Importancia biológica de la microcistina en aguas de riego y alternativas en su detección y degradación / Biological importance of microcistin in irrigation and alternative waters in its detection and degradation

Introducción: Las cianobacterias son microrganismos fotosintéticos, con capacidad de sintetizar una gran diversidad de metabolitos secundarios de interés para la industria, pero también han llamado la atención en las últimas décadas las toxinas denominas cianotoxinas, metabolitos que causan distintas alternaciones fisiológicas hasta llegar ocasionar la muerte de diferentes especies. Metodología: La determinación del estado de arte para el tema de cianobacterias se basó en una búsqueda bibliográfica en la base de datos especializada como Elservier, Springer, Google académico y MDPI basadas en palabras clave en español e inglés "microcistinas", "degradación de MC" y "cuantificación y detección de MC". Resultados: En la presente revisión considera dos áreas de caracterización de la microcistinas (MCs) las propiedades fisicoquímicas y propiedades biológicas, para entender su comportamiento e importancia tóxica en los sembradíos agrícolas y en la salud humana. Además de comprender alternativas para su degradación, por métodos fisicoquímicos como fotocatálisis y la gradación biológica por bacterias. Finalmente se mencionará algunos métodos actuales y en desarrollo, para la detección y cuantificación de estas MCs en ambientes acuáticos. Conclusiones: Las MCs tienen el potencial contaminar fuentes de agua como ríos y lagunas, causando daños a la salud humana y a las plantas agrícolas, tienen la capacidad de tolerar distintos cambios drásticos en factores fisicoquímicos y biológicos. Entre las alternativas reportadas la degradación bacteriana promete ser la más confiable. Finalmente, entre los distintos métodos para la detección de MCs, entre los métodos más aplicados son los inmunoensayos, debido a su versatilidad y estabilidad del método.

Introduction: Cyanobacteria are photosynthetic microorganisms, with the capacity to synthesize a great diversity of secondary metabolites of interest to the industry, but toxins called cyanotoxins have also attracted attention in recent decades, metabolites that cause different physiological alterations until they cause the death of different species. Methodology: The determination of the state of the art for the subject of cyanobacteria was based on a bibliographic search in specialized databases such as Elservier, Springer, Google Scholar and MDPI based on keywords in Spanish and English "microcystins", "MC degradation " and "quantification and detection of MC". Results: In the present review, two areas of characterization of microcystins are considered: the physicochemical properties and biological properties of microcystins (MC), to understand their behavior and toxic importance in agricultural crops and in human health.In addition to understanding alternatives for their degradation, by physicochemical methods such as photocatalysis and biological grading by bacteria.Finally, some current and developing methods will be mentioned for the detection and quantification of these MCs in aquatic environments. Conclusions: MCs have the potential to contaminate water sources such as rivers and lagoon, causing damage to human health and agricultural plants, they have the ability to tolerate different drastic changes in physicochemical and biological factors. Among the reported alternatives, bacterial degradation promises to be the most reliable. Finally, among the different methods for the detection of MCs, among the most applied methods are immunoassays, due to their versatility and stability of the method.

The mechanism of microcystin leucine-arginine (MC-LR)-induced injury of Sertoli cell immune response and biological behavior / 细胞与分子免疫学杂志

Microcystin-leucine arginine (MC-LR), a potentially carcinogenic toxin, is produced by Cyanobacteria such as Microcystis and Ananabacteria during water bloom. Increasing evidence demonstrated that MC-LR induces male reproductive toxicity, mainly by inducing germ cell apoptosis, destroying cell cytoskeleton, interfering with DNA damage repair pathway, and damaging blood-testicular barrier (BTB), which eventually lead to male sterility. Testicular Sertoli cells are the somatic cells that directly contact with spermatogenic cells in seminiferous tubules. They not only regulate immune response to maintain testicular immune homeostasis by secreting a variety of cytokines and immunosuppressive factors, but also provide the protective effects of spermatogenic cells by forming BTB. MC-LR induces inflammation and apoptosis of Sertoli cells, and destroys the integrity of the BTB, and then causes spermatogenesis dysfunction.

Phytoplankton scenario and microcystin in water during extreme drought in semiarid tropical water supplies, Northeastern Brazil / Cenário de fitoplanctônico e microcistinas na água durante extrema estiagem em reservatórios tropical semiárido de abastecimento de água, nordeste do Brasil

Abstract The objective of this study was to characterize the limnological, microystin and phytoplankton community of five tropical eutrophic reservoirs located in the Brazilian northeastern semi-arid region, used for domestic use at the time of extreme drought and reduction of water volume. The study was conducted in July and August 2015, and an integrated sample of the water column was collected at three points near the dam in each reservoir. Analysis of limnological parameters, identification and quantification of phytoplankton, with emphasis on cyanobacteria were performed, as well as detection of microcystin by means of immunoassay (ELISA). The reservoirs presented ~ 90% water volume reduction. High turbidity and concentrations of nitrogen and phosphorus, as well as high cyanobacterial densities, revealed an increase in the eutrophic state for hypereutrophy. The total biovolume of phytoplankton and cyanobacterial density is high, plus an average increase in relation to previous studies of 350% and 150%, respectively. The density of cyanobacteria and microcystin concentration presented values ​​above acceptable levels for drinking water according to Brazilian legislation. A phytoplankton community was represented by 17 functional groups, including potentially toxic cyanobacteria species such as Planktothrix agardhii (S1), Microcystis aeruginosa (M), Anabaena planktonica e Anabaena spp. (H1), Cylindrospermopsis raciborskii (Sn). Our results confirm that conditions of extreme drought and reduction of the volume of the reservoirs influence the composition, biovolume of phytoplankton and water quality, but not the increase of total microcystin in the analysed, although above 1μg-1 registered a significant decrease of water quality in used for human consumption.

