Convergent evolution of [D-Leucine1] microcystin-LR in taxonomically disparate cyanobacteria

Abstract Background Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. Results Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu1] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA2 adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA2 adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu1] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met1] microcystin-LR. Conclusions Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu1] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

Remoção da biomassa algal e determinação da concentração de microcistina pelo Método ELISA em ensaios de coagulação, sedimentação, filtração e adsorção

Nessa pesquisa são relatados os resultados da determinação das concentrações de microcistina e de biomassa algal após as várias etapas de tratamento de amostras de água coletadas junto ao reservatório de Barra Bonita-SP visando obtenção de água potável. O tratamento foi realizado em escala de laboratório com e sem aplicação de carvão ativado em pó (CAP) e as etapas foram: coagulação com aplicação de cloreto férrico, sedimentação, filtração em papel de filtro. Foi possível observar que a pré-clarificação desse tipo de água por coagulação seguida de sedimentação requereu dosagens relativamente elevadas de cloreto férrico (80 mg/L), tendo sido verificada eficiência muito baixa de remoção de microcistina nas etapas de tratamento por sedimentação seguida de filtração, quando não foi aplicado CAP. Apenas com a aplicação de CAP a microcistina foi reduzida à níveis que atendessem os padrões de potabilidade previstos na Portaria 518/04 (concentração menor que 1 μg/L). A determinação de microcistina pelo método que utiliza Imunoadsorventes Ligados à Enzima (ELISA) mostrou-se uma ferramenta útil e confiável para detectar e quantificar essa toxina, embora ainda apresente custo relativamente elevado.

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these Surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 mug/l drinking, water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check. the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 mug/l were found for MCs, PSPs and CYN, respectively. However, only traces (< 1.0 mug/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety. (C) 2004 Elsevier Ltd. All rights reserved.

Biodegradation of the cyanobacterial toxin microcystin LR in natural water and biologically active slow sand filters

A bacterium (MJ-PV) previously demonstrated to degrade the cyanobacterial toxin microcystin LR, was investigated for bioremediation applications in natural water microcosms and biologically active slow sand filters. Enhanced degradation of microcystin LR was observed with inoculated (1 x 10(6) cell/mL) treatments of river water dosed with microcystin LR (> 80% degradation within 2 days) compared to uninoculated controls. Inoculation of MJ-PV at lower concentrations (1 x 10(2)-1 x 10(5)cells/mL) also demonstrated enhanced microcystin LR degradation over control treatments. Polymerase chain reactions (PCR) specifically targeting amplification of 16S rDNA of MJ-PV and the gene responsible for initial degradation of microcystin LR (mlrA) were successfully applied to monitor the presence of the bacterium in experimental trials. No amplified products indicative of an endemic MJ-PV population were observed in uninoculated treatments indicating other bacterial strains were active in degradation of microcystin LR, Pilot scale biologically active slow sand filters demonstrated degradation of microcystin LR irrespective of MJ-PV bacterial inoculation. PCR analysis detected the MJ-PV population at all locations within the sand filters where microcystin degradation was measured. Despite not observing enhanced degradation of microcystin LR in inoculated columns compared to uninoculated column, these studies demonstrate the effectiveness of a low-technology water treatment system like biologically active slow sand filters for removal of microcystins from reticulated water supplies. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Contribution of lipophilic secondary metabolites to the toxicity of strains of freshwater cyanobacterial harmful algal blooms, identified using the zebrafish (Danio rerio) embryo as a model for vertebrate development

Cyanobacteria ("blue-green algae") are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called "harmful algal blooms", particularly in freshwater systems, a number of these metabolites have been associated—as "toxins", or commonly "cyanotoxins"—with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. ^ We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.^

Development of Advanced Capillary Electrophoresis Techniques with UV and Mass Spectrometry Detection for Forensic, Pharmaceutical and Environmental Applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Composição do fitoplâncton e cianotoxinas em um reservatório utilizado para o abastecimento público: o efeito do tratamento de água convencional.

