Browsing by Author "Azevedo, Joana"
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- Absence of negative allelopathic effects of cylindrospermopsin and microcystin-LR on selected marine and freshwater phytoplankton speciesPublication . Pinheiro, Carlos; Azevedo, Joana; Campos, Alexandre; Loureiro, Susana; Vasconcelos, VítorCyanobacterial toxins have been regarded by some researchers as allelopathic substances that could modulate the growth of competitors. Nevertheless, often the concentrations of toxins used are too high to be considered ecologically relevant. In this work we tested the hypothesis that microcystin-LR (MC-LR) and cylindrospermopsin (CYN) at ecologically relevant concentrations have no allelopathic effects on some species of phytoplankton. Extracts containing the toxins as well as pure MC-LR and CYN toxins were used to assess their effects on the growth rates of Nannochloropsis sp., Chlamydomonas reinhardtii, and Chlorella vulgaris. Cyanobacterial crude extracts induced more pronounced effects on growth rates than pure toxins. Microcystis aeruginosa and Aphanizomenon ovalisporum crude extracts containing MC-LR and CYN at 0.025–2.5 mg l−1 stimulated growth rates of microalgae, whereas A. ovalisporum crude extracts containing 2.5 mg l−1 of CYN strongly inhibited growth rates of microalgae after 4 and 7 days of exposure. MC-LR and CYN at environmentally occurring concentrations were unable to affect negatively the growth of microalgae, and therefore these molecules may play roles other than allelopathy in natural ecosystems.
- Analysis of the Use of Cylindrospermopsin and/or Microcystin-Contaminated Water in the Growth, Mineral Content, and Contamination of Spinacia oleracea and Lactuca sativaPublication . Llana-Ruiz-Cabello, Maria; Jos, Angeles; Cameán, Ana; Oliveira, Flavio; Barreiro, Aldo; Machado, Joana; Azevedo, Joana; Pinto, Edgar; Almeida, Agostinho; Campos, Alexandre; Vasconcelos, Vitor; Freitas, MarisaCyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 μg/L and 50 μg/L, or CYN/MC mixture at 5 + 5 μg/L and 25 + 25 μg/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50µg/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 µg/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxins.
- Anti-pneumoscystis carinni activitiy of primaquine imidazolidin-4-onesPublication . Vale, Nuno; Ferraz, Ricardo; Azevedo, Joana; Araújo, Maria João; Moreira, Rui; Collins, Margaret S.; Cushion, Melanie T.; Gomes, PaulaPneumocystis pneumonia (PCP) is one of the most frequent causes of mortality among HIV-infected patients. Primaquine (PQ) is an antimalarial 8-aminoquinoline effective against PCP when given in combination with clindamycin. This has drawn the attention of Medicinal Chemists towards the anti-PCP activity of 8-aminoquinolines, not only confined to those exhibiting antimalarial activity [1]. It is thought that anti-PCP 8-aminoquinolines exert their anti-PCP activity by acting on the electronic transport and redox system of the P. carinii pathogen [1]. Recently, our research group has been developing imidazolidin-4-one derivatives of PQ (Scheme 1), targeting novel compounds with improved therapeutic action, namely, higher resistance to metabolic inactivation, lower toxicity and equal or higher antimalarial activity than that of the parent drug [2,3]. These imidazolidin-4-ones were seen to block the transmission of rodent malaria, caused by Plasmodium berghei on BalbC mice, to the mosquito vector Anopheles stephensi [3]. The anti-PCP activity of our PQ derivatives is now under study and preliminary in vitro assays [4] show that some of the compounds exhibit slight to moderate activity after a 72 h incubation period against P. carinii. In one case, the IC50 was comparable to that of parent PQ. Both these studies and forthcoming results from ongoing biological assays will be presented and discussed.
- Application of real-time PCR in the assessment of the toxic cyanobacterium cylindrospermopsis raciborskii abundance and toxicological potentialPublication . Moreira, Cristiana; Martins, António; Azevedo, Joana; Freitas, Marisa; Regueiras, Ana; Vale, Micaela; Antunes, Agostinho; Vasconcelos, VítorCyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.
- Assessment of Constructed Wetlands’ Potential for the Removal of Cyanobacteria and Microcystins (MC-LR)Publication . Bavithra, Guna; Azevedo, Joana; Oliveira, Flávio; Morais, João; Pinto, Edgar; Ferreira, Isabel M. P. L.V. O.; Vasconcelos, Vítor; Campos, Alexandre; Almeida, C. Marisa R.Microcystis blooms and the subsequent release of hepatotoxic microcystins (MCs) pose a serious threat to the safety of water for human and livestock consumption, agriculture irrigation, and aquaculture worldwide. Microcystin-LR (MC-LR), the most toxic variant of MCs, has been widely detected in a variety of environments such as water, sediments, plants, and many aquatic organisms.