Resumo O objetivo deste trabalho foi caracterizar a comunidade limnológica, microistalina e fitoplanctônica de cinco reservatórios eutróficos tropicais localizados no semi-árido nordestino brasileiro, utilizados para uso doméstico no período de seca extrema e redução do volume de água. O estudo foi realizado em julho e agosto de 2015, e uma amostra integrada da coluna de água foi coletada em três pontos próximos à barragem em cada reservatório. Análises de parâmetros limnológicos, identificação e quantificação do fitoplâncton, com ênfase em cianobactérias, foram realizadas, assim como a detecção de microcistina por meio de imunoensaio (ELISA). Os reservatórios apresentaram ~ 90% de redução do volume de água. A alta turbidez e as concentrações de nitrogênio e fósforo, bem como as altas densidades de cianobactérias, revelaram um aumento no estado eutrófico da hipereutrofia. O biovolume total de fitoplâncton e densidade de cianobactérias é alto, além de um aumento médio em relação a estudos anteriores de 350% e 150%, respectivamente. A densidade de cianobactérias e a concentração de microcistina apresentaram valores acima dos níveis aceitáveis ​​para água de consumo, de acordo com a legislação brasileira. Uma comunidade fitoplanctônica foi representada por 17 grupos funcionais, incluindo espécies de cianobactérias potencialmente tóxicas, como Planktothrix agardhii (S1), Microcystis aeruginosa (M), Anabaena planktonica e Anabaena spp. (H1), Cylindrospermopsis raciborskii (Sn). Nossos resultados confirmam que condições de seca extrema e redução do volume dos reservatórios influenciam a composição, o biovolume de fitoplâncton e a qualidade da água, mas não o aumento do total de microcistina no analisado, embora acima de 1μg-1 tenha registrado uma diminuição significativa da qualidade da água usado para consumo humano.

Can microcystins affect zooplankton structure community in tropical eutrophic reservoirs? / Microcistinas podem afetar a estrutura da comunidade zooplanctônica em reservatórios eutróficos tropicais?

Abstract The aim of our study was to assess whether cyanotoxins (microcystins) can affect the composition of the zooplankton community, leading to domination of microzooplankton forms (protozoans and rotifers). Temporal variations in concentrations of microcystins and zooplankton biomass were analyzed in three eutrophic reservoirs in the semi-arid northeast region of Brazil. The concentration of microcystins in water proved to be correlated with the cyanobacterial biovolume, indicating the contributions from colonial forms such as Microcystis in the production of cyanotoxins. At the community level, the total biomass of zooplankton was not correlated with the concentration of microcystin (r2 = 0.00; P > 0.001), but in a population-level analysis, the biomass of rotifers and cladocerans showed a weak positive correlation. Cyclopoid copepods, which are considered to be relatively inefficient in ingesting cyanobacteria, were negatively correlated (r2 = – 0.01; P > 0.01) with the concentration of cyanotoxins. Surprisingly, the biomass of calanoid copepods was positively correlated with the microcystin concentration (r2 = 0.44; P > 0.001). The results indicate that allelopathic control mechanisms (negative effects of microcystin on zooplankton biomass) do not seem to substantially affect the composition of mesozooplankton, which showed a constant and high biomass compared to the microzooplankton (rotifers). These results may be important to better understand the trophic interactions between zooplankton and cyanobacteria and the potential effects of allelopathic compounds on zooplankton.

Resumo Com o objetivo de avaliar se as cianotoxinas (microcistinas) podem afetar a composição da comunidade zooplanctônica, levando à dominância de formas microzooplanctônicas (protozoários e rotiferos), as variações nas concentrações de microcistina e a biomassa do zooplâncton foram analisadas em três reservatórios eutróficos na região semi-árida do nordeste brasileiro. A concentração de microcistinas na água esteve correlacionada com o biovolume de cianobactérias, indicando a contribuição de formas coloniais como Microcystis na produção de cianotoxinas. A nível de comunidade, a biomassa total do zooplâncton não apresentou correlacão com a concentração de microcistina (r2 = 0.00; P > 0.001), mas em uma análise a nível de populações, a biomassa de rotíferos e cladóceros apresentou uma fraca correlação positiva. Copépodos Cyclopoida, os quais são considerados relativamente ineficientes na ingestão de cianobactérias, estiveram negativamente correlacionados com a concentração de microcistinas (r2 = - 0.01; P > 0.01). Surpreendentemente, a biomassa de copépodos Calanoida foi positivamente correlacionada com a concentração de cianotoxinas (r2 = 0.44; P > 0.001). Os resultados indicam que mecanismos de controle alelopáticos (efeitos negativos da microcistina sobre o zooplâncton) parecem não afetar substancialmente a composição do mesozooplâncton, que apresentou uma alta e constante biomassa, quando comparada à biomassa do microzooplâncton (rotíferos). Esses resultados podem ser importantes para um melhor entendimento das interações tróficas entre o zooplâncton e cianobactérias, e do efeito potencial de compostos alelopáticos sobre o zooplâncton.

