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- 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.
- 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.
- Impact of microcystin contaminated water on quality of carrots (Daucuscarota)Publication . Machado, J.; Azevedo, J.; Freitas, Marisa; Pinto, Edgar; Vasconcelos, Vitor; Campos, AlexandreCyanobacteria blooms are often found in freshwaters and may reflect the increased eutrophication of these environments and because of this many water resources worldwide may have a limited utilization. The presence of these microorganisms may pose a serious threat to water quality because many of them produce a large diversity of toxins that can be harmful to human health (Figueiredo et a l., 2004). On the other hand contaminated waters used to agricultural purposes may pose additional concerns to food safety. The absorption of toxins by plants may induce morphological and physiological changes that can lead to a loss of productivity as well as toxin bioaccumulation in edible tissues. However, the impact of cyanotoxins on plants and its ability to enter the food chain via this pathway is not fully understood (Kitleret al ., 2012). Microcystin (MC) is the cyanotoxin most frequently present in eutrophic freshwaters causing serious problems to human health, being the liver the main target. These toxins are potent and specific inhibitors of protein phosphatases PP1 and PP2Ain both animals and higher plants (Figueiredo et al., 2004). It is known that MC affect a number of physiological processes in plants (Corbel et al ., 2013). The plant root system is usually more exposed, via soil irrigation, and therefore root-vegetables more prone to contamination. Carrots (Daucuscarota) are root-vegetables with great importance for human nourishment and economy, with extensive use all over the world (Singh et al ., 2012).
- 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.
- Toxicity testing of simvastatin, sertraline, 4-MBC propylparaben and triclocarban using zebrafish and sea urchin embryos bioassaysPublication . Torres, Tiago; Ribeiro, Sílvia; Cunha, Isabel; Martins, Rosário; Santos, Miguel MachadoIn the past decade, many emergent compounds, including some active substances and ingredients of Pharmaceuticals and Personal Care Products (PPCPs) have been detected in water at levels that can negatively impact aquatic ecosystems (Lapworth et al., 2012; Jiang et al., 2013). The recent knowledge of their occurrence has raised concerns about human health effects and ecosystem risks. Although these compounds are frequently detected at concentrations that are not likely to induce adverse effects in humans and may be too low to cause acute effects in other organisms, there is still a serious lack of information about the effects in non-target species, particularly considering chronic exposure or effects resulting from interactions between them (Lapworth et al., 2012). Pharmaceuticals and Personal Care Products enter the aquatic environment from different point and nonpoint sources and wastewater treatments plants cannot ensure complete removal of many compounds, and therefore they may be present at significant concentrations in effluents (Jiang et al., 2013). Hence, it is essential to understand the effects of these substances on aquatic organisms. Owing to the large number of new chemicals that must go through toxicity testing, short-term early-life-stages have been frequently used as an alternative to long-term exposures due to its high sensitivity and logistic advantages.
- 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.
- 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.
- 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.
- 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.
- Projeto Nasythor - novos compostos naturais e sintéticos para o tratamento de tumores hormono-resistentesPublication . Quintela Vieira, Ana Filipa; Cruz, Agostinho; Oliveira, Ana; Pinho, Cláudia; Prudêncio, Cristina; Vieira, Mónica; Barros, Piedade; Ferraz, Ricardo; Martins, Rosário; Silva, ReginaUma equipa multidisciplinar do CISA-ESS | P. PORTO em colaboração com o CI|IPOP dedicou-se ao estudo do potencial anti-tumoral de novos compostos em tumores de grande incidência em Portugal: cancro da mama e da próstata. O potencial anti-tumoral de extratos de plantas e compostos sintéticos foi avaliado em linhas celulares de cancro da próstata e da mama, sendo a resposta terapêutica prevista através de amostras biológicas. Um líquido iónico provou estar associado à atenuação do fenótipo maligno das linhas celulares testadas, mostrando-se um promissor agente terapêutico anti-tumoral. A expressão de genes que se mostrou alterada após tratamento foi validada em amostras de pacientes com cancro da próstata e da mama. Os resultados foram divulgados sob a forma de artigo científico e de várias comunicações orais e posters em congressos nacionais e internacionais.