Efeitos ecotoxicológicos das saxitoxinas em Hoplias malabaricus : estudos in vivo e in vitro

Abstract

Resumo: Floracoes de cianobacterias podutoras de saxitoxinas (STXs), como a Cylindrospermopsis raciborskii, tem se tornado um grave problema ambiental, especialmente no sul do Brasil, onde a especie ja e dominante em reservatorio de abastecimento publico. As STXs sao neurotoxinas que atuam bloquendo os canais de sodio (Na+) dos neuronios, o que pode levar a morte por parada respiratoria. Entretanto, a bioacumulacao deste tipo de substancia na cadeia trofica e seus efeitos ecotoxicologicos, principalmente na agua doce,ainda nao estao totalmente esclarecidos. Neste trabalho inicialmente objetivou-se elucidar os efeitos toxicos e a bioacumulacao da STX no peixe dulcicola Hoplias malabaricus atraves de bioensaio com exposicao trofica (0,08 ƒÊg/100 g de equivalentes de STX . STXeq obtido atraves da lise celular de C. raciborskii) utilizando exemplares de Astyanax sp. como veiculo, por 20 dias com alimentacao a cada 5 dias. Os cerebros dos animais foram avaliados atraves dos biomarcadores de contaminacao ambiental: atividades da acetilcolinesterase (AChE), da superoxido dismutase (SOD), da catalase (CAT), da glutationa S-transferase (GST) e da glutationa peroxidase (GPx). Concentracao de glutationa (GSH), lipoperoxidacao (LPO), carbonilacao de proteina (PCO), e ensaio cometa tambem foram analisados. O musculo foi coletado para analise quimica de STX. Estabeleceu-se tambem um protocolo de cultivo primario de neuronios de H. malabaricus para possibilitar a avaliacao de neurotoxinas diretamente na celula alvo. Em seguida, as celulas cultivadas foram expostas em meio de cultivo contendo extrato de STXs a 3ƒÊg/L (valor toleravel no Brasil de equivalentes de STXeq na agua de abastecimento publico) e 0,3ƒÊg/L por 24h. O bioensaio sugeriu a geracao de especies reativas de oxigenio (EROs), com aumento da atividade especifica da SOD e das concentracoes de LPO e PCO, enquanto as atividades especificas da CAT, GST e GPx diminuiram, o que pode ter levado ao estresse oxidativo e aos danos genotoxicos observados pelo ensaio cometa. Entretanto, nao foi observada a presenca de STXs nos musculos analisados. O protocolo de cultivo celular de neuronios desenvolvido a partir das celulas cerebrais mostrou que um meio de cultura adequado enriquecido com fator de crescimento fibroblasto (bFGF) e suplementos nutricionais como B27, e uso de enzimas que nao causem injurias celulares como a papaina, alem de substrato de aderencia como a poli-L-lisina, e segregacao celular atraves de gradientes celulares, permitiu um cultivo com boa viabilidade celular, aproximadamente 2.5 } 0.35 x 104 cel/mg de tecido cerebral (media } desvio padrao). Portanto, aptas para ensaios toxicologicos. Apos os neuronios serem expostos as STXs, o que correspondeu a cerca de 87% das celulas isoladas, observou-se citotoxicidade atraves do ensaio de sal de tetrazolio (MTT), aumento da atividade especifica da GPx e dos nives de LPO, e genotoxicidade (ensaio cometa). No entanto, nao foi observado apoptose nos grupos testados. Os resultados obtidos sao de grande importancia e podem servir de ferramenta a gestao de recursos hidricos uma vez que, embora os efeitos da STX devido ao bloqueio do canal de Na+ sejam reportados como reversiveis, o tempo de exposicao e a concentracao das STXs sugerem injurias celulares, o que pode levar a neuropatologias. Alem disso, o estabelecimento do protocolo de cultivo primario de neuronios de peixes possibilita novas aplicacoes em ecotoxicologia na avaliacao de substancias neurotoxicas em celulas alvo.

Abstract: Cyanobacteria blooms saxitoxins (STXs) producers such as Cylindrospermopsis raciborskii, has become a serious environmental concern, especially in southern Brazil, where the species has already been dominant in public water supply reservoir. STX is a neurotoxin which blocks neurons sodium (Na+) channels, leading to death by respiratory fail. However, the bioaccumulation in trophic chain and their ecotoxicological effects, mainly in freshwater, are not completely known. First the aim this work was to elucidate the toxic effects an bioaccumulation of STXs in freshwater fish Hoplias malabaricus through trophic bioassay (0.08 ìg/100 g of STX equivalents – STXeq obtained by lysis of C. raciborskii cells) using Astyanax sp. as vehicle during 20 days feeding every 5 days. The animals brains were assessed using biomarkers of environmental contamination: activities of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPx). The concentration of glutathione (GSH), lipoperoxidation (LPO), protein carbonylation (PCO), and comet assay were also carried out. Muscles were collected for STX chemical analysis. An in vitro protocol was established using primary culture of neurons from H. malabaricus to evaluate neurotoxins directly on target cell. Then, cultured cells were exposed to culture medium containing STXs extract of 3ì/L (tolerable concentration of STXeq for public water supply in Brazil) and 0.3ì/L for 24h. The results of the bioassay suggested generation of reactive oxygen species (ROS), with increased specific activity of SOD and concentration of LPO and PCO, while the specific activities of CAT, GST and GPx decreased, which may have caused oxidative stress and genotoxic damage measured by comet assay. However, it was not observed STXs contents in muscle samples. The neurons protocol developed showed that a suitable culture medium enriched with fibroblast growth factor (bFGF) and nutritional supplements such as B27, and the use of enzymes that do not provoke cellular injury such as papain, as well substract coating appropriate such as poly-L-Lysine and segregation gradients cells, allows cellular culture with good cell viability. About 2.5 ± 0.35 x 104 cell/mg of brain tissue (mean ± standard deviation), suitable for toxicology assay. After STXs exposure neurons, which corresponds to about 87% of isolated cells, cytotoxicity, was observed evidenced by tetrazolium salt (MTT) cell viability assay, increase in the specific activity of GPx and LPO levels and genotoxicity (comet assay). Apoptosis was not observed in the exposed group. The results are important and can be used as a tool to hidric resources management. Although the STX effects due the blockage of Na+ channels is reported to be reversible, the time exposure and concentration of STXs suggests cellular injuries which can lead to neuropathology. In addition, the establishment of neurons culture primary protocol enables new applications in fish ecotoxicology in the assessment of neurotoxic substances in the target cells.