Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil

Background Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 g.L1 to 0.05 g.L1. Besides, an increase in the bacterial countfrom 71×105 bacteria.mL1 to 117×105 bacteria.mL1was observed along the incubation. Conclusions The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as a source of bacteria capable of degrading cyanobacterial toxins. This is the first Brazilian report on a bacteriumof the genus Pseudomonasthat can degrade [D-Leu1] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters.(AU)

Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil

Background Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 g.L1 to 0.05 g.L1. Besides, an increase in the bacterial countfrom 71×105 bacteria.mL1 to 117×105 bacteria.mL1was observed along the incubation. Conclusions The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as a source of bacteria capable of degrading cyanobacterial toxins. This is the first Brazilian report on a bacteriumof the genus Pseudomonasthat can degrade [D-Leu1] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters.

Estudo da presença da toxina microcistina na água de reservatório deMundaú (Garanhuns-PE) pelas metodologias ELISA e CLAE / Study on the occurrence of microcystin toxin in water reservoir ofMundaú (Garanhuns-PE) by ELISA and HPLC methodologies

As cianobactérias são responsáveis pela produção de cianotoxinas que, uma vez acumuladas, podem causar sérios danos à saúde humana e animal. As microcistinas são o tipo mais comum de cianotoxinas e são promotoras de tumores hepáticos. O reservatório de Mundaú, localizado no município de Garanhuns-PE, foi o local escolhido por apresentar histórico de florações de cianobactérias produtoras de microcistinas. Neste trabalho foi investigada a presença de microcistinas em amostras de água bruta do reservatório do rio Mundaú utilizando-se as técnicas de Enzyme Linked Immunosorbent Assay (ELISA) e Cromatografia Líquida de Alta Eficiência (CLAE). Durante quinze meses consecutivos, as amostras de água foram coletadas em duplicata no ponto de captação deste manancial e analisadas por ambas metodologias ELISA e CLAE. A presença de microcistinas foi detectada em 100 % das amostras, confirmando-se a relevância do monitoramento de microcistinas em águas de abastecimento público, pois assim como o rio Mundaú, vários mananciais de Pernambuco apresentam florações de cianobactérias que podem ser tóxicas. Este trabalho deixou como legado a implantação da referida análise no Laboratório Central de Saúde Pública (LACEN-PE), e demonstrou sua importância como metodologia complementar à contagem das cianobactérias, para fornecer subsídios às ações preventivas de vigilância à saúde.(AU)

Cyanobacteria are responsible for the production of cyanotoxins, and once accumulated, it might cause serious harm to the human and animals health. Microcystins are the most common type of cyanotoxins and they cause liver tumor. The Mundaú reservoir, located in the municipality of Garanhuns-PE, was the selected site for presenting historical blooms of microcystins-producing cyanobacteria. This study analyzed the occurrence of microcystins in raw water samples from the Mundaú reservoir by using Enzyme Linked Immunosorbent Assay (ELISA) and High Performance Liquid Chromatography (HPLC) methodologies. During the consecutive fifteen months, water samples were collected in duplicate at the intake point of this water source, and they were analyzed by both ELISA and HPLC techniques. The presence of microcystins was detected in 100 % of analyzed samples, confirming the relevance of performing the microcystins monitoring in public water supplies. And as well as the Mundaú river, various water sources of Pernambuco show cyanobacterial blooms, which might be toxic. The techniques used in this study were implemented in the Central Laboratory of Public Health of Pernambuco (LACEN-PE), and they showed to be a relevant tools as a complementary methodology to the cyanobacteria counting assay for providing preventive subsidies to the health surveillance.(AU)

A toxic cyanobacterial bloom in an urban coastal lake, Rio Grande do Sul state, Southern Brazil

Reports of cyanobacterial blooms developing worldwide have considerably increased, and, in most cases, the predominant toxins are microcystins. The present study reports a cyanobacterial bloom in Lake Violão, Torres, Rio Grande do Sul State, in January 2005. Samples collected on January 13, 2005, were submitted to taxonomical, toxicological, and chemical studies. The taxonomical analysis showed many different species of cyanobacteria, and that Microcystis protocystis and Sphaerocavum cf. brasiliense were dominant. Besides these, Microcystis panniformis, Anabaena oumiana,Cylindrospermopsis raciborskii, and Anabaenopsis elenkinii f. circularis were also present. The toxicity of the bloom was confirmed through intraperitoneal tests in mice, and chemical analyses of bloom extracts showed that the major substance was anabaenopeptin F, followed by anabaenopeptin B, microcystin-LR, and microcystin-RR.

