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- Quantification and viability analyses of Pseudokirchneriella subcapitata algal cells using image-based cytometryPublication . Machado, Manuela D.; Soares, Eduardo V.This work aims to evaluate the feasibility of using image-based cytometry (IBC) in the analysis of algal cell quantification and viability, using Pseudokirchneriella subcapitata as a cell model. Cell concentration was determined by IBC to be in a linear range between 1 × 105 and 8 × 106 cells mL−1. Algal viability was defined on the basis that the intact membrane of viable cells excludes the SYTOX Green (SG) probe. The disruption of membrane integrity represents irreversible damage and consequently results in cell death. Using IBC, we were able to successfully discriminate between live (SG-negative cells) and dead algal cells (heat-treated at 65 °C for 60 min; SG-positive cells). The observed viability of algal populations containing different proportions of killed cells was well correlated (R 2 = 0.994) with the theoretical viability. The validation of the use of this technology was carried out by exposing algal cells of P. subcapitata to a copper stress test for 96 h. IBC allowed us to follow the evolution of cell concentration and the viability of copper-exposed algal populations. This technology overcomes several main drawbacks usually associated with microscopy counting, such as labour-intensive experiments, tedious work and lack of the representativeness of the cell counting. In conclusion, IBC allowed a fast and automated determination of the total number of algal cells and allowed us to analyse viability. This technology can provide a useful tool for a wide variety of fields that utilise microalgae, such as the aquatic toxicology and biotechnology fields.
- Toxicological effects induced by the biocide triclosan on Pseudokirchneriella subcapitataPublication . Machado, Manuela D.; Soares, Eduardo V.Triclosan, a widely used biocide broadly found in aquatic environments, is cause of concern due to its unknown effects on non-targets organisms. In this study, a multi biomarker approach was used in order to evaluate the 72 h-effect of triclosan on the freshwater alga Pseudokirchneriella subcapitata (Raphidocelis subcapitata). Triclosan, at environmental relevant concentrations (27 and 37 μg L−1), caused a decrease of proliferative capacity, which was accompanied by an increase of cell size and a profound alteration of algae shape. It was found that triclosan promoted the intracellular accumulation of reactive oxygen species, the depletion of non-enzymatic antioxidant defenses (reduced glutathione and carotenoids) and a decrease of cell metabolic activity. A reduction of photosynthetic pigments (chlorophyll a and b) was also observed. For the highest concentration tested (37 μg L−1), a decrease of photosynthetic efficiency was detected along with a diminution of the relative transport rate of electrons on the photosynthetic chain. In conclusion, triclosan presents a deep impact on the microalga P. subcapitata morphology and physiology translated by multiple target sites instead of a specific point (cellular membrane) observed in the target organism (bacteria). Additionally, this study contributes to clarify the toxicity mechanisms of triclosan, in green algae, showing the existence of distinct modes of action of the biocide depending on the microalga.
- Short- and Long-Term Exposure to Heavy Metals Induced Oxidative Stress Response in Pseudokirchneriella subcapitataPublication . Machado, Manuela D.; Soares, Eduardo V.Algal cells can be exposed to toxicants for a short-term due to accidental discharges or, more commonly, for a long-term. The present work aimed to assess the ability of Cd, Cr, Cu, and Zn to induce accumulation of reactive oxygen species (ROS) in the alga Pseudokirchneriella subcapitata after a short (6 h) or a long (72 h) exposure time. The relationship between the ROS induction and the content of reduced glutathione (GSH) was also examined. For this purpose, three nominal concentrations of each metal were used corresponding approximately to 72 h-EC10 and 72 h-EC50 values and a high concentration (>72 h-EC90 values). Intracellular ROS accumulation and GSH content were evaluated using a fluorescent-based approach. A long-term (chronic) exposure of algal cells to Cd, Cu, and Zn, at the highest concentrations tested, induced an increase of intracellular ROS and GSH content. The increase of GSH content might be a form of algal cells to redress the imbalance caused by the oxidative stress. However, the increase of GSH was not enough to protect the algal cells against the long-term exposure to oxidative stress. The exposure of algal cells to low or intermediate metals concentrations induced a modification of GSH content; however, no increase of ROS production was detected, which indicates that the toxic symptoms exhibited by algal cells, under these conditions, cannot be attributed to intracellular ROS accumulation.