- Assessment of cyanobacterial biomass as sustainable agricultural fertilizer: soil experiment with plants in Pot †Publication . Massa, Anabella; Azevedo, Joana; Azevedo, Rui; Pinto, Edgar; Costa, Anabela; Vasconcelos, Vitor; Campos, Alexandre; Freitas, MarisaProviding food to the growing human population in a sustainable way is one of the greatest challenges of modern society. In this context, cyanobacterial biomass (CB) can function as a source of macronutrients to increase soil productivity. These organisms can be collected from the environment in considerable amounts, since they tend to grow in large blooms. However, some of these cyanobacterial strains produce toxins that need to be carefully monitored to avoid food accumulation. The objective of this work was to evaluate the possible use of toxic and non-toxic strains of CB as fertilizer supplement in the growth of economically relevant vegetables. One-month old Raphanus sativus (radish) and Spinacia oleracea (spinach) plants were grown in pots in indoor controlled conditions. Six experimental conditions were set: (1) a control with no nutrient addition, (2) a recommended dose of a NK commercial fertilizer (CF), 0.6g of lyophilized CB of (3) a non-toxic strain of Cylindrospermopsis raciborskii, (4) a toxin-producing strain of C. raciborskii, (5) Microcystis aeruginosa, and (6) Anabaena sp. Several variables were estimated: in CB, this included the NPK dose addition, and in plants, the height, dry weight (dw) of the shoot and root, and the mineral content of plant edible parts. The mineral content in CB was estimated and compared with the recommended dose of CF, according to the information given by the fabricant label. We found no significative differences in N composition; nevertheless, there was a significative higher content in P and significative lower content in K in the CB. In the plants, we found no significative statistical differences between the treatments for the dw of radish root and spinach height. In spinach, the dw of the shoot in the M. aeruginosa treatment was significantly lower than the control, CF, and both the toxic and non-toxic C. raciborskii biomass. Additionally, in radish, the plant height and dw of the shoot M. aeruginosa treatment were significantly lower than in the toxic strain of C. raciborskii treatment. When analyzing mineral content in edible parts, we found that spinach treated with control and CF showed a higher content of Ca, Mo, N, P, and K, while in radish, the same two treatments plus the C. raciborskii toxic had higher Co and Fe content. M. aeruginosa amendment seems to impair shoot growth in both plant species. On the contrary, the toxic C. raciborskii CB seems to have a beneficial effect on growth and in mineral uptake on radish plants.
- Bioaccessibility and changes on cylindrospermopsin concentration in edible mussels over storage and processing time.Publication . Freitas, Marisa; Azevedo, Joana; Carvalho, António Paulo; Mendes, Vera; Manadas, Bruno; Campos, Alexandre; Vasconcelos, VítorThe cyanotoxin cylindrospermopsin has been recognized of increased concern due to the global expansion of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that aquatic organisms, especially bivalves, can accumulate high levels of cylindrospermopsin. Based on the potential for human health risks, a provisional tolerable daily intake of 0.03 µg/kg body-weight has been recommended. However, human exposure assessment has been based on the cylindrospermopsin concentration in raw food items. This study aimed to assess the changes on cylindrospermopsin concentration in edible mussels over storage and processing time as well as cylindrospermopsin bioaccessibility. Mussels, (Mytilus galloprovincialis) fed cylindrospermopsin-producing C. raciborskii, were subjected to the treatments and then analyzed by LC-MS/MS. Mussels stored frozen allowed a significantly higher recovery of cylindrospermopsin (52.5%/48 h and 57.7%/one week). The cooking treatments did not produce significant differences in cylindrospermopsin concentration in mussel matrices (flesh), however, cylindrospermopsin was found in the cooking water, suggesting that heat processing can be used to reduce the availability of cylindrospermopsin in this food item. The in vitro digestion with salivary and gastrointestinal juices considerably decreased the cylindrospermopsin availability in uncooked and steamed mussels, highlighting the importance in integrating the bioaccessibility in the human health risk assessment.
- Bioaccessibility and changes on cylindrospermopsin concentration in edible mussels with storage and processing timePublication . Freitas, Marisa; Azevedo, Joana; Carvalho, António Paulo; Mendes, Vera M.; Manadas, Bruno; Campos, Alexandre; Vasconcelos, VitorThe alkaloid cylindrospermopsin has been recognized of increased concern due to the global expansion of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that bivalves can accumulate high levels of cylindrospermopsin. Based on the potential for human health risks, a provisional tolerable daily intake of 0.03 μg/kg-body weight has been recommended. However, the human exposure assessment has been based on the cylindrospermopsin concentration in raw food items. Thus, this study aimed to assess the changes on cylindrospermopsin concentration in edible mussels with storage and processing time as well as cylindrospermopsin bioaccessibility. Mussels, (Mytilus galloprovincialis) fed cylindrospermopsin-producing C. raciborskii, were subjected to the treatments and then analyzed by LC-MS/MS. Mussels stored frozen allowed a significantly higher recovery of cylindrospermopsin (52.5% in 48 h and 57.7% in one week). The cooking treatments did not produce significant differences in cylindrospermopsin concentration in the mussel matrices (flesh), however, cylindrospermopsin was found in the cooking water, suggesting that heat processing can be used to reduce the availability of cylindrospermopsin. The in vitro digestion considerably decreased the cylindrospermopsin availability in uncooked and steamed mussels, highlighting the importance in integrating the bioaccessibility of cylindrospermopsinin in the human health risk assessment.
