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- 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.
- Applications of Proteomics in AquaculturePublication . Rodrigues, Pedro M.; Schrama, Denise; Campos, Alexandre; Osório, Hugo; Freitas, MarisaAquaculture is one of the fastest growing world industries due to the increased demand of fishery products for human consumption and capture restrictions as a result of aquatic ecosystems exploitation. Aquaculture is therefore an extremely competitive business with major challenges to keep a high quality farmed fish through a sustainable production system. These challenges imposed quite important changes in this more traditional market, namely at the level of integrating scientific knowledge and research. Proteomics presents itself as a powerful tool not only for a better understanding of the marine organisms biology but also to provide solutions to deal with changes and the increasing demand in the system’s production line to ensure the required supply. In this book chapter we will give an overview of aquaculture nowadays, its challenges and describe relevant proteomics studies in several areas of this industry. A brief description of the proteomics technical approaches applied to aquaculture will also be addressed.
- Presence of microcystis sp. and microcystins in alqueva reservoirs assessed by chemical and molecular methodsPublication . Azevedo, Tomé; Azevedo, Joana; Martins, Joana; Freitas, Marisa; Vasconcelos, Vitor; Campos, AlexandreThe Alqueva reservoir, located in the Alentejo region, in the south of Portugal, is considered the largest artificial lake in Europe. It has been in operation since 2002, and it is used to produce energy and supply water for agriculture and to the populations in this region of Portugal. The water distribution system, starting from the main reservoir, includes 19 reservoirs of smaller capacity and a network of waterways totaling 382 km in length. Furthermore, the occurrence of cyanobacteria in water reservoirs has been recognized as an environmental concern due to the potential presence of their related toxins that can cause severe health effects. This work aimed to monitor the presence of cyanobacteria and the commonly associated cyanotoxin microcystin (MC) in three reservoirs belonging to the Alqueva water system, namely São Pedro, Magra, and Pisão, located in the district of Beja. These reservoirs were selected considering the historical data of phytoplankton provided by EDIA, the entity in charge of the management of this infrastructure. The field work was carried out in July, August, and September, the months with the highest risk of outgrowth of cyanobacteria, in the year 2020. Two or three samples of water (5 L) were collected in different locations of the reservoirs, once per month, by boat. Samples were collected at different depths in the photic zone using a Van Dorn bottle and pooled. The water samples were then processed in the laboratory. Molecular biology techniques were used to detect the presence of cyanobacteria (16S rRNA) and MC-related genes. Moreover, chemical analysis techniques based on liquid chromatography and mass spectrometry (MS) were used to identify and quantify MCs. The results revealed the presence of MCs in the three reservoirs in the three months monitored. Concentrations of this toxin varied between 0.01 µg/L and 0.1 µg/L, with São Pedro being the reservoir displaying the highest concentrations of MCs in all of the months monitored. These results are consistent with the molecular study based on the analysis of Microcystis sp. 16 rRNA and MC biosynthetic genes (mcya–mcyG), suggesting the presence of putative toxic Microcystis sp. strains in the three reservoirs. Despite the low concentrations of MCs detected in these reservoirs, their recurrent presence in Alqueva waters serve as a reminder of the need to monitor cyanobacteria and cyanotoxins on a regular basis.
- 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.
- Effects of Chrysosporum (Aphanizomenon) ovalisporum extracts containing cylindrospermopsin on growth, photosynthetic capacity, and mineral content of carrots (Daucus carota)Publication . Guzmán-Guillén, Remedios; Campos, Alexandre; Machado, Joana; Freitas, Marisa; Azevedo, Joana; Pinto, Edgar; Almeida, Agostinho; Cameán, Ana M.; Vasconcelos, VitorNatural toxins produced by freshwater cyanobacteria, such as cylindrospermopsin, have been regarded as an emergent environmental threat. Despite the risks for food safety, the impact of these water contaminants in agriculture is not yet fully understood. Carrots (Daucus carota) are root vegetables, extensively consumed worldwide with great importance for human nourishment and economy. It is, therefore, important to evaluate the possible effects of using water contaminated with cyanotoxins on carrot cultivation. The aim of this work was to investigate cylindrospermopsin effects on D. carota grown in soil and irrigated for 30 days, with a Chrysosporum ovalisporum extract containing environmentally relevant concentrations of cylindrospermopsin (10 and 50 μg/L). The parameters evaluated were plant growth, photosynthetic capacity, and nutritional value (mineral content) in roots of carrots, as these are the edible parts of this plant crop. The results show that, exposure to cylindrospermopsin did not have a clear negative effect on growth or photosynthesis of D. carota, even leading to an increase of both parameters. However, alterations in mineral contents were detected after exposure to crude extracts of C. ovalisporum containing cylindrospermopsin. A general decline was observed for most minerals (Ca, Mg, Na, Fe, Mn, Zn, Mo, and P), although an increase was shown in the case of K and Cu, pointing to a possible interference of the cyanobacterial extract in mineral uptake. This study is the first to evaluate the effects of C. ovalisporum extracts on a root vegetable, however, more research is necessary to understand the effects of this toxin in environmentally relevant scenarios.
