CFW and COD and with Chu and Nieuwenhuijsen (2002) which also obtained correlations between TTHMs water concentration and Tw. In conclusion, there appears to be good water and air quality in the studied Lisbon swimming pools. Regarding THMs levels in water, some pools presented high TTHMs and CF concentrations. Moreover, CF air concentrations were also high in some swimming pools. These results clearly demonstrate that THMs monitoring is particularly important and that, in a near future, this should be extended to other DBPs. Furthermore, for a correct and conscious use of swimming pools, appropriate and targeted information about safe practices should be provided to pool users.
- Monitorização de cianobactérias no centro e sul de Portugal: identificação de albufeiras de risco de ocorrência de cianotoxinasPublication . Dias, Elsa; Paulino, Sérgio; Churro, Catarina; Menezes, Carina; Alverca, Elsa; Sam-bento, Filomena; Pereira, PauloO nosso laboratório tem vindo a monitorizar a ocorrência de cianobactérias e toxinas associadas em albufeiras do centro e sul de Portugal. Iremos apresentar os resultados obtidos nos últimos 10 anos de monitorização relativamente à frequência de ocorrência e densidade celular de espécies tóxicas e aos níveis de microcistinas detectados na água bruta e água tratada (quando aplicável). Identificámos situações muito distintas, em que algumas albufeiras nunca apresentaram cianobactérias potencialmente tóxicas e em que noutras o crescimento cianobacteriano foi persistente, por vezes associado também à persistência de microcistinas na água bruta e à ocorrência de células na água tratada. Iremos discutir os critérios de identificação de albufeiras de risco para a saúde pública.
- Genotoxicidade da MCLR em vários modelos biológicos: linhas celulares, eritrócitos de murganho e linfócitos humanosPublication . Dias, Elsa; Louro, Henriqueta; Pereira, Paulo; Silva, Maria JoãoO conhecimento sobre as propriedades toxicológicas das microcistinas é ainda bastante limitado, em particular no que respeita à sua actividade cancerígena. É globalmente aceite que as microcistinas são promotores tumorais, mas é ainda ambígua a sua eventual genotoxicidade. Alguns autores defendem que as microcistinas são genotóxicas e que induzem quebras no ADN, outros defendem que as microcistinas não são genotóxicas. Alguns destes resultados são dificilmente comparáveis porque foram obtidos através de metodologias distintas, nomeadamente no que respeita ao tipo de toxina usado, ao modelo celular testado e ao método para avaliação de efeito genotóxico aplicado. A nossa equipa tem vindo a estudar as propriedades genotóxicas da MCLR em diversos modelos biológicos: linha celular Vero-E6 (rim de macaco), linha celular HepG2 (hepatoma humano) e eritrócitos de murganho. Para tal temos usado dois tipos de abordagem metodológica: o Teste do Cometa, que avalia danos ao nível da cadeia de ADN, e o Ensaio do Micronúcleo, que avalia a actividade clastogénica (quebra cromossómica) ou aneugénica (perda de cromossomas). Naqueles modelos demonstrámos que a MCLR não induz danos no ADN pelo teste do Cometa mas induz o aumento da frequência de micronúcleos in vitro e in vivo. Nesta apresentação iremos discutir os nossos resultados mais recentes acerca do efeito da MCLR na indução de micronúcleos em linfócitos humanos e comparar com os resultados publicados por outros autores. Iremos também discutir o mecanismo de acção genotóxica da MCLR e a sua importância para a avaliação do risco de exposição humana a água contaminada com cianobactérias produtoras de microcistinas.