Florações de cianobactérias nocivas ocorrem frequentemente em reservatórios brasileiros, devido ao incremento de nutrientes pela eutrofização e pelas mudanças climáticas, como o aquecimento global. Estas florações alteram a qualidade dos corpos hídricos, produzindo compostos de gosto e odor e cianotoxinas, que representam um problema para as Estações de Tratamento de Água (ETAs). Estes compostos, quando dissolvidos na água dificultam os tratamentos convencionais. Além das cianobacérias, um dinoflagelado exótico tem ocorrido em águas doces brasileiras, incluindo reservatórios utilizados para o abastecimento público. Os reservatórios de Caxias do Sul (RS – Brasil) são gerenciados pelo Serviço Autônomo Municipal de Água e Esgoto (SAMAE) e apresentam um histórico de florações de cianobactérias nocivas, como Dolichospermum Bory de Saint-Vincent ex Bornet & Flahault e Microcystis (Kützing) ex Lemmermann, dentre outras. Além disso, desde 2012, tem ocorrido nestes reservatórios florações de Ceratium furcoides. Este organismo quando em extensas florações tem sido relacionado à perda da qualidade dos corpos hídricos. O reservatório Maestra foi construído entre os anos de 1965-1970 e abastece 22% da população de Caxias do Sul. Este reservatório fornece água para a ETA Celeste Gobatto, que utiliza o método convencional de tratamento da água. Este trabalho esta estruturado em três capítulos. O primeiro consiste de uma revisão bibliográfica de assuntos relavantes acerca do histórico do monitoramento da qualidade dos reservatórios no Brasil, da biologia de algas e cianobactérias, e as principais cianotixinas e acidentes devido à intoxicação no Brasil. Além disso, é feita uma breve revisão sobre o tratamento convencional da água, mostrando a importância de cada etapa para a remoção das impurezas, de acordo com os padrões de potabilidade da Portaria 2914. O segundo capítulo é manuscrito na forma de artigo científico intítulado “Composição de algas, cianobactérias e cianotoxinas no reservatório Maestra – Caxias do Sul, RS – Brasil”. Este estudo foi realizado entre janeiro de 2012 a abril de 2013. O terceiro capítulo consta de um manuscrito na forma de artigo científico, intitulado “Efeito do tratamento de água convencional na remoção de algas, cianobactérias e cianotoxinas em uma Estação de Tratamento de Água Convencional”. A eficiência de remoção foi avaliada em escala piloto, em uma Estação de Tratamento de Água de Caxias do Sul – RS a qual utiliza o método convencional de tratamento.