Degradation of cyanotoxins (microcystin) in drinking water using photoelectrooxidation / Degradação de cianotoxinas (microcistinas) em água potável aplicando fotoeletrooxidação

The discharge of sewage and industrial effluents containing high concentrations of pollutants in water bodies increases eutrophication. Cyanobacteria, some of the organisms whose growth is promoted by high nutrient concentrations, are resistant and produce several types of toxins, known as cyanotoxins, highly harmful to human beings. Current water treatment systems for the public water supply are not efficient in degradation of toxins. Advanced oxidation processes (AOP) have been tested for the removal of cyanotoxins, and the results have been positive. This study examines the application of photoelectrooxidation in the degradation of cyanotoxins (microcystins). The performance of the oxidative processes involved was evaluated separately: Photocatalysis, Electrolysis and Photoelectrooxidation. Results showed that the electrical current and UV radiation were directly associated with toxin degradation. The PEO system is efficient in removing cyanotoxins, and the reduction rate reached 99%. The final concentration of toxin was less than 1 µg/L of microcystin in the treated solution.

A descarga de esgotos e efluentes industriais contendo altas concentrações de poluentes nos corpos d'água aumenta a eutrofização. As cianobactérias, são organismos cujo crescimento é promovido por concentrações elevadas de nutrientes, são resistentes e produzem vários tipos de toxinas conhecidas, como cianotoxinas, altamente prejudiciais para os seres humanos. Os sistemas atuais de tratamento de água para o abastecimento público de água não são eficientes na degradação destas toxinas. Processos oxidativos avançados (POA) foram testados para a remoção de cianotoxinas, e os resultados têm sido positivos. Este estudo avalia o processo de fotoeletrooxidação (FEO) na degradação de cianotoxinas (microcistinas). Foi avaliado o desempenho dos processos envolvidos separadamente: fotocatalisis, eletrólise e fotoeletrooxidação. Os resultados mostram que a potencia da radiação UV e da corrente elétrica estão diretamente associados com a degradação de toxinas. O sistema de FEO é eficiente na remoção de cianotoxinas e a redução foi de 99%. A concentração final de toxina foi inferior a 1 g / L de microcistina na solução tratada.

Antimycobacterial and cytotoxicity activity of microcystins

Background: The present work aimed to evaluate the antimycobacterial activity and cytotoxicity of Microcystis aeruginosa toxins, the MC-LR variant and purified extract of [D-Leu1] microcystin-LR. Methods: The antimicrobial activity of M. aeruginosa extract and microcystin was evaluated by resazurin microtiter assay against Mycobacterium tuberculosis, M. terrae, M. chelonae and M. kansasii. The cytotoxicity assay was performed by trypan blue exclusion against the HTC cell line. Results: Antimicrobial activity was observed in the hexanic extract of M. aeruginosa (RST 9501 strain) against M. tuberculosis, including sensitive and resistant strains with minimal inhibitory concentrations (MIC) between 1.93 μM and 0.06 μM. The high activity of M. aeruginosa hexanic extract could be attributed to the major presence of the toxins MC-LR and [D-Leu1] MC-LR that showed activity at MIC between 53 and 0.42 μM against tested mycobacterial strains. Even at the highest concentration tested, no toxicity of M. aeruginosa extracts was identified against HTC cells. Conclusions: These preliminary results suggest that [D-Leu1] MC-LR is a promising candidate for the development of a new antimycobacterial agent.

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 L−1 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 μg.L−1 to < 0.05 μg.L−1. Besides, an increase in the bacterial count–from 71 × 105 bacteria.mL−1 to 117 × 105 bacteria.mL−1–was 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 ...

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.

Cylindrospermopsis raciborskii (Cyanobacteria, Nostocales) productora de microcistinas en Corrientes, Argentina / Cylindrospermopsis raciborskii (Cyanobacteria, Nostocales) producing microcystins in Corrientes, Argentina

