Departamento de Saúde Ambiental
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- Micronucleus analysis in a portuguese population exposed to pesticides:preliminary surveyPublication . Costa, Carla; Silva, Susana; Coelho, Patrícia; Roma-Torres, Joana; Teixeira, João PauloThe general population is exposed in their everyday life to different chemicals namely to pesticides. Many of these compounds are capable of inducing mutations in DNA and lead to several diseases including cancer. With this study we intended to evaluate DNA damage inflicted by pesticide exposure in a population occupationally exposed to those chemicals by means of the micronucleus (MN) test. The obtained results showed a significant increase in MN frequency in occupationally exposed individuals (po0.001) compared with the control group. Higher frequencies of MN were associated with a specific workplace (greenhouses) and the lack of protective measures (gloves) during labour activities. These results reinforce that conditions in workplace should be improved to minimize exposure to these chemicals. This study also emphasizes the need to reinforce the good practices campaigns in order to enlighten those who work with pesticides on the potential hazard of occupational exposure and the importance of using protective measures.
- Prevalência da Legionella pneumophila em águas de diferentes proveniências das regiões norte e centro de Portugal no período de 2000 a 2006Publication . Mansilha, Catarina; Coelho, Carla; Reinas, Alcina; Heitor, AnaLegionella é uma bactéria amplamente distribuída capaz de sobreviver em condições ambientais hostis por longos períodos, o que contribui para a sua fácil disseminação, daí resultar uma elevada probabilidade de exposição do Homem a este agente. O seu habitat natural consiste em reservatórios de água, nomeadamente: rios, lagos, nascentes, fontes hidrotermais e solos húmidos, onde sobrevive como parasita intracelular de alguns protozoários que funcionam como seu reservatório natural. Também pode ser encontrada em sistemas artificiais de circulação de água, como os circuitos de água quente sanitária, de água fria para consumo humano, de rega por aspersão, filtros de aparelhos de ar condicionado e suas condutas, sistemas de refrigeração, condensadores de evaporação, nebulizadores e humidificadores. Nestes últimos a Legionella encontra-se frequentemente associada a biofilmes que providenciam os nutrientes e o ambiente necessário à sua manutenção e proliferação. Esta capacidade peculiar de adaptação a novas condições ambientais é responsável pela frequente contamicontaminação de sistemas de distribuição de água artificiais e pela dificuldade de erradicação da Legionella de estruturas contaminadas. A Legionella pneumophila está associada a duas doenças: a Doença dos Legionários e a febre de Pontiac, cuja incidência depende do grau de contaminação dos reservatórios de água, da susceptibilidade da pessoa exposta e da intensidade da exposição. A infecção transmite-se por via respiratória a partir de bioaerossóis de água contaminada com este agente. Pela importância que representa em Saúde Pública, a Doença dos Legionários, e porque a sua monitorização apenas pelo sistema de notificação de doenças transmissíveis de declaração obrigatória (DDO) se tem mostrado insuficiente, considera-se fundamental a pesquisa e quantificação da Legionella nas amostras ambientais para uma melhor vigilância e prevenção desta patologia.