Studies on the synthesis of novel PP2A inhibitors and a GPCR detergent

Chapter 1 While targeting kinases in oncology research has been explored extensively, targeting protein phosphatases is currently in its infancy. However, a number of pharmaceutical companies are currently looking to expand their research efforts in this area. PP2A has been shown to down-regulate ERK5, a mitogen-activated protein kinase (MAPK) that has been shown to be important in driving the invasive phenotype of prostate cancer. Fostriecin and its related structural analogues PD 113,270 and 113,271 have been shown to inhibit a mitotic entry checkpoint in cell growth through the potent and selective inhibition of protein phosphatases PP1, PP2A, and PP4 (IC50 of 45 μM, 1.5 nM, and 3 nM respectively). Fostriecin is one of the most selective protein phosphatase inhibitors disclosed to date with a 104 fold selectivity for PP2A/PP4 versus PP1. Unfortunately, fostriecin and its analogues are very unstable, and this instability has effectively prevented them from being used as effective therapeutic leads. The microcystins and nodularins on the other hand, exhibit significant inhibitory activity against PP1 and PP2A (IC50 = 26 pM and 1.8 nM respectively), but their high toxicity has prevented any therapeutic application. Truncation of the ADDA chain from these polypeptides completely attenuates PP inhibitory activity. Simpler analogues incorporating the N-acylated ADDA chain and D-Ala retain moderate activity against PP1 and PP2A (IC50 = 1.0 μM and 0.17 μM respectively). The generation of a new series of fostriecin analogues to further expand its structure-activity relationship is envisaged with a view to creating new more stable PP2A inhibitors. It was hoped that by incorporating some of the more stable structural features of ADDA into fostriecin that stability and activity could be reconciled. With that in mind a series of PP2A inhibitors were synthesised and biologically evaluated. Chapter 2 GPCRs are an important area of research and are the targets of a quarter of the drugs on the market (2005). As a result, GPCRs continue to be at the forefront of research in both small and large drug companies. However one of the difficulties in studying this diverse class of membrane proteins is their tendency to denature in aqueous solution. As a result there is a pressing need to develop new detergents to solubilise, stabilise and crystallise GPCRs in their native form for further study. Cholesterol analogues have been shown to be important for stabilising membrane proteins and preventing their thermal inactivation. In addition the β2-adrenergic receptor, a GPCR membrane protein, has been crystallised in the active state with two cholesterol molecules bound between the I, II, III and IV helices of the protein. This appears to represent a distinct cholesterol binding pocket on the membrane protein that is speculated to be conserved across up to 44% of the rhodopsin class of GPCRs. CHOBIMALT is a cholesterol-based detergent that has been shown to exhibit promising GPCR-stabilising properties. When benchmarked against other cholesterol based detergents it was found to be superior to all others tested except for cholesteryl hemisuccinate.1 CHOBIMALT has an aggregation number of roughly 200 and forms 210 ± 30 kDa micelles, which are significantly larger than those of most detergents used for biological systems which is likely due to the packing constraints associated with CHOBMALT’s large polar headgroup.2 As a result, CHOBIMALT is used mostly as an additive to other commercially available detergents in order to decrease micelle size. A branched dimaltoside motif is common in recently synthesised detergents by Chae and co-workers. These detergents have shown promising detergent properties, for example the maltose neopentyl glycol (MNG) detergent synthesised by Chae. This branched dimaltoside detergent was shown to be able to solubilise and stabilise the very labile light harvesting complex I (LHI) from Rhodopsin capsulatus in its active form for 20 days with little loss of protein conformation.3 A cholesterol-based detergent was envisaged that combines the cholesterol framework of CHOBIMALT but replaces its linear tetrasaccharide with a branched dimaltoside. This detergent would then be investigated to assess its ability to solubilise, stabilise and crystallise GPCR proteins. This cholesterol-based detergent (shown below) was eventually synthesised in 9 linear steps from cholesterol.

Avaliação da toxicidade de florações naturais e de cultura de cianobactérias: efeitos sobre Ceriodaphnia silvestri (Crustacea, Cladocera)

The incidence of toxic cyanobacterial blooms is one of the important consequences of eutrophication in aquatic ecosystems. It is a very common phenomenon in reservoirs and shrimp ponds in the State of Rio Grande do Norte (RN), Brazil. Cyanobacterias produce toxins which can affect aquatic organisms and men trough the food chain. Aiming to contribute to the studies of cyanobacterias in RN, we propose: a) to evaluate the toxicity of isolated cyanobacterias in important fresh-water environments; and b) to verify the effects of both natural and cultured blooms occurred in reservoirs for human supply and in the cladoceran Ceriodaphnia silvestrii. This study was carried out using samples of natural blooms occurred between March and October of 2004 in Gargalheiras Dam (08º L e 39º W), in July of 2004 in Armando Ribeiro Gonçalves Dam (06o S e 37o W) and in commercial shrimp ponds (Litopenaeus vannamei) located in fresh-water environments. The samples were collected with plankton net (20µm.) for identification, isolation and obtaining of phytoplanktonic biomass for liophilization and later toxicity bioassays. The toxicity of cultured samples and natural blooms was investigated through bioassays in Swiss mice. Quantification of cyanobacteria in samples was conducted following the Ütermol method, with 300mL samples fixed with lugol. The toxicity test with Ceriodaphnia silvestrii followed ABNT, 2001 recommendations, and were accomplished with natural hepatotoxic bloom s samples and cultured samples of both non-toxic and neurotoxic C. raciborskii. In this test, five newborns, aged between 6 and 24 hours, were exposed to different concentrations (0 a 800 mg.L-1) of crude cyanobacterial extracts during 24 and 48 hours. Three replicates were used per treatment. The pH, temperature and dissolved oxygen at the beginning and after 24 and 48hours from the test were measured. We estimated the CL50 through the Trimmed Spearman-Karber method. The blooms were constituted by Microcystis panniformis, M. aeruginosa, Anabaena circinalis, Cylindrospermopsis raciborskii and Planktothrix agardhii, producers of mycrocistin-LR confirmed with HPLC analysis. Samples of hepatotoxic blooms registered toxinogenic potential for C. silvestrii, with CL50-24h value of 47.48 mg.L-1 and CL5048h of 38.15 mg.L-1 for GARG samples in march/2005; CL50-24h of 113,13 mg.L-1 and CL5048h of 88,24 mg.L-1 for ARG July/2004; CL50-24h of 300.39 mg.L-1 and CL50-48h of 149.89 mg.L-1 for GARG October/2005. For cultured samples, values of CL50-24h and CL50-48h for C. raciborskii toxic strains were 228.05 and 120.28 mg.L-1, respectively. There was no mortality of C. silvestrii during the tests with non-toxic C. raciborskii strain. The toxicity test with C. silvestrii presented good sensitivity degree to cyanotoxins. The toxicity of natural hepatotoxic blooms samples (microcystins) and cultured neurotoxic saxitoxins producer samples analyzed in this study give us strong indications of that toxin s influence on the zooplanktonic community structure in tropical aquatic environments. Eleven cyanobacteria strains were isolated, representing 6 species: Anabaenopsis sp., Cylindrospermopsis raciborskii, Chroococcus sp., Microcystis panniformis, Geitlerinema unigranulatum e Planktothrix agardhii. None presented toxicity in Swiss mice. The strains were catalogued and deposited in the Laboratório de Ecologia e Toxicologia de Organismos Aquáticos (LETMA), in UFRN, and will be utilized in ecotoxicológical and ecophysiological studies, aiming to clarify the causes and control of cyanobacterial blooms in aquatic environments in RN. This state s reservoirs must receive broader attention from the authorities, considering the constant blooms occurring in waters used for human consumption