- Cylindrospermopsin and glyphosate accumulation in lettuce (Lactuca sativa) simultaneously exposed to both toxicants in hydroponic and soil systemsPublication . Sengupta, S.; Freitas, Marisa; Pinto, Edgar; Ferreira, I.; Oliveira, F.; Azevedo, J.; Prieto, A.; Diez-Quijada, L.; Jos, A.; Cameán, A.M.; Campos, A.; Vasconcelos, V.In nature, the simultaneous occurrence of multiple emergent contaminants such as cyanotoxins (e.g., cylindrospermopsin (CYN)) and herbicides (e.g., glyphosate (GLY)), is highly expectable and it can be anticipated, mainly in the aquatic and terrestrial environments. The use of contaminated water for irrigation can be hazardous to the agricultural sector and some studies have reported that, individually, these contaminants can be accumulated in the edible tissues exerting a negative influence on crop plants safety and ultimately in human health. Furthermore, recent studies have suggested that some cyanotoxins (e.g., microcystins) can change the membrane permeability of roots, resulting in changes in the accumulation rates of other contaminants in plants. Since edible plants are exposed to a wide variety of substances through irrigation water, there is increasing concern in the potential adverse effects of the interactions between those substances when present simultaneously, especially when this can have potential public health consequences. The aim of this study was to determine the accumulation of CYN in Lactuca sativa simultaneously exposed to GLY at environmentally relevant concentrations. Lettuce plants were exposed for 15 days to 50 μg/L or kg of CYN-containing crude extract (Chrysosporum ovalisporum culture - LEGE X001) and 750 μg/L or kg of GLY, in hydroponic and soil systems, respectively. The resins (HP20, SP700, and XAD18) were evaluated for MC-LR uptake kinetics, capacities, and extraction efficiencies and simple procedures were developed for determining MC-LR concentration in binding disc extracts by Adda-ELISA (U.S. EPA Method 546). The XAD18-DGT/Adda-ELISA method had a 7-d deployment time detection limit of ≈0.05 μg/L and capacity of > 250 μg/L of MC-LR in water samples which encompass U.S. EPA and WHO advisory concentrations for drinking and recreational waters. The XAD18DGT/Adda-ELISA method determined timeaveraged MC-LR concentrations in waters with wide ranging pH (4.9–8.3) and ionic strength (0.04–0.8 M) under well-stirred and quiescent conditions with 90–101% accuracy. In addition to high sensitivity and accuracy, the method is simple, inexpensive, and applicable for determining MC-LR and related MCs concentrations in waterbodies with wide ranging chemical characteristics and hydrodynamic conditions. 4.11.07 Cylindrospermopsin and Glyphosate Accumulation in Lettuce (Lactuca sativa) Simultaneously Exposed to Both Toxicants in Hydroponic and Soil Systems S. Sengupta, School of Bio Sciences and Technology Vellore Institute of Technology, Vellore - 632 014, Tamil Nadu, India; M.M. Freitas, School of Health, Polytechnic Institute of Porto; E. Pinto, I. Ferreira, LAQV/REQUIMTE, Departament of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge de Viterbo Ferreira 228, 4050313 Porto, Portugal; F. Oliveira, J. Azevedo, CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450–208 Porto; A. Prieto, L. Diez-Quijada, A. Jos, A.M. Cameán, Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n◦2, 41012 Seville, Spain / Toxicology; A. Campos, Interdisciplinary Centre of Marine and Environmental Sciences, CIIMAR; V. Vasconcelos, CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450–208 Porto In nature, the simultaneous occurrence of multiple emergent contaminants such as cyanotoxins (e.g., cylindrospermopsin (CYN)) and herbicides (e.g., glyphosate (GLY)), is highly expectable and it can be anticipated, mainly in the aquatic and terrestrial environments. The use of contaminated water for irrigation can be hazardous to the agricultural sector and some studies have reported that, individually, these contaminants can be accumulated in the edible tissues exerting a negative influence on crop plants safety and ultimately in human health. Furthermore, recent studies have suggested that some cyanotoxins (e.g., microcystins) can change the membrane permeability of roots, resulting in changes in the accumulation rates of other contaminants in plants. Since edible plants are exposed to a wide variety of substances through irrigation water, there is increasing concern in the potential adverse effects of the interactions between those substances when present simultaneously, especially when this can have potential public health consequences. The aim of this study was to determine the concentration of CYN and GLY in lettuce plants (roots and leaves) was determined by LC/MSMS. The results show that, at the described conditions, CYN was accumulated in roots (0.06-7.62 μg CYN/g Dw) and leaves (0.13-1.1 μg CYN/g Dw) of lettuce, especially when plants were exposed in hydroponic system. However, interestingly, when lettuce plants were exposed simultaneously to both toxicants the concentration of CYN assimilated by lettuce plants (roots and leaves) was respectively, 1.5fold and 1-3-2.2-fold lower than in the exposure to isolated CYN. Conversely, the plants exposed to the mixture in soil system, showed that the concentration of GLY incorporated by lettuce (roots and leaves) was higher than in the exposure to the isolated compound (0.04 - 0.21 µg GLY/g and < LOQ - 0.84 µg GLY/g, respectively). This finding highlights the potential for the enhancement of GLY accumulation in lettuce plants due to their cooccurrence with CYN, and it underlines the importance of further research regarding the mechanism involved.