Cylindrospermopsis raciborskii (Woloszyn´ ska) Seenayya et Subba Raju 1972 es una especie capaz de generar diversas toxinas que impactan negativamente sobre la calidad del agua destinada al consumo humano así como a otros usos de la misma. Cepas del hemisferio norte producen la hepatotoxina cilindrospermopsina, mientras que las sudamericanas generan las toxinas neurotóxicas saxitoxinas. No se ha reportado hasta el momento la producción de microcistinas por parte de esta especie El objetivo del presente estudio fue analizar la presencia de microcistinas en el agua del Arroyo Yatay en Corrientes, Argentina, y de cilindrospermopsina, saxitoxinas y microcistinas en cepas de C. raciborskii aisladas del mismo, mediante el ensayo por Inmuno Absorción Ligado a Enzimas (ELISA). Los resultados dieron negativos para microcistinas en el agua cruda, así como de cilindrospermopsina y saxitoxinas en el cultivo algal. Sin embargo, en el agua del cultivo se detectaron concentraciones de 2,34-2,50 µg. L-1 de microcistina-LR, confirmándose posteriormente su presencia mediante su análisis por Cromatografía Líquida de Alta Resolución (HPLC). En estudios posteriores se deberán realizar análisis moleculares a los fines de establecer la caracterización genética de la cepa en estudio e investigar su relación filogenética con otras cepas provenientes de diversos ambientes a nivel mundial.

Cylindrospermopsis raciborskii (Woloszyn´ ska) Seenayya et SubbaRaju 1972 is a species capable of generating toxins that negatively impact on drinking water quality as well as other water uses. Northern strains can produce the hepatotoxin cylindrospermopsin, while southern strains can produce the neurotoxin saxitoxins. Microcystins production by this species has not been reported to date.The aim of this study was to analyze the presence of microcystins in raw water of Steam Yatay in Corrientes, Argentina, and cylindrospermopsin, saxitoxins and microcystins in strains of C. raciborskii isolated from that stream, by means of Enzyme-Linked Immuno Sorbent Assay (ELISA).Results were negative for microcystins in raw water, as well as for cylindrospermopsin and saxitoxins in algal culture. Microcystins were detected in algal culture by ELISA test, at concentrations of 2.34-2.50 µg. L-1 of microcystin-LR, and subsequent confirmation by means of High Performance Liquid Chromatography (HPLC). Molecular studies should be carried out in the future to establish the genetic characterization of the strain under study and investigate their phylogenetic relationship with other strains from diverse environments.

Phytotoxicity associated to microcystins: a review / Fitotocixidade associada às microcistinas: uma revisão

Microcystins (MC) are the most studied toxins of cyanobacteria since they are widely distributed and account for several cases of human and animal poisoning, being potent inhibitors of the serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A). The phosphatases PP1 and PP2A are also present in plants, which may also suffer adverse effects due to the inhibition of these enzymes. In aquatic plants, biomass reduction is usually observed after absorption of cyanotoxins, which can bioaccumulate in its tissues. In terrestrial plants, the effects caused by microcystins vary from inhibition to stimulation as the individuals develop from seedling to adult, and include reduction of protein phosphatases 1 and 2A, oxidative stress, decreased photosynthetic activity and even cell apoptosis, as well as bioaccumulation in plant tissues. Thus, the irrigation of crop plants by water contaminated with microcystins is not only an economic problem but becomes a public health issue because of the possibility of food contamination, and this route of exposure requires careful monitoring by the responsible authorities.

Microcistinas (MC) são as toxinas de cianobactérias mais estudadas, uma vez que são amplamente distribuídas e responsáveis por vários casos de intoxicação humana e animal. São potentes inibidoras das proteínas fosfatases serina/treonina 1 (PP1) e 2A (PP2A). As fosfatases PP1 e PP2A também estão presentes em plantas, as quais podem sofrer efeitos adversos devido à inibição dessas enzimas. Em plantas aquáticas, a redução da biomassa é geralmente observada após absorção de cianotoxinas que podem bioacumular em seus tecidos. Em plantas terrestres, os efeitos causados pelas microcistinas variam de inibição ao estímulo, como no desenvolvimento de plântulas ao estádio adulto, e incluem a redução de proteínas fosfatases 1 e 2A, estresse oxidativo, diminuição da atividade fotossintética e até mesmo apoptose celular, bem como a bioacumulação em tecidos de plantas. Assim, a irrigação de plantas cultivadas com água contaminada com microcistina não é apenas um problema econômico, mas torna-se um problema de saúde pública, devido à possibilidade de contaminação dos alimento, sendo uma via de exposição que requer um monitoramento cuidadoso por parte das autoridades responsáveis.

Estudo da presença da toxina microcistina na água de reservatório de Mundaú (Garanhuns-PE) pelas metodologias ELISA e CLAE / Study on the occurrence of microcystin toxin in water reservoir of Mundaú (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 ELISAe 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...

Isolation and cultivation of a wild microcytin-RR-producing cyanobacterium and verification of its toxin by high performance liquid chromatography and acute oral toxicity / 中华预防医学杂志

