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- Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimanePublication . Machado, Manuela D.; Soares, Eduardo V.The green alga Pseudokirchneriella subcapitata has been extensively used for the assessment of adverse impacts of pollutants. Glutathione is involved in antioxidant defence and drug detoxification. Intracellular reduced glutathione (GSH) concentration can be used as an indicator of the health of cells. This work describes a simple and fast fluorescent cell-based assay for the evaluation of intracellular GSH in the alga P. subcapitata, using monochlorobimane (mBCl). Metabolically active algal cells incubated with 50 μmol L−1 mBCl form fluorescent bimane–glutathione (B-SG) adducts that can be measured fluorometrically. The distribution of GSH (B-SG adducts) in whole cells can be observed by epifluorescence microscopy, in the form of blue fluorescent spots. Depletion of cellular GSH with iodoacetamide, inhibition of glutathione S-transferase with ethacrynic acid or heat-induced death of the cells inhibited the formation of fluorescent adducts in the presence of mBCl. The fluorometric assay, using the 96-well microplate format, was able to detect GSH depletion in algal cells. This cell-based assay can be used to evaluate decreases in GSH content due to exposure to toxicants. This assay is amenable to automation and may be useful in high-throughput toxicity screening using the alga P. subcapitata.
- Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitataPublication . Machado, Manuela D.; Soares, Eduardo V.Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5 μmol/l SYTOX Green for 40 min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6 h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC10 and EC50 values were 7,617 mg/l 1-pentanol (95 % confidence limits 4,670–9,327) and 12,818 mg/l 1-pentanol (95 % confidence limits 10,929–15,183), respectively. The developed microplate-based short-term assay can be useful in the highthroughput screening of toxics or environmental samples using the alga P. subcapitata.
- Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitataPublication . Machado, Manuela D.; Soares, Eduardo V.The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmolL−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The shortterm assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μgL−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.