O número de relatos de ocorrências de florações de cianobactérias em todo o mundo vem aumentando consideravelmente e na maioria desses episódios, as toxinas dominantes são as microcistinas. O presente estudo relata a ocorrência de floração na Lagoa do Violão, município de Torres, RS, em janeiro de 2005. As amostras coletadas em 13/01/2005 foram submetidas a estudos taxonômicos, toxicológicos e químicos. O exame microscópico do fitoplancton mostrou a dominância das espécies Microcystis protocystis e Sphaerocavum cf. brasiliense; foram observadas, também, Microcystis panniformis, Anabaena oumiana,Cylindrospermopsis raciborskii e Anabaenopsis elenkinii f. circularis. A toxicidade da floração foi confirmada através de ensaio intraperitonial em camundongos e a análise química de extratos obtidos da biomassa liofilizada mostrou que a substância majoritária era a anabaenopeptina F, seguida por anabaenopeptina B, microcistina-LR e microcistina-RR.

Hepatotoxicity associated with microcystin / Hepatotoxicidade associada à microcistina

Urban and industrial discharges, intense agricultural exploitation and fisheries have been causing the eutrophication in both drinking and recreational waters. A frequent consequence of eutrophication in waters is the massive development of cyanobacteria. The occurrence of these blooms induces a severe problem, as Microcystis aeruginosa, the most widespread distributed cyanobacteria, can produce microcystins (MC). Toxic effects of MC have been described in liver, lungs, stomach, and intestine. Deaths in wildlife, livestock and human beings were also associated with MC exposition. MC exposition can occurs directly by ingestion, inhalation, contact, intravenous inoculation of contaminated water (hemodialysis) or indirectly, by the consumption of animals, as fish and mollusks, the majors ingestors of cyanobacteria and its toxins. The most toxic MC, an also the most common is microcystin-LR (MC-LR), that has the liver as the main target organ. Microcystin is taken up specifically into the liver by bile acid transporters and, after entering the cytoplasm, inhibit protein phosphatases 1 and 2A, which leads to the increase in protein phosphorylation. This effect has two main consequences: the destruction of cytoskeleton directly causing cytotoxic effects, and deregulation of cell division, leading to tumor-promoting activity. Acute exposition to MC induces severe intrahepatic hemorrha

Efluentes industriais e urbanos e a intensa exploração agrícola e de pescado têm levado à eutrofização de muitos mananciais de água, destinados ao consumo e às atividades recreacionais. A eutrofização das águas, freqûentemente, tem como conseqûência o desenvolvimento expressivo de cianobactérias. Estas florações induzem a sérios problemas, visto que a ocorrência de Microcystis aeruginosa, uma das cianobactérias mais difundidas, pode produzir microcistinas (MCs). O efeito citotóxico da MC tem sido descrito no fígado, pulmões, estômago e intestino. Mortes de seres humanos, de animais silvestres e domésticos têm sido associadas à exposição a MC. Esta pode ocorrer diretamente por ingestão, inalação, contato, inoculação intravenosa (hemodiálise) ou indiretamente, pelo consumo de animais, dentre os quais os peixes e moluscos, que podem ingerir as cianobactérias e suas toxinas. A mais tóxica e também mais comum das MCs é a microcistina-LR (MC-LR), cujo órgão alvo é o fígado. A MC chega ao fígado especificamente por transporte dos ácidos biliares e, uma vez no citoplasma, inibe as proteínas fosfatases 1 e 2A, induzindo ao aumento da fosforilação protéica. Esta reação tem duas conseqûências: destruição do citoesqueleto, causando efeitos citotóxicos e descontrole da divisão celular, levando à promoção tumoral. A exposição agu

Hepatotoxicity associated with microcystin / Hepatotoxicidade associada à microcistina

