DSA - Artigos em revistas internacionais
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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.
- 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.
- 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.
- 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.
- 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.
- The Estela Sousa e Silva Algal Culture Collection: a resource of biological and toxicological interestPublication . Paulino, Sérgio; Sam-Bento, Filomena; Churro, Catarina; Alverca, Elsa; Dias, Elsa; Valério, Elisabete; Pereira, PauloThe Estela Sousa e Silva Algal Culture Collection (ESSACC) is the legacy of nearly 40 years of the National Institute of Health Dr. Ricardo Jorge (INSA) scientific and public activity in marine and freshwater phytoplankton biology and toxicology. The living isolates maintained in the ESSACC are mostly represented by marine dinoflagellates and freshwater cyanobacteria strains isolated from bloom occurrences in Portugal. More than 170 isolates comprising the most frequently found species have been obtained from environmental samples and are successfully cultured at INSA facilities. Moreover, new isolates are continuously being added to the collection, following new detection of natural blooms. Although not intended to represent the entire range of taxonomical different species occurring in Portuguese environments, the ESSACC includes a wide array of geographical, morphological, physiological, and ecological relevant isolates. So far, laboratory up-scaled culturing has been put in place for several purposes, including the production of secondary metabolites, purification of toxins, detection of toxin producing strains by molecular methods, screening for cytotoxic and genotoxic effects of purified compounds, testing for antialgal activity of organic compounds, and evaluating the combining effects of environmental factors on biomass and toxin production. We are disseminating information about this collection since it is an important wide source of readily available and easy to manipulate biological material for research purposes within the scientific community.
- Variation in the measurement of DNA damage by comet assay measured by the ECVAG† inter-laboratory validation trialPublication . Forchhammer, Lykke; Johansson, Clara; Loft, Steffen; Möller, Lennart; Godschalk, Roger, W. L.; Langie, Sabine A. S.; Jones, George D. D.; Kwok, Rachel W. L.; Collins, Andrew R.; Azqueta, Amaya; Phillips, David H.; Sozeri, Osman; Stepnik, Maciej; Palus, Jadwiga; Vogel, Ulla; Wallin, Hakan; Routledge, Michael N.; Handforth, Catherine; Allione, Alessandra; Matullo, Giuseppe; Teixeira, João Paulo; Costa, Solange; Riso, Patrizia; Porrini, Marisa; Møller, PeterThe comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0–10 Gy) and used to construct a calibration curve to calculate the number of lesions per 106 base pair. All laboratories detected dose–response relationships in the coded samples irradiated with ionizing radiation (1.5–7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.
- Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samplesPublication . Valério, Elisabete; Chambel, Lélia; Paulino, Sérgio; Faria, Natália; Pereira, Paulo; Tenreiro, RogérioThe aim of this study was to develop a PCR-based method of gene-directed multiplex PCR to rapidly identify microcystins producing cyanobacteria, regardless of their taxa, that could be applied in routine freshwater monitoring. Instead of using the amplification of only one or two mcy gene fragments, a multiplex PCR that simultaneously amplifies mcyA-cd, mcyAB, and mcyB fragments of the microcystin gene cluster was validated with DNA from 124 cyanobacterial isolates and applied in 37 environmental samples. The toxicological status of the isolates was assessed by high-performance liquid chromatography also used as the "gold standard" for the evaluation of multiplex mcy genes-based PCR, where a sensitivity of 92.3% and a specificity of 100% have been obtained. For the environmental samples, a rapid protocol for their direct use in the PCR reaction has been developed and, by using ELISA results as "gold standard" for the presence of microcystins in these samples, a sensitivity of 80% and a specificity of 100% were achieved, showing that this multiplex PCR test is a rapid, reliable, and economical way of assessing the microcystin-producing potential of cyanobacteria in freshwaters, regardless of their taxa or microcystins variant produced.
- Salmonella: the forgotten pathogen: health hazards of compliance with European bathing water legislation.Publication . Mansilha, Catarina; Coelho, Carla; Reinas, Alcina; Moutinho, Ana; Ferreira, Sónia; Pizarro, Cristina; Tavares, AntónioThe increasing demands on recreational waters have made microbial contamination a matter of public and scientific concern. This study aimed to search for Salmonella spp. in waters classified according EU Directive 2006/7/EC, in order to assess associations between its prevalence and the concentration of the non-pathogenic new faecal indicators: Escherichia coli and intestinal enterococci. Although a statistically significant association was observed Salmonella was detected on beaches classified as "Good" (9.3%) and "Excellent" (14.4%) which compromises the idea that faecal indicators can be predictors of pathogens. Attending the high prevalence of Salmonella found (23.1%) it seemed important to improve the efficiency of the conventional analytical method (ISO 6340:1995), comparing its draft with SML-VIDAS Salmonella and two new chromogenic media: AES Salmonella Agar Plate (ASAP) and Simple Method Salmonella (SMS). ASAP showed the higher efficiency and can be recommended for a faster detection and presumptive identification of salmonellae in bathing waters.