Avaliação transcricional de Glutationas -transferases em PEIXE-ZEBRA (Danio rerio) e alterações causadas pela exposição á Microcistina-LR

As Microcistinas são heptapeptídios cíclicos produzidos como metabólitos secundários por diferentes espécies de cianobactérias, sendo relevantes pelo seu potencial hepatotóxico. Peixes apresentam estratégias bioquímicas para detoxificar contaminantes ambientais, incluindo a ativação de enzimas de fase II de biotransformação, que incluem as isoformas de glutationa S-transferase (GST). As GST catalizam a conjugação de glutationa reduzida (GSH) com uma variedade de xenobióticos, incluindo as microcistinas. O presente estudo avaliou os níveis transcricionais de quinze isoformas de GST a fim de identificar isoformas possivelmente envolvidas na detoxificação de contaminantes ambientais como a microcistina-LR (MC-LR) em Danio rerio. A técnica de PCR em tempo real (RT-qPCR) foi utilizada para avaliação dos níveis transcricionais, permitindo análise das GST em diferentes órgãos, abundância e a ativação/repressão das isoformas de GST pela exposição à MC-LR. Foram avaliados os possíveis efeitos causados em brânquia e fígado após exposição por 24 hs às concentrações de 5 µg.L-1 e 50 µg.L-1 de MC-LR. Baseado nos scores de estabilidade para oito genes normalizadores, foram selecionados glicose-6-fosfato desidrogenase (g6pdh), β-actina1 e beta-2-microglobulina (b2m); b2m, alfa-tubulina 1 (tuba) e β- actin1; e tuba, b2m e g6pdh, para normalização dos níveis trancricionais de GST para distribuição órgão-específica, abundância e efeito da MC-LR em brânquia e fígado, respectivamente. A avaliação transcricional da distribuição órgão-específica revelou níveis significativos de gstal e gstk1.1 no fígado; gstp1 e gstp2 em brânquia; mgst3a, gstr1, gstm2, gstm33, gstp1, gstp2 e gstk1.1 no intestino; gstm2, gstm3 e gstal no olho e gstt1a e gsta2.1 no cérebro. Considerando os níveis de transcritos para um dado órgão, gstk1.1, gstal, gstp1 e gstt2 foram mais abundantes nos órgãos de detoxificação, tais como o fígado, brânquias e intestino, enquanto gstt1a e gsta2.1 foram mais abundantes no rim. Em brânquia, gsta2.1 e gstt1b foram reprimidas por 5 µg.L-1 de MC-LR e mgst1.1 foi reprimida em 50 µg.L-1 de MC-LR. No fígado, as isoformas gst2.2 e gstp2 foram reprimidas em ambas as concentrações, gstal foi reprimida em 5 µg.L-1, e gstt1a e gstk1.1 foram reprimidas em 50 µg.L-1 de MC-LR. As isoformas gstal, gstr1, gstp1, mgst3a, gstm1, gstm2 e gstm3 não foram alteradas pela exposição a MC-LR. Os resultados obtidos fornecem informações para a escolha de isoformas específicas de GST possivelmente envolvidas na detoxificação/toxicidade de MC-LR, a serem melhores caracterizadas ao nível protéico e também contribui para a escolha de genes normalizadores a serem utilizados em outros estudos da mesma natureza