- OMICs approaches in diarrhetic shellfish toxins researchPublication . Campos, Alexandre; Freitas, Marisa; Almeida, André M. de; Martins, José Carlos; Domínguez-Pérez, Dany; Osório, Hugo; Vasconcelos, Vitor; Costa, Pedro ReisDiarrhetic shellfish toxins (DSTs) are among the most prevalent marine toxins in Europe’s and in other temperate coastal regions. These toxins are produced by several dinoflagellate species; however, the contamination of the marine trophic chain is often attributed to species of the genus Dinophysis.
- Exposure of Lycopersicon Esculentum to Microcystin-LR: Effects in the Leaf Proteome and Toxin Translocation from Water to Leaves and FruitsPublication . Gutiérrez-Praena, Daniel; Campos, Alexandre; Azevedo, Joana; Neves, Joana; Freitas, Marisa; Guzmán-Guillén, Remédios; Cameán, Ana María; Renaut, Jenny; Vasconcelos, VítorNatural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 μg/L) or Microcystis aeruginosa crude extracts containing 100 μg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 μg/kg fw), green tomatoes (5.15–5.41 μg/kg fw), mature tomatoes (10.52–10.83 μg/kg fw), and leaves (12,298.18 μg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination.
- Effects of proteolytic digestion on the cyanotoxins microcystin-LR and cylindrospermopsin: the importance in integrating the bioaccessibility in human health risk assessmentPublication . Freitas, Marisa; Azevedo, J.; Carvalho, A.P.; Mendes, V.M.; Manadas, B.; Campos, A.; Vasconcelos, V.The occurrence and proliferation of toxic cyanobacterial blooms are an emergent environmental concern worldwide. Microcystin-LR (MC-LR), a potent hepatotoxin, is the most documented and studied cyanotoxin. The cytotoxin cylindrospermopsin (CYN) has been recognized of increased concern due to the invasive nature of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that edible aquatic organisms, especially bivalves, can accumulate high levels of these cyanotoxins. MC-LR and CYN are stable at a wide range of temperatures and pHs, thus the knowledge of the influence of human digestion on its concentration in food is required to achieve a more accurate health risk assessment. The aim of this study was to assess the MC-LR and CYN bioaccessibility in edible bivalves. Clams (C. fluminea) fed MC-LR-producing M. aeruginosa and mussels (M. galloprovincialis) fed CYN-producing C. raciborskii were subjected to an in vitro digestion model adapted from Maulvault et al. (2011) and Versantvoort et al. (2005). Bioaccessibility of MC-LR and CYN were then assessed by LC-MS/MS. The bioaccessibility of MC-LR after proteolytic digestion was reduced to 83%, potentially because of its degradation by pancreatic enzymes. The in vitro digestion with salivary and gastrointestinal juices considerably decreased the CYN availability in uncooked and steamed mussels. Our results suggest that risk assessment based on MC-LR and CYN concentration in raw products might not be representative of true human exposure, once bioaccessibility strongly reduces the potential toxicological risks. Thus, the incorporation of the bioaccessibility of these cyanotoxins in the human exposure estimation would be of particular relevance to the application of more forceful management measures.
- Management of food allergens in contract catering: evaluation of the knowledge of employees, verification of labeling compliance and validation of sanitation plansPublication . Freitas, Marisa; Seixas, Andrea; Rebelo, Andreia; Costa, Natália; Costa, CarmenFood allergies have been increasing in recent years, with gluten being one of the most prevalent allergens. Since there is no cure for food allergies, eviction is the best strategy for allergic individuals. Thus, labeling is particularly relevant, enabling susceptible consumers making safe choices without compromising their nutrition. Regulation (EU) No.1169/2011 imposes the allergen declaration of unpackaged food to all food operators. However, the management of allergens in contract catering is difficult due to the random nature of the developed activities, where cross-contamination is probable to occur.