DSA - Apresentações orais em encontros internacionais
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- 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.
- Avaliação da influência da intensidade de luz na expressão do gene mcyA e na produção de microcistina em Microcystis aeruginosa e Planktothrix agardhiiPublication . Salvador, Daniel; Churro, Catarina; Valério, ElisabeteAs cianobactérias são frequentemente associadas à produção de toxinas, nomeadamente microcistinas. A sua síntese é não ribossomal, e acontece utilizando complexos multienzimáticos (genes mcy). Diversos estudos têm demonstrado que os fatores ambientais podem influenciar a produção de toxina. O objetivo deste estudo foi avaliar a influência da intensidade da luz na transcrição do gene mcyA e correspondente produção de microcistina em isolados tóxicos de Microcystis aeruginosa e Planktothrix agardhii. Para esse fim, as culturas foram expostas a três diferentes intensidades de luz (4, 20 e 30 µmol fotões m-2 s-1) durante 18 dias a 20 ± 1ºC. O crescimento foi seguido diariamente espectrofotometricamente. O nível de transcritos foi quantificado por RT-qPCR e a expressão relativa determinada usando três genes de referência - rRNA 16S, gltA e rpoc1. Os resultados mostraram a existência de uma correspondência entre a taxa de crescimento e a intensidade de luz em ambas as espécies. As taxas de crescimento foram menores a 4 e maiores a 30 µmol fotões m-2 s-1. Em M. aeruginosa a concentração de microcistina por célula foi semelhante entre intensidades de luz e ao longo do tempo, enquanto que em P. agardhii a concentração foi mais elevada na fase estacionária a 4 µmol fotões m-2 s-1. Existiram diferenças na expressão de mcyA entre as duas espécies. Em M. aeruginosa, a expressão foi máxima a 4 µmol fotões m-2 s-1 na fase de adaptação, já em P. agardhii foi máxima a 4 µmol fotões m-2 s-1 na fase exponencial de crescimento.
- Beach sand as a source of faecal indicator organismsPublication . Brandão, JoãoMicrobial life in beach sands research began in Bacteriology with Roger Fujioka of the University of Hawai'i and in Mycology with Laura Rosado, of the Portuguese National Institute of Health. Since then, many reports and scientific papers have been published on the subject. The number of publications in this area is currently increasing exponentially, most of which addressing methodological approaches and weaving considerations on the microbiome characterization and influence on human health. Levels of bacteriological indicators of bathing water quality are of concern due to run-offs and tide retractions. Clear and precise guidance on the subject, however, has been lost in time. It should serve as a working basis for regulators and research. Global warming and climate change also bring a regional de-characterization of the microbiota, due to the geographic expansion of endemic microbes. These bring along unexpected illnesses diagnosed with some degree of unexpectedness or difficulty by local clinicians. The following three points are currently of high relevance in sand contaminants: A. Sampling, representativeness and analytical methods: sand is patchy, not a homogeneous or a fluid system. In 2015, an open letter was published, as a result of a meeting of experts, from several scientific areas converging in one posture towards the issue of sand contaminants. During this meeting, it was decided that sampling should be evaluated, and that levels of relevance and clinical expression or influence of the quality of adjacent waters should stratify analytical methods. A basic analytical snapshot of the level of contamination of sand (sanitation), is inexpensive and its feasibility leads to easy transfer between laboratories. This allows it to become current practice and to integrate the process of quality regulation and public health protection. However, in pathogen detection situations, epidemiological outbreaks or over-dominance of unexpected and persistent microbial agents, the health risk or socio-economic implications should be assessed, and the causes identified, in order to be controlled or eradicated. There is present need to define levels of analysis in order to adopt the recommendation of the open letter, excluding neither classic nor molecular methodologies (namely, next generation sequencing, which allows the characterization of full microbiomes). B. Biological groups indigenous to beach sand, the definition or relevance of indicators and pathogens, and their forms of detection and counting, are being published without any methodological equivalence or biological relevance. The lack of regulation and international standards for the evaluation of contaminants in sand is generating a logistical nightmare for those who want to make interregional comparisons. A quantitative microbial risk assessment based on the clinical and safety aspects of several agents and biological groups is in order to pre-structure international regulation. Fecal indicator and other pathogenic or opportunistic bacteria from intertidal sands, as well as dry sand contamination resulting from animal shedding or direct sand / soil life, will be included in these groups; Enteric and respiratory viruses propagated through the environment (intertidal sands); Pathogenic, opportunistic and allergenic fungi; Parasites; and forms of allochthonous life as a consequence of sand nourishment. Considerations on environmental resistome of sand should not be excluded. C. Implications of climate change in sand / soil microbiome Extreme weather events, global warming and migration of endemic species are current factors that will dictate future risk assessments.
