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- Contributions of TOXICROP project for the assessment of the impacts of toxic cyanobacteria in agriculture †Publication . Campos, Alexandre; Freitas, Marisa; Oudra, Brahim; Vasconcelos, VítorWater contaminated with microcystins (MCs) or other cyanotoxins is recurrently used in agriculture and for crop irrigation. Several deleterious effects of MCs in plants that may impair crop productivity, including a decrease in growth and tissue necrosis, as well as an inhibition of photosynthesis and metabolic changes, have been reported. Studies also revealed a significant accumulation of MCs in edible tissues and plant organs, which raise concerns related to food safety. The European project TOXICROP precisely tackles this environmental problem. The main aims of the project are to map agricultural risk areas of cyanotoxin occurrence, to assess the fate of cyanotoxins in crops, and evaluate the impacts of using low-quality water for crop irrigation. The project also develops research on water remediation, exploring nature-based technologies. Here, we review part of the research carried out in the project, concerning the toxicity of cyanotoxins in crops. The research from TOXICROP Consortium has revealed for instance that adult strawberry or faba bean plants are susceptible to moderate concentrations of MCs (10 to 20 µg MCs/L). Furthermore, experiments with faba bean and common wheat grown in sterile (microorganism-free) and non-sterile (microorganism-rich) soil, watered with 100 μg MCs/L, revealed that native rhizospheric microbiota play an important role in the mitigation of the phytotoxic impact of MCs on plant growth, reducing toxin accumulation in both soils and plant tissues. Our studies also revealed that leaf vegetables, such as lettuce and spinach, growing in hydroponics are more susceptible to MCs than to the toxin, cylindrospermopsin (CYN). The lowest toxin concentrations affecting spinach and lettuce growth were 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, respectively. The results also reveal that the accumulation of MCs and CYN in plants depends on the conditions in which plants grow and concentrations of toxins in the irrigation water. In some cases, MCs are accumulated in plant tissues and exceed the tolerable daily intake proposed by the World Health Organization. We highlight the importance and contributions of this research to the definition and implementation of regulatory limits for cyanotoxins in irrigation waters.
- Cytotoxicity of marine cyanobacteria extracts on osteosarcoma cellsPublication . Costa-Rodrigues, João; Costa, M. M.; Costa, S.; Garcia, M.; Fernandes, M. H.; Vasconcelos, V.; Barros, Piedade; Martins, RosárioMarine cyanobacteria have been identified as a rich source of secondary metabolites with potential pharmacological applications. Anti-inflammatory, antibacterial and anticancer activities are some examples of properties described for cyanobacteria compounds, being the cytoxicity against cancer cell lines one of the most documented. The Laboratory of Ecotoxicology Genomics and Evolution (LEGE) — CIIMAR, Porto, Portugal, possesses a large collection of cyanobacteria strains isolated from the Portuguese coast. In order to investigate the interest of genera such as Cyanobium, Synechocystis, Synechococcus, Leptolyngbya and Pseudoanabaena which are genera that have been largely overlooked in terms of bioactivity, we have been screening their ability to induce cytotoxicity on human cancer cell lines. Assays have been conducted with a crude extract obtained by a dichloromethane and methanol extraction of freeze dried biomass and three fractions obtained using Si column chromatography with a gradient from 100% hexane, to 100% ethyl acetate to 100% methanol. The cytotoxicity of cyanobacteria crude extract and fractions has been evaluated by the MTT assay at 24, 48 and 72 h. Here we present the results concerning the cytotoxicity of 24 cyanobacteria strains on the osteosarcoma cell line MG63. The results show both inhibitory and stimulatory effects on cell growth within the same cyanobacteria strain. However, five cyanobacteria strains were found to induce a decrease in cell viability that reached the 80% within the ethyl acetate fraction, which makes this fraction interesting for the isolation and characterization of new bioactive compounds.
- Cytotoxity induced by extracts of Pisolithustinctorius spores on human cancer and normal cell lines – evaluation of the anticancer potentialPublication . Alves, Ricardo; Preto, Marco; Oliveira, Rui S.; Martins, RosárioCancer is one of the most important causes of death and morbidity worldwide. According to the World Health Organization 8.2 million people have died from cancer in 2012. The treatment of this fast growing disease is regarded as a major challenge in health for the XXI century. Conventional anti-cancer therapy is usually aggressive to the patients due to systemic toxicity caused by lack of specificity of the chemotherapy drugs (Kidd, 2000). This has triggered the interest of the scientific community to search for new and more effective therapies, less aggressive than conventional treatments. Used since antiquity in oriental civilizations, fungi, in particular mushrooms have been shown to limit or reverse tumor growth (Kidd, 2000). Pisolithustinctorius (Basidiomycota) is a soil fungus with world-wide distribution that it known to form ectomycorrhizal symbioses in forest ecosystems (Kope & Fortin, 1990). The sporocarps of this fungus contain Pisosterol, a triterpene that has been shown to have antitumor activity against some cancer cell lines (Montenegro et al., 2004). Nevertheless, no studies have focused on the anticancer potential of other structures such as spores and thus the anticancer potential of P. tinctorius , remains largely unknown.