- Cyanobacterial biomass used as biofertilizer in lettuce plants: effects on growth and cyanotoxin accumulation †Publication . Santos, Érica; Massa, Anabella; Azevedo, Joana; Martins, Diogo; Reimão, Mariana; Vasconcelos, Vitor; Campos, Alexandre; Freitas, MarisaThe use of cyanobacterial biomass as a biofertilizer is promising in terms of sustainable agriculture. Nevertheless, cyanobacteria can be considered a threat to human and environmental health due to the potential presence of cyanotoxins, since some studies report that the use of contaminated water for agricultural irrigation can impair plant growth and lead to contamination of food products. Interestingly, at environmentally relevant concentrations, cylindrospermopsin (CYN) seems to cause no deleterious effects in plants, and it might even promote their yield. However, studies assessing CYN accumulation in the edible tissues at environmental concentrations are lacking. The objective of this work was to evaluate the effects of cyanobacterial biomass CYN producing or non-producing on lettuce plant growth, and that of CYN accumulation in edible tissues. This study consisted of growing lettuce plants, under controlled conditions, for 25 days in soil (1) with no extra nutrient addition (control) and supplementation with (2) cyanobacterial biomass that did not produce CYN, (3) cyanobacterial biomass that produced CYN (~10 µg of dissolved CYN), and (4) cyanobacterial biomass that produced CYN, treated by boiling for 5 min (~25 µg of dissolved CYN). At the end of the exposure, lettuce growth was assessed, as well as CYN accumulation in tissues and soil. The results showed that leaf growth was significantly increased (p < 0.05) in lettuce plants supplemented with cyanobacterial biomass, especially at condition (3), which was five-fold higher compared with the control group. Regarding CYN accumulation, for conditions (3) and (4), the toxin was detected in the tissues of plants, as well as in soil at the following decreasing order of concentrations: soil > roots > leaves. Interestingly, the concentration determined in lettuce leaves in condition (4) was three-fold lower when compared with the condition (3). Nevertheless, for both conditions, although CYN has been detected in lettuce leaves, the concentration in the edible part did not exceed the proposed provisional tolerable daily intake (TDI) of 0.03 µg/kg/BW. In conclusion, these results suggest that the use of cyanobacterial biomass as lettuce biofertilizer, even containing CYN at environmentally relevant concentrations, can positively influence plant growth and development without compromising the safety of edible tissues.
- Early physiological and biochemical responses of rice seedlings to low concentration of microcystin-LRPublication . Azevedo, Catarina; Azevedo, Joana; Osório, Hugo; Vasconcelos, Vítor; Campos, AlexandreMicrocystin-leucine and arginine (microcystin- LR) is a cyanotoxin produced by cyanobacteria like Microcystis aeruginosa, and it’s considered a threat to water quality, agriculture, and human health. Rice (Oryzasativa) is a plant of great importance in human food consumption and economy, with extensive use around the world. It is therefore important to assess the possible effects of using water contaminated with microcystin-LR to irrigate rice crops, in order to ensure a safe, high quality product to consumers. In this study, 12 and 20-day-old plants were exposed during 2 or 7 days to a M. aeruginosa extract containing environmentally relevant microcystin-LR concentrations, 0.26–78 lg/L. Fresh and dry weight of roots and leaves, chlorophyll fluorescence, glutathione S-transferase and glutathione peroxidase activities, and protein identification by mass spectrometry through two-dimensional gel electrophoresis from root and leaf tissues, were evaluated in order to gauge the plant’s physiological condition and biochemical response after toxin exposure. Results obtained from plant biomass, chlorophyll fluorescence, and enzyme activity assays showed no significant differences between control and treatment groups. How- ever, proteomics data indicates that plants respond to M. aeruginosa extract containing environmentally relevant microcystin-LR concentrations by changing their metabolism, responding differently to different toxin concentrations. Biological processes most affected were related to protein folding and stress response, protein biosynthesis, cell signalling and gene expression regulation, and energy and carbohydrate metabolism which may denote a toxic effect induced by M. aeruginosa extract and microcystin- LR. Theimplications of the metabolic alterations in plant physiology and growth require further elucidation.
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