Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.18/506
Título: Unveiling the Microcystin-LR Toxicity Mechanisms- A Microscopical Approach
Autor: Alverca, Elsa
Menezes, Carina
Dias, Elsa
Paulino, Sérgio
Leão-Martins, J. M.
Gago-Martínez, Ana
Pereira, Paulo
Palavras-chave: Microcystin-LR
HepG2 Cell Line
Vero Cell Line
Água e Solo
Data: 20-Out-2011
Editora: Instituto Nacional de Saúde Doutor Ricardo Jorge, IP
Resumo: Microcystin-LR (MCLR) is a potent toxin produced by freshwater cyanobacteria, being responsible for acute and chronic hazards to animal and human health [1]. Although considered a hepatotoxin, MCLR also targets other organs such as kidneys and intestines [2]. Despite the increasing recognition of its toxic effects, the cellular basis of MCLR-induced toxicity is still poorly understood, particularly in non-liver cells. In this study, morphological, ultrastructural and biochemical analyses were performed to evaluate the effects of a semi-purified MCLR-containing cyanobacterial extract on hepatic (HepG2) and renal (Vero) cell lines. In addition, its subcellular localization was analyzed through immunofluorescence labeling with a monoclonal antibody against MCLR. Our results showed that there was a marked viability decrease in both cell lines, after a 24h exposure to MCLR concentrations higher than 25μg.ml-1, which was particularly evident in HepG2 cells. The ultrastructural observations revealed that at subcytotoxic concentrations MCLR induced the formation of large cytoplasmic vacuoles. These vacuoles proved to be enriched in LC3B protein, suggesting that autophagy is an early cellular response of HepG2 and Vero cells to MCLR. At cytotoxic MCLR concentrations, lysossomal dysfunction in both cell lines occurred prior to mitochondrial disruption, as demonstrated by the specific labeling with Acridine Orange and Rhodamine-123, suggesting that lysossomes may be an MCLR-early target. Additionally, immunolocalization and western blot analysis of the endoplasmic reticulum protein GRP94 showed that MCLR induced a re-localization of GRP94 within Vero cells and a decrease of GRP94 expression in the HepG2 cell line. Most HepG2 cells presented positive anti-MCLR labeling, consisting of a green signal dispersed throughout the cytoplasm, together with numerous bright foci. On Vero cell line a smaller proportion of positively labeled cells was observed and the anti-MCLR labeling appeared in a discrete punctuated pattern, with only few cytoplasmic foci. The data presented reinforces the involvement of an organelle crosstalk in the cellular response to MCLR, although the role of each intervenient, the sequence of cellular events and their relation to different toxin concentrations has to be clarified. Further challenges relate to the quantification and dynamics of toxin uptake within the two cell lines and to determine if the MCLR accumulation foci co-localize with a particular cellular structure. [1] Falconer IR, Acta Hydrochem Hydrobiol, 33 (2005) 64-71. [2] Wang Q, Xie P, Chen J, Liang G, Toxicon, 52 (2008) 721-727.
URI: http://hdl.handle.net/10400.18/506
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