- Cianobactérias e Saúde: um problema ou uma oportunidade?Publication . Dias, Elsa; Paulino, Sérgio; Pereira, PauloMuito se tem escrito e discutido sobre o impacto negativo das toxinas produzidas por cianobactérias na saúde humana. No entanto, muitas dúvidas persistem sobre os efeitos reais destes compostos nas populações. A comunidade científica depara-se com dificuldades na avaliação do risco de exposição humana a cianotoxinas, o que dificulta a regulamentação dos níveis de toxinas no ambiente, bem como a gestão do risco associado às ocorrências de cianobactérias tóxicas. Em países com Portugal e Espanha o risco de intoxicação aguda por cianotoxinas será provavelmente reduzido. Nestes países, os sistemas de tratamento de água e os programas de vigilância em águas de consumo e recreativas garantem que as populações não serão expostas a níveis elevados de toxinas. No entanto, desconhecemos quais os riscos de exposição humana prologada a baixas doses decorrente da utilização de reservatórios frequentemente contaminados com cianobactérias tóxicas, onde eventualmente persistem níveis residuais de cianotoxinas, inferiores ao limite legal e até ao limite de deteção analítico. O potencial cancerígeno de algumas cianotoxinas tem vindo a ser discutido. É amplamente aceite que as microcistinas induzem hepatotoxicidade aguda, que actuam pela inibição das fosfatases proteicas PP1 e PP2A e do stress oxidativo, que são promotores tumorais. São toxinas classificadas pela IARC como “possivelmente cancerigenicas para os humanos (classe 2B)”. No entanto, a evidência epidemiológica não é suficientemente sustentada, especula-se que as microcistinas sejam agentes genotóxicos e que, portanto, possam ser também iniciadores tumorais. Mas ainda não sabemos se as microcistinas são cancerígenas. Ultimamente tem sido dada maior atenção à cilindrospermopsina, descrita como genotóxica e cuja ocorrência em países não tropicais tem vindo a ser descrita, embora muito se desconheça sobre a sua toxicocinética e os seus efeitos no Homem, em particular os efeitos a longo termo. Outro exemplo é o do BMAA, conhecido há várias décadas e “re-descoberto” recentemente na Europa e na Península Ibérica, que ilustra bem o paradigma da incerteza toxicológica. Assumimos, portanto, que as cianotoxinas são prejudiciais à saúde humana, mas ainda não sabemos como avaliar e gerir os seus riscos. Por outro lado, a ligação das cianobactérias à saúde humana não se resume à toxicidade e efeitos nocivos. Nos últimos anos temos vindo a assistir a um crescente interesse na actividade antibiótica, antiviral, anti-inflamatória e enzimática de bioprodutos cianobacterianos com potencial aplicação médica e biotecnológica, nomeadamente, na terapia de patologias tão diversas como o cancro, a dor crónica, a acalásia e a doença de Alzheimer, entre outras. Podemos, ainda, encontrar referências à aplicação de metabolitos cianobacterianos no combate a agentes infeciosos ou sobre o envolvimento das cianobactérias no fenómeno de disseminação de resistência a antibióticos no ambiente. Estas são novas áreas de intersecção cianobactérias-saúde, nas quais Portugal e Espanha começam já a dar o seu importante contributo.
- 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.
- Deciphering a potential toxic synergy between persistent organic pollutantsPublication . Puskar, Ljiljana; Jordão, LuísaPlastic, massively used in everyday life, inevitably accumulates in the environment, becoming a persistent pollutant due to its reduced and/ or extremely slow recyclability1. Plastic particles in the micro and nano range, known as micro (MP) and nanoplastics (NP), respectively represent a huge ecotoxicological challenge. Due to their high surface areas, they might ad/absorb other persistent pollutants with similar chemical properties, such as polycyclic aromatic hydrocarbons (PAHs), with unpredictable effects on persistence and distribution in the environment2. MPs/NPs, with or without pollutants ad/absorbed, could enter the trophic chain at the level of invertebrates, inflicting toxicological effects through all levels of the ecosystem3. Polystyrene MPs and PAHs previously found in water samples (e.g. fluoranthene, phenanthrene)4 and known to be adsorbed by MPs (Pyrene, benzo(a) pyrene)5 will be used in the study. PAH’s mixtures will be used to mimic environmental samples and single compounds will be used in order to understand the individual contribution of each compound for the observed toxic effect. This experimental approach will also allow to evaluate a potential synergy between compounds with effect on toxicity. Since PAHs and MPs have similar chemical properties, they are known to adsorb to each other. We will use PAHs adsorbed to MPs to test higher concentrations of PAHs (not soluble in aqueous solutions such as cell culture medium) and to document the intracellular distribution of adsorbed versus free PAHs. Concerning MPs only one material will be used at this stage. Polystyrene was selected because it is commercially available in spheres suitable for internalization by HepG2 cells and was previously isolated from environmental samples 4. This study could contribute to identify differences in toxicity and contribute to the elucidation of the underlying toxicity mechanisms using molecular biology protocols, light/electron microscopy and FTIR micro/spectrometry.