<p><b>OBJECTIVE</b>Attempting to isolate and cultivate the microcytin-RR-producing cyanobacteria from natural blooms as well as to further investigate some characteristics of their growth and metabolite toxicity.</p><p><b>METHODS</b>Capillary-pipette method was used to isolate wild Microcystis strains collected from eutrophicated lakes. The isolated strains were cultured in BG11 media at (25 ± 1) °C, under 2 000 lx illumination of fluorescent light with a light-dark rhythm of 12-12 h. The growth curve was observed by measuring optical density of culture suspension, toxin-related genes and the metabolite toxins were identified separately by PCR and HPLC, and its acute toxicity was carried out by orally administered toxins to Kunming (KM) mice.</p><p><b>RESULTS</b>One of five toxigenic strains from 198 collected samples was confirmed to be a MC-RR producing blue-green alga by existing two specific toxin-synthesized enzyme genes and showing specific chromatographic peak of the toxin compared with standard MC-RR through both PCR and HPLC methods. The toxic strain was classified as Microcystin aeruginosa by morphologic and phylogenetic tree analysis. The growth length of the strain lasted nearly 81 days with 55-60 days' exponential phase and the maximal concentration of 5.52 × 10⁷ cell/ml. The LD50 of the MC-RR to the KM mice ranged from 10.75 mg/kg to 13.45 mg/kg of body weight. As a result of the acute toxicity, the enzymatic indexes in serum such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) were significantly higher than those in the control group. The levels of ALT, AST, ALP and LDH in the treated group at 45 min were (157.08 ± 20.38), (333.00 ± 68.53), (392.70 ± 89.59) and (1 071.13 ± 160.22) U/L respectively, and at 4 h were (514.68 ± 156.87), (593.15 ± 40.41), (618.55 ± 208.76) and (2 281.72 ± 866.67) U/L respectively, and meanwhile the values of ALT, AST, ALP and LDH in the control group were (40.30 ± 4.89), (142.70 ± 26.59), (56.90 ± 11.89) and (509.50 ± 94.75) U/L separately (t values at 45 min were -11.20, -5.77, -7.38, -6.60 respectively, and at 4 h were -6.04, -20.21, -5.35, -4.07 respectively, P values were all <0.01). The liver coefficient in the treated group at 45 min and 4 h were 6.855 ± 0.225 and 8.409 ± 0.276, significantly higher than that (5.784 ± 0.286) in the control group (t values were -3.96 and -12.22, P values were both <0.01). The histopathological changes of liver were hyperemia obviously.</p><p><b>CONCLUSION</b>Isolated from the bloom waters, a strain of Microcystis aeruginosa is obtained with characteristics of longer growth duration, positive microcystin synthetase genes, and dominant production of MC-RR. The LD50 of the extracted MC-RR administered by oral route to mice is (12.10 ± 1.35) mg/kg of body weight, and liver is the target organ of MC-RR. The existence and potential risk of MC-RR in China cannot be ignored.</p>

Cyanobacterial blooms and their toxicity in Vojvodina lakes, Serbia

The presence of cyanobacteria [blue-green algae] is a significant indicator of water quality, having in mind that several genera have the quality of producing cyanotoxins, which are harmful to animals, plants and humans. Seven lakes in Vojvodina [Republic of Serbia] were examined in May and September 2007 for cyanobacterial presence and cyanotoxins content, Chl a concentration and index of phosphatase activity as the indicators of water quality. In the spring period, cyanobacteria species were not dominant in only 2 of 14 water samples. Microcystins were detected in all of the lakes examined. The concentrations of microcystins ranged from 6.66 Micro g/L [Provala Lake] to 199.9 and 238 Micro g/L [Palic and Ludas Lake respectively]. The investigations conducted on 7 water ecosystems in Vojvodina, regarding the basic parameters of eutrophication, Chl a concentration and phosphatase activity index, have rendered the examined ecosystems immensely endangered due to significant water quality deterioration. It was determined that the intensification of eutrophication processes has been linked to the increased presence and abundance of cyanobacteria as well as significant toxin production

The influence of microcystin-LR on monocytes and lymphocytes of mice / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the influence of microcystin-LR (MC-LR) on monocytes and lymphocytes in blood of mice and to find a sensitive index of toxic effects.</p><p><b>METHODS</b>Specific pathogen free Kunming male mice, aging 1 month-old,were randomly divided into 5 groups by weights, 7 mice for each group. The mice in 5 groups were exposed to MC-LR through intraperitoneal injection at 0, 3.125,6.250, 12.500 and 25.000 µg/kg respectively for 7 days. Then cytokine levels in the serum were measured by radioimmunoassay, DNA-protein crosslinks (DPC) was measured by the SDS/KCl precipitation technique, and the phagocytosis and ROS of leukocytes were detected by flow cytometry.</p><p><b>RESULTS</b>The levels of interleukin 6 in the 6.250, 12.500 and 25.000 µg·kg(-1)·d(-1) dose groups were (346.837 ± 25.536), (360.847 ± 37.886) and (434.245 ± 35.858)pg/ml respectively, which were significantly higher than those in the control group which the value was (232.775 ± 32.816) pg/ml (t values were -7.258, -6.760 and -10.966 respectively, P values were all < 0.05).While the level of tumor necrosis factor-alpha was(10.782 ± 0.966) fmol/ml in 25 µg·kg(-1)·d(-1) dose group was statistically lower than it in the control group which the value was (16.878 ± 3.378) fmol/ml (t value was 4.591, P < 0.05). The DPC levels of lymphocytes in 6.250, 12.500 µg·kg(-1)·d(-1) dose group were (242.576 ± 7.545),(241.472 ± 2.793) ng/ml,higher than it in the control group while the value was (228.657 ± 4.130) ng/ml (t value was -4.282, -6.801, P values were all <0.05). The fluorescence intensity of DCF in lymphocytes in the 4 treated groups were separately 3299.37 ± 120.54, 3281.38 ± 58.34, 3308.06 ± 136.12 and 3346.92 ± 108.69, all significantly lower than 3770.81 ± 131.39 in the control group (t values were 6.995, 9.007, 6.472 and 6.577 respectively, and P values were all <0.05). The fluorescence intensity of DCF in monocytes in the 4 treated groups (3271.51 ± 140.79, 3270.05 ± 117.92, 3326.90 ± 114.39 and 3292.49 ± 145.97 respectively) were also significantly lower than the value in the control group was 3841.72 ± 130.92 (t values were 7.847, 8.584, 7.835 and 7.411 respectively, P values were all <0.05). There was no significant difference in other index among the four experiment groups and the control group.</p><p><b>CONCLUSION</b>The MC-LR administered via intraperitoneal injection to mice induced the alterations of some cytokines of monocytes and lymphocytes in blood. By comparison, the ROS of leukocyte was the most sensitive index.</p>

