Avaliação dos efeitos tóxicos e genéticos de microcistinas sob sistemas testes animais e vegetais
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Data
2017-11-16
Autores
Silva, Maria Tereza Pamplona [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
As cianobactérias são organismos procarionte fotossintetizantes que apresentam capacidade de produzir metabólitos secundários de alto efeito tóxico para seres vivos, denominados de cianotoxinas. A presença desses organismos nos ambientes aquáticos se dá, inicialmente, pelo excesso de nutrientes, derivados, principalmente, de excessivo descarte de efluentes urbanos e industriais e de poluentes oriundos das atividades agrícolas. As cianobactérias produzem cianotoxinas do tipo endotoxinas, cuja toxicidade se dá, geralmente, quando a toxina é liberada para o meio extracelular, em decorrência de lise celular, devido ao envelhecimento ou morte das cianobactérias. São substâncias presentes nas paredes celulares de muitas cianobactérias; e exotoxinas, que apresentam uma potente ação tóxica e estão relacionadas com o metabolismo de manutenção da cianobactéria. Neste trabalho, foram desenvolvidos ensaios biológico com o organismo Allium cepa, um organismo teste muito utilizado em avaliações de ação de drogas e em ensaios ambientais, e com células hepáticas de peixe (ZFL) e humanas (HepG2) mantidas em cultura. Neste estudo foram avaliados os efeitos citotóxico, genotóxico e mutagênico da microcistina purificada, adquirida comercialmente; de amostras ambientais coletadas em um ambiente com recorrentes florações por cianobactérias; com biomassa de Microcistis aeruginosa, obtidas em laboratório, para inferir efeitos das endotoxinas produzidas por essa espécie; e com o meio de cultivo da M. aeruginosa, para inferir os efeitos das exotoxinas desses procariontes. Para esta avaliação, foram realizados teste in vivo com Allium cepa, e testes in vitro com as culturas celulares. Com o organismo teste A. cepa, foram aplicados testes de aberrações cromossômicas (AC) e de micronúcleo (MN) em células meristemáticas e de micronúcleo (MN) em células F1. Neste organismo foram testadas as concentrações de 1 μg/L (concentração considerada como alerta de nível 1 pela OMS); 1,5 μg/L (concentração 50% maior que a de alerta de nível 1), simulando um início de floração; e 2 μg/L (concentração duas vezes maior que a concentração de alerta), simulando uma floração de cianobactéria em estado avançado. As amostras ambientais e o meio de cultivo das M. aeruginosa passaram pelo processo de extração de fase sólida (EFS). Os extratos foram utilizados na diluição 1/10. Foi observado nos testes realizados com A. cepa, que nas concentrações e nas condições a que as células foram expostas, a microcistina purificada possui ação mutagênica e genotóxica e que sua ação pode estar relacionada com a inibição do sistema de reparo da célula e com uma ação clastogênica sobre o DNA. Ainda pode-se observar que em amostras ambientais, consideradas complexas, o efeito tóxico da microcistina pode ser amplificado, em decorrência de efeito sinergístico da microcistina com outras substâncias presentes na amostra, levando a efeitos adicionais, como indução de processos apoptóticos. Pelo teste do micronúcleo com bloqueio de citocinese (MNBC), realizados com as linhagens celulares HepG2 e ZFL, pode-se observar um alto índice de mutagenicidade e genotoxicidade, porém não foi observado resultados significativos de genotoxicidade pelo ensaio do cometa. Esta contradição entre os testes infere que os danos no DNA, observados no teste MNBC, estariam relacionados a um estágio inicial de apoptose e não com uma ação genotóxica da substância. Os resultados também sugerem que a indução de MNBC pode ser decorrente de ação direta da microcistina sobre o citoesqueleto celular, uma vez que existem estudos que evidenciaram que essas toxinas agem sobre os microtúbulos, microfilamentos e filamentos intermediários das células.