- 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 a Changing Earth on Microbial Dynamics and Human Health Risks in the Water/Sand ContinuumPublication . Weiskerger, Chelsea; Brandão, João; Robinson, Clare; Staley, Chris M.; Kleinheinz, Greg; Nshimyimana, Jean Pierre; Kinzelman, Julie; Nevers, Meredith B; Sadowsky, Michael Jay; Phanikumar, Mantha S; Whitman, Richard; Edge, Tom Andrew; Piggot, Alan; Boehm, Alexandria; Aslan, Asli; Badgley, Brian; Heaney, Christopher; Symonds, Erin; Solo-Gabriele, Helena; Fleisher, Jay; Harwood, Jody; Yamahara, Kevan; Vogel, Laura; Jordão, Luisa; Avolio, Lindsay; Merilainen, Paivi; Pitkanen, Tarja; Warish, Ahmed; Staley, Zachery; Klaus, JamesHumans may be exposed to microbial pathogens at recreational beaches via environmental sources such as water and sand. Although infectious disease risk from exposure to waterborne pathogens, and the fecal indicator bacteria (FIB) used to monitor water quality are active areas of research, sand is a relatively unexplored reservoir of pathogens and FIB. Sand and water at beaches experience continuous exchange of microorganisms, and these habitats provide unique advantages and challenges to pathogen introduction, growth, and persistence. Models of FIB and pathogen fate and transport in beach habitats can aid prediction of the risk of infectious disease from recreational water use, but filling knowledge gaps is necessary for accurate modeling. Climate change predictions estimate an increase in global temperatures of 2.5 – 10° F, sea level rise, and intensification of storms and precipitation in some regions. Other global change factors like population growth and urbanization may exacerbate predicted impacts. These changes can alter microbial population dynamics in beach habitats, and may consequently affect the assumptions and relationships used in numerical models. We discuss literature on microbial population and transport dynamics in sand/beach habitats, with an emphasis on how climate change and other anthropogenic influences (e.g., land use, urbanization) should be considered when using and developing models.
- Exploring the interplay between microplastics, polyciclic aromatic hidrocarbons and biofilms in freshwaterPublication . José, Sílvia; Jordão, LuísaWater pollution resulting from domestic and industrial use of non-biodegradable materials such as plastics is a major concern. Most plastics are only fragmentable giving rise to small fragments that will persist within the environment and interact with all elements that make up the ecosystem. To date, research has focused largely on the pollution of the oceans with plastics, but the impact of this of pollution on freshwater bodies and soils is as large or as relevant.In the present work, we investigated the ability of five different plastics to adsorb PAHs and functioning as a surface for biofilm assembly with the ultimate goal of evaluating the impact on human health. The ability of polyethylene terephthalate (PET), high desinty polyethylene (HDPE), low density polyethylene (LDPE), polypropylene (PP) and polystyrene (PS) to adsorb benzo-(a)-pyrene (BaP), a group 1 carcinogenic compound, and pyrene (Pyr) a less toxic compound that we detected in different water samples in a previous study was evaluated by HPLC-MS. All the plastics were able to adsorb BaP and Pyr from the water. The extent of this retention was ruled out by the period of contact, plastic and PAH characteristics. In paralel, the ability of bacterial species isolated from freshwater responsible for infections in humans (E.coli, K. pneumoniae and Aeromonas sobria) to assemble biofilms on plastics was evaluated by scanning electron microscopy (SEM) and viable colony forming units (CFU) were evaluated by platting on selective media after 1 and 3 months. More than 300 per mL were recovered after 1 and 3 months. Bacteria assembled biofilms on different plastics showing special tropism to LDPE as shown in figure 1. The present results show that plastics can adsorb PAH present in water and function as surfaces for biofilm assembly by different human pathogens. The impact of PAH adsorbed in plastics on bacterial biofilm assembly is a work on progress, as well as, the toxicity effects on human cells.
- Exposure to fungal particles in sand. Why only water quality is assessed for safety in recreational settings?Publication . Brandão, JoãoFungi were never introduced to water quality regulations, the incidence of fungal infections worldwide is growing, and changes in antimicrobial resistance patterns are taking place. The presence of fungi in different types of water has been thoroughly investigated during the past 30 years only in Europe, and more than 400 different species were reported from ground-, surface-, and tap-water. The most frequently reported fungi, however, were not waterborne, but are frequently related to soil, air, and food.