Presença da toxina microcistina em água, impactos na saúde pública e medidas de controle / Microcystin toxin in the presence of water, public health impact and control measures

As cianobactérias constituem um grupo de micro-organismos procariontes encontrado em ambientes aquáticos, os quais vêm sendo pesquisado devido à capacidade de produzir toxinas que causam grandes impactos à saúde pública e ambiental. A ocorrência de florações desses micro-organismos em reservatórios de água utilizada para abastecimento público tem sido frequente e prejudicado os usos múltiplos da água. Algumas cepas produzem florações nocivas liberando compostos neurotóxicos, hepatotóxicos ou irritantes à pele de seres humanos e animais, podendo ocasionar intoxicação e morte. A literatura reporta a ocorrência de um acidente em Caruaru, Pernambuco, onde faleceram 65 pacientes que faziam hemodiálise, devido à presença de microcistina-LR na água que consumiram. Esse estudo objetivou realizar uma revisão sobre a presença da toxina microcistina em água, os impactos e medidas de controle. Foram relatados os principais métodos utilizados para a remoção e quantificação da microcistina em água, bem como sua relação com a saúde humana.

Cyanobacteria are a group of prokaryotic microorganisms found in aquatic environments, which have been researched because of the ability to produce toxins that cause major public health impacts and environmental. The occurrence of seaweed buddings in reservoirs used for public supplying has been frequent, and has affected the multiple uses of the drinking water. Some strains of seaweed, in special the cyanobacterial, can produce toxins as the microcystins, which are responsible for the intoxication and death of animals and humans.The literature reports the occurrence of an accident in Caruaru, Pernambuco, where he died 65 hemodialysis patients who were due to the presence of microcystin-LR in water. This study aimed to perform a review of the presence of the toxin microcystin in water, impacts and control measures. Reported were the main methods used for the removal and quantification of microcystins in water and its relation to human health.

Identificação do gene mcyA em florações naturais de Radiocystis fernandoi, em um tributário do reservatório de Rosana, Brasil / Identification of the mcyA gene in natural blooms of Radiocystis fernandoi from a tributary of the Rosana reservoir, Brazil

As cianobactérias são conhecidamente produtoras de toxinas. Dentro de uma mesma espécie, podemos encontrar variedades tóxicas e não-tóxicas, impossíveis de diferenciação apenas pela morfologia. A principal toxina produzida pelas cianobactérias é a microcistina. Esta proteína é biossintetizada por um grupo de genes conhecidos como mcy. A detecção destes genes a partir de PCR permite a distinção das variedades tóxicas e não-tóxicas. Desse modo, o objetivo desse trabalho foi investigar a ocorrência de florações produtoras de toxinas em um rio tributário do reservatório de Rosana, via amplificação do gene mcyA por PCR. Foram coletadas duas amostras de água da subsuperfície. As duas amostras coletadas no rio do Corvo foram dominadas pela espécie Radiocystis fernandoi e apresentaram resultados positivos para a presença do gene mcyA, confirmando o potencial tóxico dessa espécie. Os resultados representam alerta sobre a qualidade da água do rio do Corvo. A técnica PCR foi eficiente para a rápida detecção de cianobactérias produtoras de toxinas, inclusive podendo ser utilizada antes mesmo do agravamento das condições ambientais pela produção de toxinas, além de apresentar baixo custo.

Cyanobacterias are known as toxin producers. Within the same species, toxic and non-toxic varieties can be found and it is impossible to differentiate them only by morphology. The most important toxin produced by cyanobacteria is microcystin. This protein is synthesized by a cluster of genes known as mcy. The detection of these genes by PCR allows the differentiation of the producing toxin strain from the non-producing toxin strain. Thus, the goal of this work was to investigate the occurrence of toxigenic blooms of cyanobacteria in the Corvo River through PCR amplification of mcyA gene. For this, two samples of blooms of cyanobacteria were collected in Corvo River. Both samples were dominated by Radiocystis fernandoi and presented positive results for the presence of the mcyA gene, which may confirm the potential toxigenicity for that species. These results are an alert about water quality in the Corvo River. Here we demonstrate that amplification of the mcyA gene by PCR is a fast, cheap and efficient method for the detection of toxin- producing cyanobacteria.