- Determinação de alergénios de amendoim e amêndoa em alimentos comercializados não pré-embaladosPublication . Soares, Renata; Sousa, Carolina; Soares, Sílvia; Moreira, Andreia; Vieira da Silva, Manuela; Freitas, MarisaA sustentabilidade ambiental é um tema de destaque a nível mundial. Considerando os princípios da hierarquia da gestão de resíduos e a maior sensibilização da população para evitar o uso de embalagens descartáveis, é inegável o ressurgimento e expansão das lojas de venda de alimentos a granel (Beitzen-Heineke et al., 2017).
- Effects of interaction and bioaccessibility of the cyanotoxins microcystin and cylindrospermopsin in aquatic and terrestrial speciesPublication . Freitas, Marisa; Azevedo, Joana; Carvalho, António Paulo; Planchon, Sébastien; Renaut, Jenny; Mendes, Vera; Manadas, Bruno; Pinto, Edgar; Barreiro, Aldo; Neves, Joana; Campos, Alexandre; Vasconcelos, VitorThe occurrence and proliferation of toxic cyanobacteria blooms as a potential consequence of eutrophication and climate change are an emergent environmental concern worldwide. Microcystin-LR (MC-LR), mainly produced by Microcystisaeruginosa is the most documented and studied cyanotoxin. Cylindrospermopsin (CYN) has been recognized of increased concern due to the invasive nature of its main producer, Cylindrospermopsisraciborskii. Recent studies support the hypothesis that MC-LR and CYN exert harmful effects on crop plants. Lettuce, Lactuca sativa, is an important commercial leafy vegetable, which supplies important components for a healthy diet (e.g., fibers, minerals and antioxidants). Therefore, it is of particular interest the knowledge of lettuce sensitivity to ecologically relevant concentrations of cyanotoxins, inclusively mixtures. Proteomic technologies seem to be suitable to investigate the effects of MC-LR and CYN and may allow the identification of early stress responses, which are not perceptible by traditional endpoints. Proteomics may also provide new insights of protein biomarkers of exposure and the identification of allergenic proteins, which may be of interest for human health risk assessment. However, human health problems due to MC-LR and CYN are most likely associated to its chronic exposure by drinking water and contaminated food. Previous studies have shown that aquatic organisms, especially bivalves (filter-feeding organisms), can accumulate high levels of cyanotoxins without lethal effect. Based on the potential for human health risks, a provisional tolerable daily intake (TDI) of 0.04 and 0.03μg/kg-body weight, has been established for MC-LR and CYN, respectively. However, the risks associated to the consumption of contaminated food may increase if the consumers use storage and processing practices that enhance the concentration of cyanotoxins and their bioaccessibility. It has been reported that MCs are stable at high concentration of cyanotoxins and their bioaccessibility. It has been reported that MCs are stable at high temperatures (above 300 °C) and they can with stand several hours boiling. Likewise, CYN is highly watersoluble and stable to extreme temperatures and pHs, thus the knowledge of the influence of storage and cooking practices as well as human digestion on MC-LR and CYN concentration in food is required to a more accurate human risk assessment.
- 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.
- Effects of the irrigation with Microcystin contaminated water on the quality of carrots (Daucus carota)Publication . Machado, J.; Azevedo, J.; Freitas, Marisa; Pinto, Edgar; Vasconcelos, V.; Campos, A.Cyanobacteria blooms are often found in freshwaters and may reflect the increased eutrophication of these environments. Microcystin-LR (MC-LR), mainly produced by Microcystis aeruginosa, is the most documented and studied cyanotoxin causing serious problems to human health. Contaminated waters are commonly used for agriculture purposes and may represent a risk to food safety and crop’s quality. The plant root system is usually more exposed via soil irrigation, and therefore, rootvegetables are more predisposed to contamination. Is important to evaluate the possible negative effects of the use of water containing MC in the physiology and quality of carrots (Daucus carota) due to its importance for human nourishment and economy.