Urban and industrial discharges, intense agricultural exploitation and fisheries have been causing the eutrophication in both drinking and recreational waters. A frequent consequence of eutrophication in waters is the massive development of cyanobacteria. The occurrence of these blooms induces a severe problem, as Microcystis aeruginosa, the most widespread distributed cyanobacteria, can produce microcystins (MC). Toxic effects of MC have been described in liver, lungs, stomach, and intestine. Deaths in wildlife, livestock and human beings were also associated with MC exposition. MC exposition can occurs directly by ingestion, inhalation, contact, intravenous inoculation of contaminated water (hemodialysis) or indirectly, by the consumption of animals, as fish and mollusks, the majors ingestors of cyanobacteria and its toxins. The most toxic MC, an also the most common is microcystin-LR (MC-LR), that has the liver as the main target organ. Microcystin is taken up specifically into the liver by bile acid transporters and, after entering the cytoplasm, inhibit protein phosphatases 1 and 2A, which leads to the increase in protein phosphorylation. This effect has two main consequences: the destruction of cytoskeleton directly causing cytotoxic effects, and deregulation of cell division, leading to tumor-promoting activity. Acute exposition to MC induces severe intrahepatic hemorrha

Efluentes industriais e urbanos e a intensa exploração agrícola e de pescado têm levado à eutrofização de muitos mananciais de água, destinados ao consumo e às atividades recreacionais. A eutrofização das águas, freqüentemente, tem como conseqüência o desenvolvimento expressivo de cianobactérias. Estas florações induzem a sérios problemas, visto que a ocorrência de Microcystis aeruginosa, uma das cianobactérias mais difundidas, pode produzir microcistinas (MCs). O efeito citotóxico da MC tem sido descrito no fígado, pulmões, estômago e intestino. Mortes de seres humanos, de animais silvestres e domésticos têm sido associadas à exposição a MC. Esta pode ocorrer diretamente por ingestão, inalação, contato, inoculação intravenosa (hemodiálise) ou indiretamente, pelo consumo de animais, dentre os quais os peixes e moluscos, que podem ingerir as cianobactérias e suas toxinas. A mais tóxica e também mais comum das MCs é a microcistina-LR (MC-LR), cujo órgão alvo é o fígado. A MC chega ao fígado especificamente por transporte dos ácidos biliares e, uma vez no citoplasma, inibe as proteínas fosfatases 1 e 2A, induzindo ao aumento da fosforilação protéica. Esta reação tem duas conseqüências: destruição do citoesqueleto, causando efeitos citotóxicos e descontrole da divisão celular, levando à promoção tumoral. A exposição aguda à MC induz severa hemorragia intra-hepática, necrose e apopt

Toxicidade em camundongos devido à exposição prolongada a uma floração de cianobactérias contendo microcistinas: avaliações comportamental, hematológica, bioquímica e anatomopatológica / Toxicity in mice due to prolonged exposure to a cyanobacterial bloom containing microcystins: behavioral, hematological, biochemical and anatomopathological evaluations