The effects of microcystin-LR on the mRNA expression levels of base excision repair genes and genes related to apoptosis / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the effects of microcystin-LR (MCLR) on the expression of base excision repair genes and genes related to apoptosis.</p><p><b>METHODS</b>The BRL-3A cells were exposed to different concentrations of MCLR for various periods of time and the cell viability was measured by MTT. The mRNA expression was determined with the quantitative real-time polymerase chain reaction (QRT-PCR).</p><p><b>RESULTS</b>The viability of BRL-3A cells significantly reduced in a concentration- and time-dependent manner. In 30 µg/ml group, the mRNA expression level (1.327 ± 0.028) of p53 increased significantly at 24 h after exposure, as compared with the other groups (1.005 ± 0.117, 0.862 ± 0.154, 1.028 ± 0.056 and 1.015 ± 0.091) (P < 0.05). The mRNA expression levels (5.080 ± 0.729, 5.820 ± 0.373, 6.018 ± 0.359 and 6.183 ± 0.515) of Bax in all exposure groups were significantly higher than that (1.024 ± 0.277) in control group at 24 h after exposure. However, the Bax mRNA expression level (0.604 ± 0.146) in the 30 µg/ml group at 72 h after exposure was significantly lower than those (1.004 ± 0.107, 0.811 ± 0.142, 0.855 ± 0.101 and 0.814 ± 0.056) in other groups (P < 0.05). When compared with control group (1.006 ± 0.132) and 1 µg/ml group (1.034 ± 0.241), the mRNA expression level (0.488 ± 0.147) of PARP1 in 30 µg/ml group at 48 h after exposure decreased significantly (P < 0.05). Furthermore, the mRNA expression levels (0.594 ± 0.180, 0.491 ± 0.015 and 0.305 ± 0.091) of JWA, XRCC1 and PARP1 in 30 µg/ml group at 72 h after exposure decreased significantly, as compared with the other groups (P < 0.05).</p><p><b>CONCLUSION</b>The induction of gene expression is a transient phenomenon that occurred at different times of exposure for different genes. Inhibition of MCLR on the base excision repair gene expression may play important role in the course of MCLR promoting liver tumor.</p>

Investigação da biossíntese de toxinas produzidas por cepas de cianobactérias / Investigation on the cyanobacterial strains toxins biossinthesys

A demanda crescente de água doce de boa qualidade são problemas atuais e mundiais, além do descaso com os dejetos lançados nos ambientes aquáticos que comprometem a qualidade dos recursos hídricos. Um dos parâmetros que atesta a potabilidade da água é a presença de cianobactérias e cianotoxinas. Cianobactérias são microrganismos procariontes aeróbicos fotoautróficos que sintetizam as cianotoxinas. Estes compostos podem ser classificados de acordo com seus mecanismos de ação em hepatotóxicos, neurotóxicos e dermatotóxicos. Por sua diversidade, representam diferentes riscos não só ao ecossistema e a outros organismos dos ambientes aquáticos, como também aos seres humanos. Esse projeto visou o isolamento e cultivo de cepas de cianobactérias produtoras de toxinas para a investigação da biossíntese desses compostos. Com este intuito, foram realizadas coletas de água em três reservatórios no estado de São Paulo e um no Paraná. Cepas de cianobactérais foram isoladas, identificadas e analisadas quanto à produção de toxinas. Uma cepa de Microcystis aeruginosa (LTPNA 02) produtora de microcistinas (MC-LR, MC-RR, MC-YR, MC-LF, MC-LW e desm-MC-LR e desm- MC-RR) foi escolhida para ser estudada frente diferentes condições de cultivo e ter o seu crescimento, produção de toxinas e expressão gênica estudados. Foram utilizados os meios de cultura já referidos na literatura: ASM-1 (N:P=1, 10 e 20), MLA (N:P=10), Bold 3N (N:P=16) e BG-11 (N:P=10 e 100). Para acompanhar o crescimento, dois métodos foram utilizados: contagem de células e espectrofotometria. As toxinas foram quantificadas por LC-MS - QTrap. A análise da expressão gênica foi realizada por reação de PCR em tempo real pelo método de quantificação relativa ΔΔCt. Foi observada diferença no crescimento da cepa estudada nos diferentes meios de cultivo empregados. A contagem das células permitiu a identificação das fases logarítmica e total de crescimento. Durante a fase logarítmica, três experimentos demonstraram...