- Cyanobacterial blooms in natural waters in southern Portugal: a water management perspectivePublication . Galvão, Helena M.; Reis, Margarida P.; Valério, Elisabete; Domingues, Rita B.; Costa, Cristina; Lourenço, Dulce; Condinho, Sílvia; Miguel, Rute; Barbosa, Ana; Gago, Conceição; Faria, Natália; Paulino, Sérgio; Pereira, PauloThis synthesis of 3 studies from 2 regions of southern Portugal (Alentejo and Algarve) was part of a workshop focusing on cyanobacteria held at the SAME 10. The first study monitored impacts of the large Alqueva dam on the Guadiana estuary since 1996, revealing changes in sediment load, nutrient regime and phytoplankton succession. Prior to dam construction, dense cyanobacterial blooms occurred in the upper estuary during summer and fall. After dam construction, chlorophyll concentration, phytoplankton diversity and abundances of cyanobacteria decreased, contrary to predictions. Mycrocystins remained at low levels in the seston and undetectable in water samples, except during summer 2003 when the particulate fraction contained 1 µg l–1, while chlorophyll concentrations and abundances of potentially toxic cyanobacteria remained low. Algarve reservoirs studied since 2001 revealed differences in phytoplankton dynamics. In the western mesotrophic reservoirs (Bravura and Funcho), 40 to 50% of surface samples contained cyanobacterial concentrations of ≥2000 cells ml–1, while over 80% of samples from the eastern oligotrophic reservoirs (Odeleite and Beliche) exceeded this value. Spring blooms were dominated by Oscillatoriales in Odeleite and Beliche and by Chroococcales in Bravura and Funcho. Bloom composition seemed to depend on water temperature and management strategies, while toxin concentrations reflected the increased biomass of toxic species. Finally, phytoplankton communities and microcystin production in 5 Alentejo freshwater reservoirs were studied from May to December 2005 and April to July 2006. Cyanobacterial blooms occurred, with varying intensities, not only during summer but also occasionally in winter. Microcystins were detected in 23% of the samples (n = 51), but without correlation with cyanobacterial biomass. Although Microcystis aeruginosa seemed to be the major producer of microcystins, other potentially toxic species were found. In summary, the varying pattern of cyanobacterial bloom occurrence and toxicity requires a systematic approach to monitoring programs for adequate risk assessment.
- Toxic cyanobacteria blooms in Portuguese freshwaters – a summarized overviewPublication . Paulino, Sérgio; Faria, Natália; Valério, Elisabete; Pereira, Paulo; Tavares, AntónioToxic cyanobacteria are common in Portuguese freshwaters and are a cause of concern, given that exposure to subacute levels of cyanobacterial toxins through drinking and recreational water might have deleterious effects on human health. Since 1996 several laboratories have been involved in the screening of cyanotoxins in different freshwater bodies, some on regularly basis and others more sporadically. Here we summarized the main data obtained by our laboratory during the last years, in order to give a simplified overview of the quality and diversity of our freshwater resources in terms of cyanobacteria occurrence.
- Detection of Planktothrix rubescens (Cyanobacteria) associated with microcystin production in a freshwater reservoirPublication . Paulino, Sérgio; Valério, Elisabete; Faria, Natália; Fastner, Jutta; Welker, Martin; Tenreiro, Rogério; Pereira, PauloSo far, the presence of microcystins in Portuguese freshwater resources has always been attributed to the bloom-forming cyanobacteria Microcystis aeruginosa. In 2005, however, microcystins were detected at the Beliche reservoir (Algarve, South Portugal), following the development of a bloom dominated by Planktothrix rubescens. The identity of the causative organism was confirmed by combining both morphological and 16S rRNA gene sequence analysis. Its ability to produce microcystins was confirmed by HPLC and MALDI-TOF MS. Unlike M. aeruginosa that usually accumulates near the water surface, P. rubescens found at the Beliche reservoir accumulated only at deep water levels. Being invisible from the surface, the occurrence of toxic P. rubescens in freshwater resources requires special attention when designing site inspection and sampling procedures for the correct risk assessment and management of cyanobacterial blooms in the field.
- 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.