As cianobactérias (algas azuis) são organismos procariontes, fotossintéticos, encontrados em praticamente todo tipo de habitat líquido e que podem formar grandes massas superficiais de cor verde intensa denominadas florações. O aparecimento de florações na superfície da água ocorre com uma incidência cada vez mais elevada e é considerado um risco potencial à saúde, devido à natureza tóxica de algumas algas. As cianobactérias podem produzir hepatotoxinas, neurotoxinas e dermatotoxinas, que já causaram reações adversas e morte de animais e seres humanos. A maioria das hepatotoxinas são microcistinas (MCs), uma família de toxinas produzidas por espécies de cianobactérias de água doce. A MC-LR foi a primeira a ser identificada é a mais estudada, pois tem sido associada à maioria dos casos de intoxicação envolvendo cianotoxinas. O presente trabalho visou avaliar a toxicidade causada em camundongos pela exposição prolongada ao extrato aquoso de uma floração de cianobactérias contendo microcistinas. Para tanto, camundongos machos Swiss receberam o extrato (300 ou 900 mg/kg) por quatro semanas, sendo avaliados: ganho de peso, consumo de ração e água, comportamento no campo aberto (CA) e no labirinto em cruz elevado (LCE), hemograma, bioquímica sanguínea, achados anatomopatológicos e presença de MC no plasma e fígado. Além disso, quantificou-se as MCs presentes no extrato e avaliou-se in vitro a morte celular de leucócitos expostos a esse extrato e à MC-LR. A análise química do extrato mostrou que este possui, além das MCs, anabaenopeptinas; dentre as MCs foram encontradas a MC-LR, MC-RR, MC-YR e [D-Asp3] MC-LR. A exposição prolongada ao extrato por 28 dias não alterou o ganho de peso e o peso relativo de órgãos. Na necropsia também não foram observadas alterações macroscópicas, contudo alterações histopatológicas foram encontradas no fígado e no rim. No fígado foram observados focos discretos de necrose lítica hepatocelular associada a infiltrado de neutrófilos e células mononucleares. No rim, o tratamento com o extrato aumentou significativamente a intensidade de nefrose tubular, sendo esta mais acentuada nos animais tratados com a maior dose. O tratamento prolongado com o extrato em ambas doses causou efeitos comportamentais dosedependentes no CA: aumento significativo da distância percorrida, do número de entradas, do tempo em movimento e do número de levantamentos no centro do CA, além de um aumento do tempo de permanência no centro, com conseqüente diminuição deste parâmetro na periferia. No entanto, nenhum dos parâmetros avaliados no LCE foi alterado. A exposição prolongada ao extrato causou alterações sutis em parâmetros hematológicos e bioquímicos, caracterizadas por tendência de diminuição de leucócitos e de aumento dos níveis de creatinina sérica nos animais tratados com a maior dose do extrato. O tratamento prolongado com o extrato permitiu a detecção de MC apenas no fígado. Tanto a MC-LR como o extrato promoveram aumento das taxas de apoptose em leucócitos de camundongo in vitro. Finalizando, observou-se que a exposição prolongada a uma floração contendo MCs causou baixa toxicidade e efeitos comportamentais dose-dependentes, mostrando que o comportamento animal é bastante útil para avaliar efeitos mais sutis de agentes tóxicos sobre o sistema nervoso central

Cyanobacteria (blue-green algae) are prokaryotic and photosynthetic organisms found in practically every type of liquid habitat and that can form great superficial masses of intense green color called blooms. The development of algal blooms on water surface occurs with a higher incidence and is recognised as being a potential health hazard due to the toxic nature of some algae. Cyanobacteria can produce hepatotoxins, neurotoxins and dermatotoxins, which already caused adverse effects and death of animals and human beings. Most of the hepatotoxins are microcystins (MCs), a family of toxins produced by species of freshwater cyanobacteria. MC-LR was the first to be identified and is the more studied, therefore, it has been associated to most of the cases of intoxication involving cyanotoxins. The present study was performed to evaluate the toxicity in mice after prolonged exposure to a cyanobacteria bloom extract containing microcystins. For this, male Swiss mice were treated with the extract (300 or 900 mg/kg) for four weeks and was evaluated: weight gain, food and water consumption, behavior in the open field (OF) and in the elevated plus-maze (EPM), blood count, clinical biochemistry, anatomopathological findings and presence of MC in the plasma and liver. Besides, the MCs present in the extract were quantified and the cellular death of leukocytes exposed in vitro to MC and to the extract was evaluated. The chemical analysis of the extract showed that it presented, besides MCs, anabaenopeptins; the MCs found were MC-LR, MC-RR, MC-YR and [D-Asp3] MC-LR. The prolonged exposure to the extract for 28 days did not alter the weight gain and the relative weight of organs. At necropsy, macroscopic alterations were not observed, however, histopathological alterations were found in the liver and in the kidney. In the liver, discreet necrosis focuses associated with neutrophils and mononuclear cells were found. In the kidney, the treatment with the extract increased significantly the intensity of tubular nephrosis, and this was more accentuated in the animals treated with the largest dose. The prolonged treatment with both doses caused dose-dependent behavioral effects in the OF: significant increase of moved distance, number of entries, time moving and rearing in the center of the OF, besides an increase of time spent in the center, with consequent decrease of this parameter in the periphery. None of the parameters evaluated in EPM were altered. The prolonged exposure to the extract caused subtle alterations in hematological and biochemical parameters, characterized by tendency for leukocyte decrease and increase of the levels of serum creatinine in the animals treated with the largest dose of the extract. The prolonged treatment with the extract allowed the detection of MC just in the liver. As much MC-LR as the extract promoted increase of the apoptosis levels in mouse leukocytes in vitro. Concluding, it was observed that the prolonged exposure to the cyanobacteria bloom containing MCs caused low toxicity and dose-dependent behavioral effects, showing that animal behavior is useful to evaluate subtler effects of toxic agents on the central nervous system