- Ocorrência de Planktothrix spp. em águas doces Portuguesas: identificação e caracterização molecularPublication . Churro, Catarina; Menezes, Carina; Dias, Elsa; Pereira, Paulo; Valério, Elisabete; Vasconcelos, VitorPlanktothrix é um género de cianobactéria potencialmente produtor de microcistinas frequentemente encontrado em albufeiras Portuguesas. Com o objectivo de caracterizar genética e morfologicamente espécies deste género foram usados neste estudo dezoito estirpes de Planktothrix. As estirpes foram previamente isoladas de várias albufeiras e em diferentes anos e mantidas em culturas monoclonais na colecção de culturas Estela Sousa e Silva. Cada estirpe foi identificada e caracterizada através da análise morfológica e taxonómica, através da técnica de “PCR-fingerprinting” com a sequência repetitiva “STRR (short tandemly repeated repetitive sequences)”, e com base em análise filogenética do gene rpoC1. O potencial tóxico de cada estirpe foi avaliado através da amplificação por PCR do gene mcyA. A diversidade genética das diferentes estirpes foi também avaliada por meio do padrão de bandas de sequências repetitivas de DNA do bacteriófago M13. Os perfis de “PCR-fingerprinting” gerados permitiram a discriminação de estirpes pertencentes a diferentes bacias hidrográficas e revelou similaridade genética entre estirpes de mesma bacia hidrográfica.
- 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.
- Analysis and Identification of Trihalomethanes in Lisbon Indoor Swimming Pools: Distribution, Determinants and Human ExposurePublication . Silva, Z.; Rebelo, H.; Silva, M.M.; Alves, A.; Cabral, C.; Almeida, A.C.; Aguiar, F.; Oliveira, A.; Nogueira, A.; Pinhal, H.; Matos, A.; Ramos, C.D.; Pacheco, P.; Aguiar, P.; Cardoso, A.S.Water disinfection methods are used in swimming pools to ensure an effective protection of users against microbiological pathogens, being chlorination the most common disinfection method used worldwide. The use of chlorine based treatment techniques has one strong drawback, which is the generation of disinfection by-products (DBPs), potentially harmful products that have been associated with respiratory and ocular symptoms, bladder cancer and adverse reproductive effects. Amongst DBPs, the most better characterized are trihalomethanes (THMs): chloroform (CF), bromoform (BF), bromodichloromethane (BDCM), and chlorodibromomethane (DBCM). In Portugal a specific legislation to assess the quality of swimming pools is inexistent. The guideline value used to evaluate total THMs in water (TTHMsW) is the one established in the Law 306/2007 - 100 μg/L. Some other water/air parameters were assessed by Law 5/97, WHO guidelines for safe recreational water environments (2006) and Standard 62.1 (2006) from American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASRHAE). The main goal of this investigation was to study the occurrence, distribution and determinants of THMs in indoor swimming pools. This will also enable an evaluation of the exposure of users to THMs. In order to achieve this goals, the characterization of water quality in 30 Lisbon indoor swimming pools, using chorine based treatment techniques, was made during a six month period. Several parameters such as TTHMs, CF, BDCM, DBCM, BF levels in water, free residual chlorine (FrCl), pH, TW, Tair, Hu, chemical oxygen demand (COD), and other, were determined in each pool, once a month. Statistical analysis was performed using the SPSS 17.0 software. Descriptive analysis was applied to all variables. Student’s t-test, Mann-Whitney tests and Spearman’s rank correlation coefficient were also used. Large variation in TTHMs and CF water levels between the pools was obtained, being CF the THM obtained in higher concentrations, with sporadic higher values than the allowed. In air, CF level (CFair) also presented occasional higher levels. There was a clear positive linear correlation between CFW and TTHMsW (R>0.95, p<0.01), CFW and CFair (R> 0.5; p<0.05), CFW and FrCl (R>0.2; p<0.05) and, CFw and Tw (R>0.2; p<0.05). Good correlations were also obtained between other THMs: BDCM and DBCM (R>0.5, p<0.01) and BF and DBCM (R>0.6; p<0.05). The strong positive correlation obtained between CFW and TTHMsW was expected, since, usually, CFW has the higher contribution to TTHMsW. CFW is often considered as a good indicator of TTHMs concentration in water. Therefore correlations between TTHMs and CFair, FrCl, and Tw were expected and observed: TTHMsW vs CFair (R>0.5; p<0.05), TTHMsW vs FrCl (R>0.2; p<0.05) and TTHMsW vs TW (R>0.2; p<0.01). In conclusion, reasonable water and air quality was obtained in the studied Lisbon swimming pools, although some pools presented high TTHMsW, CFW and CFair. These results clearly demonstrate that THMs monitoring is particularly important and that, in a near future, this should be extended to other DBPs. Furthermore, for conscious use of swimming pools, appropriate and targeted information about safe practices should be provided to pool users.
- Controlo AmbientalPublication . Cano, M.