- Comparative study of the cytotoxic effects of microcystin-LR in mammalian cell lines: Vero, HepG2, Caco2 and MDCKPublication . Menezes, Carina; Amorim, Ana; Alverca, ElsaMicrocystin-LR (MCLR) is a natural occurring freshwater cyanotoxin, recognized as one of the most toxic microcystin variants. It is thought to be responsible for cases of livestock and human intoxication due to consumption of toxic cyanobacteria-contaminated water. Although considered a hepatotoxin, MCLR also targets other organs such as the kidneys and intestines. In spite the cellular mechanisms associated with the toxicity of MCLR are still unclear, a previous work in a monkey kidney cell line suggested that the endoplasmic reticulum was an early target of MCLR toxicity and that autophagy was triggered as a cell defense mechanism at subcytotoxic concentrations of MCLR. In the present work, cytotoxic, morphological and ultrastructural effects of MCLR were compared in HepG2 (human liver), Vero (monkey kidney), MDCK (dog kidney) and Caco2 (human intestine) cell lines. MCLR induced a concentration-dependent decrease in cell viability by the NR assay in all cell lines, with HepG2 and Vero showing the lowest cytotoxic thresholds of 25 and 50 μM MCLR, respectively. In these cells, MCLR exposure induced lysosomal damages previously to mitochondrial disruption, reinforcing the role of lysosomes in MCLR-induced toxicity. Immunolabelling and ultrastructural visualization of autophagosomes, showed that autophagy was a response transversal to both cell lines, triggered at subcytotoxic MCLR concentrations, confirming its importance as a defense mechanism to early damages inflicted by the toxin. The analysis of GRP94, an ER stress protein, did not undoubtedly demonstrate that MCLR targets the ER. However, together with the ultrastructural data, suggested that in both HepG2 and Vero cells, the ER has a role in autophagy induction. Additionally, in HepG2 cells, GRP94 down-regulation with increasing MCLR concentrations supported the ER role in the triggering of apoptosis. At high toxin concentrations, ultrastructural alterations consistent with apoptosis were observed for all four cell lines, proving that this is a general MCLR-induced mechanism.
- Morphological and ultrastructural effects of microcystin-LR from Microcystis aeruginosa extract on a kidney cell linePublication . Alverca, Elsa; Andrade, Mariana; Dias, Elsa; Sam Bento, Filomena; Batoréu, Maria Camila; Jordan, Peter; Silva, Maria João; Pereira, PauloThe aim of this study was to examine the toxic effects of a microcystin-LR (MCLR)-containing cyanobacteria extract on the subcellular organization of a kidney cell line (Vero-E6). Cells were exposed to different MCLR concentrations (1.3-150 microM) for 24, 48 and 72h and two cytotoxicity assays were performed. This information was combined with the analysis of lysosomal, mitochondrial and cytoskeleton integrity and with an ultrastructural study. Biochemical and microscopic data revealed a good agreement and demonstrated that cellular response to MCLR is dependent on the dose/exposure time. Cell viability decayed markedly after 24h of exposure to toxin concentrations greater than 30 microM. Furthermore, it was demonstrated that lysosome destabilization precedes mitochondria dysfunction. The ultrastructural analysis showed that mild toxin incubation conditions induce endoplasmic reticulum (ER) vacuolization and assembly of large autophagic vacuoles, suggesting that autophagy is an early cellular response to the toxin. After exposure to higher MCLR doses, the number of apoptotic cells increased, as identified by microscopic observations and confirmed with TUNEL assay. Additionally, drastic exposure conditions induced the increase of necrotic cells. These results suggest that the ER is the primary microcystin target in Vero cells and that autophagy, apoptosis and necrosis are induced in a dose- and time-dependent manner.
- 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.
- Bathing waters: new directive, new standards, new quality approachPublication . Mansilha, Catarina; Coelho, Carla; Heitor, Ana; Amado, João; Martins, Joaquim; Gameiro, PaulaBathing water quality is an important public health issue, mainly because of fecal contamination. In 2006, the European Commission (EC) adopted a new directive with respect to recreational bathing waters that calls for stricter standards and reduces the number of laboratory tests done in routine beach monitoring from nineteen to two bacterial indicators, namely, Escherichia coli and intestinal enterococci, replacing policies of the EC Bathing Water Directive that have existed since 1976. Our practice in Portugal is in line with this international development, and this study demonstrates the equivalency of the new bacteriological parameters with the old. The water quality of 25 coastal beaches was surveyed using both new and old microbiological indicators of fecal contamination. Statistical analysis demonstrated equivalency of the results obtained for fecal coliforms with Escherichia coli and for fecal streptococci with intestinal enterococci.