- Elucidating the mechanisms Involved in the cytotoxicity induced by marine cyanobacteria strains against the RKO colon carcinoma cell linePublication . Freitas, S.; Urbatzka, R.; Osório, H.; Costa, M.; Barros, Piedade; Vasconcelos, V.; Martins, RosárioCyanobacteria are known to synthesize secondary metabolites that may have potential as drugs for the treatment of human diseases such as cancer. Previous studies on marine cyanobacteria isolated from the Portuguese coast revealed strains of the picoplanktonic genera Cyanobium and Synechocystis as potential sources of anticancer compounds. The ethyl acetate fraction of the strains Cyanobium sp. LEGE06113 and the Synechocystis salina LEGE06155 was found to reduce cell viability of cancer cell lines. This work aimed to elucidate the mechanisms involved in the cytotoxicity of this strains in the colon adenocarcinoma cell line RKO by employing real-time PCR (RT-PCR) for genes involved in cell cycle and apoptosis, by flow cytometry for cell cycle and by two-dimensional gel electrophoresis for protein expression. RT-PCR results revealed differences in mRNA expression of genes CCNB1 (cell cycle) and BCL-2 (apoptosis). Flow cytometry results revealed a decrease in the G0/G1 and S phase and increased its number in the G2/M phase, which is in accordance with the lower expression of CCNB1. The proteomic results demonstrated different protein patterns comparing treatment groups with control. Proteins differentially regulated in exposed RKO cells were involved in cell cycle regulation, apoptosis, cell structure, protein regulation and cell metabolism. Although several of these proteins were identified in cells exposed to both cyanobacterial extracts, the data provides an indication that the cytotoxicity is induced by different toxic mechanisms in LEGE06113 and LEGE06155 fractions.
- Evaluating the effect of the Ionic liquid [C16Pyr] [Amp] in hormone-resistant tumors using an in vivo zebrafish assayPublication . Barros, Piedade; Martins, Rosário; Cruz, Agostinho; Silva, ReginaThe NASYTHOR project, aimes to study the potential of novel natural and synthetic compounds as anti-cancer drugs on hormone-resistant tumors. One of the objectives is the evaluation of the in vivo efficacy of the anticancer activity of the compounds using Danio rerio, zebrafish embryo as a model. In this work we describe the general methodology, advantages and disadvantages. Among a large range of advantages in the use of zebrafish in cancer research, the high level of genetic and physiologic homology with humans, including brain, digestive tract, musculature and vasculature can be highlighted. Also, the immature immune system of the embryos favors xenotransplantation of human cancer cells, and makes these animals a promising experimental model to tumorigenesis, angiogenesis, invasion and metastasis. The ionic liquid cetylpyridinium ampicillin [C16Pyr] [Amp] induces cytotoxicity in hormone-resistant breast and prostate cancer cell lines. To study the possible use of [C16Pyr] [Amp] as an anticancer, the toxicity towards zebrafish embryos should be evaluated first by an acute toxic assay that should be carried out following the OECD 236 Guideline.
- Exploring the antiradical potential of species from Lamiaceae family: implications for functional food development in the context of neurodegenerative and neuropsychiatric diseasesPublication . Campinho, Ana; Alves, Joana; Martins, Rosário; Vieira, Mónica; Grosso, Clara; Delerue-Matos, CristinaNeurodegenerative and neuropsychiatric diseases have become highly significant in Western societies. Unfortunately, these diseases currently lack a cure, and existing treatments merely manage the symptoms. Thus, it is imperative to explore new alternatives for either preventing these disorders or treating them effectively. One promising avenue for prevention lies in the development of neuroprotective and antioxidant functional foods. To this end, a study focused on ten species from the Lamiaceae family, which have attracted attention due to their well-known antioxidant, anti-inflammatory, anti-obesity, and anti-cancer properties, among others. The interest in their pharmacological applications has grown significantly in recent years. In order to uncover the biological potential of these species, the study involved performing decoctions and evaluating both the total phenolic content (TPC) and antiradical activity. The results revealed that TPC values ranged from 59.97 ± 6.18 (Ocimum basilicum L. var minimum) to 374.0 ± 16.9 (Salvia officinalis L.) mg gallic acid equivalents (GAE)/g of dry extract (dw). Additionally, the IC50 values for DPPH• and ABTS•+ scavenging activities varied between 21.55 ± 1.18 (Origanum vulgare L.) and 132.0 ± 15.3 μg/mL (O. basilicum var minimum), and from 14.79 ± 0.50 (O. vulgare) to 44.65 ± 2.34 μg/mL (O. basilicum), respectively. The observed strong antiradical activity holds great promise for the future development of functional foods aimed at combating the oxidative stress implicated in these diseases and promoting overall brain health. By harnessing the potential of these species from Lamiaceae family, we may pave the way for innovative approaches to tackle neurodegenerative and neuropsychiatric conditions.