Interação da cianotoxina microcistina e o nanomaterial de carbono fulereno (C60) em brânquias do peixe Cyprinus carpio (Teleostei: Cyprinidae) sob incidência de radiação UVA

A produção mundial de nanomateriais tem aumentado nos últimos anos, em função de suas variadas aplicações tecnológicas e, como consequência do seu crescente uso e demanda, poderão existir riscos ambientais sendo a água o ambiente onde muitas destas substâncias podem exercer efeitos deletérios. Um dos nanomaterias de carbono mais utilizados é o fulereno, um composto orgânico lipofílico que pode se comportar como carreador de moléculas tóxicas, potencializando a entrada de contaminantes ambientais em órgãos específicos, fenômeno conhecido como “cavalo de Troia”. As microcistinas (MC) são cianotoxinas produzidas por cianobactérias durante episódios de floração, afetando aos organismos aquáticos e ao ser humano. Diversos estudos demonstram que organismos expostos tanto às MCs quanto ao fulereno podem causar produção excessiva de espécies ativas de oxigênio e alterar os níveis de antioxidantes. Além disso, outro fator que pode vir a intensificar o potencial tóxico de ambos é a incidência de radiação UVA. Sendo assim, procurou-se avaliar os efeitos em parâmetros de estresse oxidativo da co-exposição ex vivo da cianotoxina microcistina-LR (MC-LR) e o nanomaterial de carbono fulereno em brânquias do peixe Cyprinus carpio sob incidência de radiação UVA. Os resultados mostraram que: (a) houve uma perda da capacidade antioxidante no tratamento com MC-LR (baixa concentração) quando coexposta com fulereno no UVA em relação com o tratamento realizado sem co-exposição com fulereno; (b) o fulereno no UV diminuiu a atividade da enzima glutationa-Stransferase (GST) quando comparado com o controle no UV; (c) a MC-LR (alta concentração) co-exposta com fulereno foi capaz de diminuir as concentrações do antioxidante glutationa (GSH) quando comparado com o mesmo tratamento tanto no UVA quanto no escuro sem a co-exposição ao fulereno; (d) o tratamento MC-LR (baixa concentração) com UVA aumentou o dano oxidativo lipídico quando comparado com o controle UVA; (e) o fulereno não causou uma maior bioacumulação da microcistina no tecido. Sendo assim, pode-se concluir que o fulereno não apresentou o potencial de carregador de moléculas nessas concentrações de microcistina, porém, a co-exposição dos compostos diminuem tanto capacidade antioxidante total, como a concentração da GSH, podendo gerar problemas a longo prazo na detoxificação da toxina.

Microcystin-LR in Brazilian Aquaculture Production Systems

The growth of aquaculture production systems, mostly the sport-fishing kind, coupled with a lack of control, brings about concerns on the quality of water and food produced. The current paper determines which factors may trigger the growth of cyanobacteria, with subsequent concentrations of microcystins in collected water samples, at the surface and in the water column, from 10 aquaculture systems, during the dry and rainy seasons. The above is undertaken by measurements of biotic (counting of Chlorophyceae, cyanobacteria, and microcystin-LR [MC-LR]) and abiotic (total nitrogen and total phosphorus) factors. Because the water from the 10 aquaculture production systems had MC-LR concentrations that were highly correlated with Microcystis aeruginosa (M. aeruginosa) biomass, most MC-LR microcystins were produced by this species. The MC-LR concentrations and M. aeruginosa counting were positively correlated with nitrogen-to-phosphorus ratios and suggest that parameters may affect not only the M. aeruginosa biomass, but also MC-LR concentrations. Water Environ. Res., 82, 240 (2010).