There is a great concern these days about potable and good quality water due to the increase of the population needs and also to the arising problems with contamination caused by anthropogenic sources. The presence of cyanobacteria and cyanotoxins are some parameters that attest water potability. Cyanobacteria are prokaryotic aerobic photoautotrophic microorganisms that may synthesize cyanotoxins. These compounds can be classified as hepatotoxic, neurotoxic and dermatotoxic according to their action mechanisms. Because of their diversity, they may represent different risks, not only to their ecosystem and other aquatic living organisms, but also to human beings. The aim of this project was the isolation and cultivation of cyanotoxin-producing cyanobacteria for further investigation on the biosynthesis of these compounds. Water samples from three different reservoirs in São Paulo state and one in Paraná state were collected in order to isolate cyanobacteria strains and accomplish their identification and to evaluate the toxin production. The Microcystis aeruginosa (LTPNA 02) microcystin producer strain (MCLR, MC-RR, MC-YR, MC-LF, MC-LW, desm-MC-LR and desm-MC-RR) was chosen to be grown in different cultivation conditions and later analyzed for its growth rate, toxin production and gene expression. All culture media used in this research were chosen according to the literature: ASM-1 (N:P=1, 10 and 20), MLA (N:P=10), Bold 3N (N:P=16) and BG-11 (N:P=10 and 100). To evaluate growth rate, two techniques were used: cell counting and absorbance determination in two different wavelengths (680 nm and 750 nm). Toxins were quantified by LC-MS in a hybrid triple-quadrupole instrument (Qtrap). Gene expression was assessed by real time PCR, using the ΔΔCt relative quantification method. Cell counting allowed total growth and logarithmic phase identification. During the last, three experiments showed statistical difference from control group (p<0,05). Four experiments...

A tragédia da hemodiálise 12 anos depois: poderia ela ser evitada? / The "tragedy of hemodialysis" 12 years later: could be avoided?

Este trabalho objetiva estudar o que em 1996 a imprensa local denominou como a Tragédia da Hemodiálise. Um surto de intoxicação em pacientes renais crônicos de uma clínica de Hemodiálise localizada no município de Caruaru, situado na região semi-árida do agreste pernambucano. Para tanto, foram analisadas as condições sócio-ambientais no período do episódio, os sistemas de abastecimento da cidade e de duas clínicas de hemodiálise desse município. Trata-se de um estudo de caso empregando como triangulação metodológica um sistema de matrizes de dados (SAMAJA, 2000). A utilização desse método se deu em função da grande quantidade de variáveis de níveis diferentes. Como fonte de informação foi utilizada o processo de ação civil pública movido pelo Ministério Público de Pernambuco e os relatórios da CPI da Hemodiálise realizado pela Assembléia Legislativa do Estado de Pernambuco e anexado aos autos do processo. Contou também com informações pluviométricas e de clima para caracterização ambiental no período do evento. Os resultados do estudo apontaram que para além da Microcistina LR, como causa direta o fornecimento de água não potável a clínica através de caminhões pipa, de responsabilidade concessionária pelo abastecimento estadual, e como o contexto de vulnerabilidades, formado pela composição de condicionantes, tais como: que passam pela ausência de na gestão integrada do meio ambiente e do recursos hídricos e de Vigilância da Saúde.

This article aims to study what, in 1996, the local Newspaper Reported like “The Hemodialysis Tragedy”, an intoxication outbreak in chronical kidney patients from Caruaru, a city in Pernambuco’s semi-arid region. Chasing this goal, environmental and sociological conditions related to such episode have been analysed. It is a case study that takes in consideration a data matrix as a triangulation of methods (SAMAJA, 2000). The use of such methodology was chosen based on the wide number of variables in different levels. The process of public civil action endorsed by Pernambuco’s Public Ministry and the reports of the Congress Hemodialysis commission were used as information sources. It has also taken advantage of rainfall and weather information for environmental purposes in such period. The study’s results that more than the LR Microcystin as the outbreak’s etiological agent, other vulnerability contexts were responsible by the event like: insuficient integradted action from the public institucions; Absence the environmental management as the general cause.

Influência de fatores climatológicos na ocorrência de microcistina / Influence of climatic factors on the occurrence of microcystin

As toxinas de cianobactérias têm sido um problema de saúde pública pela capacidade de contaminar águas dos reservatórios. Microcistinas (MCs) são compostos fortemente hepatotóxicos produzidos por diferentes cianobactérias, sendo a mais comum a Microcystis aeruginosa. Este estudo, realizado em 2005, pesquisou a ocorrência de MCs na região noroeste do Estado de São Paulo, bem como sua relação com a temperatura e o índice pluviométrico. A pesquisa de MCs nas amostras de água foi realizada por meio de ELISA (kitcomercial), empregando-se anticorpo monoclonal. As concentrações de MCs mostraram variação temporal e foram relativamente menores durante as altas temperaturas. Apesar da contaminação detectada não ser considerada alta, recomenda-se a realização de constante monitoramento.

Cyanobacteria toxins have been a public health problem, due to the ability incontaminating waters ofreservoirs. Microcystins (MCs) are strongly hepatotoxic produced by various cyanobacteria, notablyMicrocystis aeruginosa. The present investigation, conducted in 2005, aimed at studying the occurrenceof MCs in the northwestern region of São Paulo State, and also its relationship with temperature and rainfalls, which favor the development of cyanobacteria. The MCs were determined in water samples by meansof commercial ELISA kit using a monoclonal antibody. Concentrations of MCs showed temporal variationand being relatively lower during the high temperatures. In spite of the contamination has not been high,a constant monitoring is recommended.