Cyanobacteria are prokaryote photosynthetic organisms that can produce secondary metabolites with a high toxic effect on living organisms, known as cyanotoxins. The presence of these organisms in aquatic environments is initially due to nutrients excess, mainly derived from excessive disposal of urban and industrial effluents and pollutants from agricultural activities. Cyanobacteria produce cyanotoxins of two types: endotoxin, the toxicity of which generally occurs when the toxin is released into the extracellular medium because of cell lysis due to the aging or death of the cyanobacteria. They are substances present in the cell walls of many cyanobacteria; and exotoxins, which present a potent toxic action and are related to the maintenance metabolism of cyanobacteria. In this work, biological assays were developed with the organism Allium cepa, a test organism widely used in evaluations of drug action and in environmental analyses, and with hepatic fish (ZFL) and human (HepG2) cells maintained in culture. In this study the cytotoxic, genotoxic and mutagenic effects of the commercially acquired purified microcystin were evaluated; of environmental samples collected in an environment with recurrent cyanobacterial blooms; with biomass of Microcystis aeruginosa, obtained in the laboratory, to infer effects of the endotoxins produced by this species; and with M. aeruginosa culture medium, to infer the effects of the exotoxins of these prokaryotes. For this evaluation, in vivo tests were performed with Allium cepa, and in vitro tests with cell cultures. With the test organism A. cepa, tests of chromosomal aberrations (AC) and micronucleus (MN) were applied in meristematic and micronucleus (MN) cells in F1 cells. In this organism, concentrations of 1 μg / L (concentration considered as level 1 alert by the WHO) were tested; 1.5 μg / L (concentration 50% higher than the alert level 1), simulating a beginning of flowering; and 2 μg / L (concentration twice as high as the alert concentration), simulating an advanced flowering of cyanobacteria. The environmental samples and culture medium of M. aeruginosa underwent solid phase extraction (SPE). The extracts were used in the 1:10 dilution. It was observed in the tests carried out with A. cepa that at the concentrations and the conditions to which the cells were exposed, the purified microcystin has a mutagenic and genotoxic action and that its action may be related to the inhibition of the cell repair system and with a clastogenic action on DNA. It can be observed that in environmental samples, considered as complex, the toxic effect of microcystin can be amplified, due to the synergistic effect of microcystin with other substances present in the sample, leading to additional effects, such as induction of apoptotic processes. A high index of mutagenicity and genotoxicity was observed by the micronucleus test with cytokinesis blockade (CBMN), carried out with the cell lines HepG2 and ZFL, but no significant results of genotoxicity were observed by the comet assay. This contradiction between the tests infers that the DNA damage observed in the MNBC test would be related to an early stage of apoptosis and not to a genotoxic action of the substance. The results also suggest that the induction of MNBC may be due to the direct action of microcystin on the cell cytoskeleton, since there are studies that showed that these toxins act on the microtubules, microfilaments and intermediate filaments of the cells.
Cyanobacteria are prokaryote photosynthetic organisms that can produce secondary metabolites with a high toxic effect on living organisms, known as cyanotoxins. The presence of these organisms in aquatic environments is initially due to nutrients excess, mainly derived from excessive disposal of urban and industrial effluents and pollutants from agricultural activities. Cyanobacteria produce cyanotoxins of two types: endotoxin, the toxicity of which generally occurs when the toxin is released into the extracellular medium because of cell lysis due to the aging or death of the cyanobacteria. They are substances present in the cell walls of many cyanobacteria; and exotoxins, which present a potent toxic action and are related to the maintenance metabolism of cyanobacteria. In this work, biological assays were developed with the organism Allium cepa, a test organism widely used in evaluations of drug action and in environmental analyses, and with hepatic fish (ZFL) and human (HepG2) cells maintained in culture. In this study the cytotoxic, genotoxic and mutagenic effects of the commercially acquired purified microcystin were evaluated; of environmental samples collected in an environment with recurrent cyanobacterial blooms; with biomass of Microcystis aeruginosa, obtained in the laboratory, to infer effects of the endotoxins produced by this species; and with M. aeruginosa culture medium, to infer the effects of the exotoxins of these prokaryotes. For this evaluation, in vivo tests were performed with Allium cepa, and in vitro tests with cell cultures. With the test organism A. cepa, tests of chromosomal aberrations (AC) and micronucleus (MN) were applied in meristematic and micronucleus (MN) cells in F1 cells. In this organism, concentrations of 1 μg / L (concentration considered as level 1 alert by the WHO) were tested; 1.5 μg / L (concentration 50% higher than the alert level 1), simulating a beginning of flowering; and 2 μg / L (concentration twice as high as the alert concentration), simulating an advanced flowering of cyanobacteria. The environmental samples and culture medium of M. aeruginosa underwent solid phase extraction (SPE). The extracts were used in the 1:10 dilution. It was observed in the tests carried out with A. cepa that at the concentrations and the conditions to which the cells were exposed, the purified microcystin has a mutagenic and genotoxic action and that its action may be related to the inhibition of the cell repair system and with a clastogenic action on DNA. It can be observed that in environmental samples, considered as complex, the toxic effect of microcystin can be amplified, due to the synergistic effect of microcystin with other substances present in the sample, leading to additional effects, such as induction of apoptotic processes. A high index of mutagenicity and genotoxicity was observed by the micronucleus test with cytokinesis blockade (CBMN), carried out with the cell lines HepG2 and ZFL, but no significant results of genotoxicity were observed by the comet assay. This contradiction between the tests infers that the DNA damage observed in the MNBC test would be related to an early stage of apoptosis and not to a genotoxic action of the substance. The results also suggest that the induction of MNBC may be due to the direct action of microcystin on the cell cytoskeleton, since there are studies that showed that these toxins act on the microtubules, microfilaments and intermediate filaments of the cells.
Descrição
Palavras-chave
Allium cepa, Ensaios in vitro, Células HepG2, Células ZFL, Teste do micronúcleo com bloqueio de citocinese, Ensaio do cometa, Cianotoxinas, Microcistinas, In vitro assays, HepG2 cells, ZFL cells, Micronucleus test with cytokinesis block, Comet assay, Cyanotoxins, Microcystins