- Palmelloid-like phenotype in the alga Raphidocelis subcapitata exposed to pollutants: A generalized adaptive strategy to stress or a specific cellular response?Publication . Machado, Manuela D.; Soares, Eduardo V.This work focuses on the formation of palmelloid-like phenotype in the freshwater alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum), when exposed to adverse conditions generated by the presence of organic [the antibiotic erythromycin (ERY) and the herbicide metolachlor (MET)] or inorganic [the heavy metals, cadmium (Cd) and zinc (Zn)] pollutants, at environmentally relevant concentrations. This alga in absence of stress or when exposed to ERY or Zn, up to 200 µg/L, essentially showed a single-nucleus state, although algal growth was reduced or stopped. R. subcapitata “switched” to a multinucleated state (palmelloid-like morphology) and accumulated energy-reserve compounds (neutral lipids) when stressed by 100–200 µg/L MET or 200 µg/L Cd; at these concentrations of pollutants, growth was arrested, however, the majority of the algal population (≥83 %) was alive. The formation of palmelloid-like phenotype, at sub-lethal concentrations of pollutants, was dependent on the pollutant, its concentration and exposure time. The multinucleated structure is a transitory phenotype since R. subcapitata population was able to revert to a single-nucleus state, with normal cell size, within 24–96 h (depending on the impact of the toxic in the alga), after being transferred to fresh OECD medium, without pollutants. The obtained results indicate that the formation of a palmelloid-like phenotype in R. subcapitata is dependent on the mode of action of toxics and their concentration, not constituting a generalized defense mechanism against stress. The observations here shown contribute to understanding the different strategies used by the unicellular alga R. subcapitata to cope with severe stress imposed by organic and inorganic pollutants.
- 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.
- Influence of the metabolic state on the tolerance of Pichia kudriavzevii to heavy metalsPublication . Mesquita, Vanessa A.; Machado, Manuela D.; Silva, Cristina F.; Soares, Eduardo V.This work aims to examine the influence of the metabolic state of the yeast Pichia kudriavzevii on the susceptibility to a metals mixture (5 mg L(-1) Cd, 10 mg L(-1) Pb, and 5 mg L(-1) Zn). Cells exposed to the metals mixture in the presence of 25 mmol L(-1) glucose displayed a higher loss of membrane integrity and proliferation capacity, compared to cells incubated in the absence of glucose. The analysis of the effect of individual metals revealed that glucose increased the toxic effect of Cd marginally, and of Pb significantly. The increased susceptibility to heavy metals due to glucose was attenuated in the simultaneous presence of a mitochondrial respiration inhibitor such as sodium azide (NaN3 ). ATP-depleted yeast cells, resulting from treatment with the non-metabolizable glucose analogue 2-deoxy-d-glucose, showed an increased susceptibility to heavy metals mixture. Pre-incubation of yeast cells with 1 or 1.5 mmol L(-1) Ca(2+) reduced significantly (P < 0.05) the loss of membrane integrity induced by the metals mixture. These findings contribute to the understanding of metals mechanisms of toxicity in the non-conventional yeast P. kudriavzevii.
- Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stressPublication . Machado, Manuela D.; Soares, Eduardo V.This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.
- Evaluation of the role of glutathione in the lead-induced toxicity in saccharomyces cerevisiaePublication . Perez, Rita R.; Sousa, Cátia A.; Vankeersbilck, Thomas; Machado, Manuela D.; Soares, Eduardo V.The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.
- Impact of fluorides on the removal of heavy metals from an electroplating effluent using a flocculent brewer’s yeast strain of Saccharomyces cerevisiaePublication . Machado, Manuela D.; Soares, Eduardo V.; Soares, Helena M.V.M.Besides several toxic heavy metals, electroplating effluents can have in solution different cations and anions, which may influence heavy metals removal by the biomass. Among them, fluorides are commonly used in the electroplating industries and thus can be found in the respective wastewaters. In the present work, the effect of the presence of fluorides in the efficiency of chromium(III), copper(II) and nickel(II) removal, from an effluent, by heat-inactivated cells of a brewing flocculent strain of Saccharomyces cerevisiae was evaluated. The presence of fluorides severely decreased (460%) the removal of chromium(III) by yeast biomass. This effect impaired the effective treatment of the effluent according to the US Environmental Protection Agency and the Portuguese law; conversely, a higher removal of copper(II) and nickel(II) was observed. This behaviour can be understood by metal speciation. In the presence of fluorides, chromium(III) was mainly complexed, becoming unavailable for yeast accumulation; this effect decreased the efficiency of chromium(III) removal. Thus, in the presence of fluorides, less chromium(III) is associated with biomass and consequently more yeast binding sites remain available for the uptake of other metals present in solution. This fact explains the increase of copper(II) and nickel(II) removal in the presence of fluorides.
- 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.