Avaliação da toxicidade de florações naturais e de cultura de cianobactérias: efeitos sobre Ceriodaphnia silvestri (Crustacea, Cladocera)

The incidence of toxic cyanobacterial blooms is one of the important consequences of eutrophication in aquatic ecosystems. It is a very common phenomenon in reservoirs and shrimp ponds in the State of Rio Grande do Norte (RN), Brazil. Cyanobacterias produce toxins which can affect aquatic organisms and men trough the food chain. Aiming to contribute to the studies of cyanobacterias in RN, we propose: a) to evaluate the toxicity of isolated cyanobacterias in important fresh-water environments; and b) to verify the effects of both natural and cultured blooms occurred in reservoirs for human supply and in the cladoceran Ceriodaphnia silvestrii. This study was carried out using samples of natural blooms occurred between March and October of 2004 in Gargalheiras Dam (08º L e 39º W), in July of 2004 in Armando Ribeiro Gonçalves Dam (06o S e 37o W) and in commercial shrimp ponds (Litopenaeus vannamei) located in fresh-water environments. The samples were collected with plankton net (20µm.) for identification, isolation and obtaining of phytoplanktonic biomass for liophilization and later toxicity bioassays. The toxicity of cultured samples and natural blooms was investigated through bioassays in Swiss mice. Quantification of cyanobacteria in samples was conducted following the Ütermol method, with 300mL samples fixed with lugol. The toxicity test with Ceriodaphnia silvestrii followed ABNT, 2001 recommendations, and were accomplished with natural hepatotoxic bloom s samples and cultured samples of both non-toxic and neurotoxic C. raciborskii. In this test, five newborns, aged between 6 and 24 hours, were exposed to different concentrations (0 a 800 mg.L-1) of crude cyanobacterial extracts during 24 and 48 hours. Three replicates were used per treatment. The pH, temperature and dissolved oxygen at the beginning and after 24 and 48hours from the test were measured. We estimated the CL50 through the Trimmed Spearman-Karber method. The blooms were constituted by Microcystis panniformis, M. aeruginosa, Anabaena circinalis, Cylindrospermopsis raciborskii and Planktothrix agardhii, producers of mycrocistin-LR confirmed with HPLC analysis. Samples of hepatotoxic blooms registered toxinogenic potential for C. silvestrii, with CL50-24h value of 47.48 mg.L-1 and CL5048h of 38.15 mg.L-1 for GARG samples in march/2005; CL50-24h of 113,13 mg.L-1 and CL5048h of 88,24 mg.L-1 for ARG July/2004; CL50-24h of 300.39 mg.L-1 and CL50-48h of 149.89 mg.L-1 for GARG October/2005. For cultured samples, values of CL50-24h and CL50-48h for C. raciborskii toxic strains were 228.05 and 120.28 mg.L-1, respectively. There was no mortality of C. silvestrii during the tests with non-toxic C. raciborskii strain. The toxicity test with C. silvestrii presented good sensitivity degree to cyanotoxins. The toxicity of natural hepatotoxic blooms samples (microcystins) and cultured neurotoxic saxitoxins producer samples analyzed in this study give us strong indications of that toxin s influence on the zooplanktonic community structure in tropical aquatic environments. Eleven cyanobacteria strains were isolated, representing 6 species: Anabaenopsis sp., Cylindrospermopsis raciborskii, Chroococcus sp., Microcystis panniformis, Geitlerinema unigranulatum e Planktothrix agardhii. None presented toxicity in Swiss mice. The strains were catalogued and deposited in the Laboratório de Ecologia e Toxicologia de Organismos Aquáticos (LETMA), in UFRN, and will be utilized in ecotoxicológical and ecophysiological studies, aiming to clarify the causes and control of cyanobacterial blooms in aquatic environments in RN. This state s reservoirs must receive broader attention from the authorities, considering the constant blooms occurring in waters used for human consumption