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- Nickel Oxide Nanoparticles Trigger Caspase- and Mitochondria-Dependent Apoptosis in the Yeast Saccharomyces cerevisiaePublication . Sousa, Cátia A.; Soares, Helena M. V. M.; Soares, Eduardo V.The expansion of the industrial use of nickel oxide (NiO) nanoparticles (NPs) raises concerns about their potential adverse effects. Our work aimed to investigate the mechanisms of toxicity induced by NiO NPs, using the yeast Saccharomyces cerevisiae as a cell model. Yeast cells exposed to NiO NPs exhibited typical hallmarks of regulated cell death (RCD) by apoptosis [loss of cell proliferation capacity (cell viability), exposure of phosphatidylserine at the outer cytoplasmic membrane leaflet, nuclear chromatin condensation, and DNA damage] in a process that required de novo protein synthesis. The execution of yeast cell death induced by NiO NPs is Yca1p metacaspase-dependent. NiO NPs also induced a decrease in the mitochondrial membrane potential and an increase in the frequency of respiratory-deficient mutants, which supports the involvement of mitochondria in the cell death process. Cells deficient in the apoptosis-inducing factor ( aif1Δ) displayed higher tolerance to NiO NPs, which reinforces the involvement of mitochondria in RCD by apoptosis. In summary, this study shows that NiO NPs induce caspase- and mitochondria-dependent apoptosis in yeast. Our results warn about the possible harmful effects associated with the use of NiO NPs.
- 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.
- Alternative chelating agents: Evaluation of the ready biodegradability and complexation propertiesPublication . Martins, J.G.; Neto, Isabel F. F.; Pinto, Isabel S.S.; Soares, Eduardo V.; Barros, Maria T.; Soares, Helena MariaThe ready biodegradability of four chelating agents, N,N -(S,S)-bis[1-carboxy-2-(imidazol-4-yl)ethyl]ethylenediamine (BCIEE), N - ethylenedi-L-cysteine (EC), N,N -bis (4-imidazolymethyl)ethylenediamine (EMI) and 2,6-pyridine dicarboxylic acid (PDA), was tested according to the OECD guideline for testing of chemicals. PDA proved to be a readily biodegradable substance. However, none of the other three compounds were degraded during the 28 days of the test. Chemical simulations were performed for the four compounds in order to understand their ability to complex with some metal ions (Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Pb, Zn) and discuss possible applications of these chelating agents. Two different conditions were simulated: (i) in the presence of the chelating agent and one metal ion, and (ii) in the simultaneous presence of the chelating agent and all metal ions with an excess of Ca. For those compounds that were revealed not to be readily biodegradable (BCIEE, EC and EMI), applications were evaluated where this property was not fundamental or even not required. Chemical simulations pointed out that possible applications for these chelating agents are: food fortification, food process, fertilizers, biocides, soil remediation and treatment of metal poisoning. Additionally, chemical simulations also predicted that PDA is an efficient chelating agent for Ca incrustations removal, detergents and for pulp metal ions removal process.
- 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.
- Promising bacterial genera for agricultural practices: An insight on plant growth-promoting properties and microbial safety aspectsPublication . Ferreira, Carlos M.H.; Soares, Helena M.V.M.; Soares, Eduardo V.In order to address the ever-increasing problem of the world's population food needs, the optimization of farming crops yield, the combat of iron deficiency in plants (chlorosis) and the elimination/reduction of crop pathogens are of key challenges to solve. Traditional ways of solving these problems are either unpractical on a large scale (e.g. use of manure) or are not environmental friendly (e.g. application of iron-synthetic fertilizers or indiscriminate use of pesticides). Therefore, the search for greener substitutes, such as the application of siderophores of bacterial source or the use of plant-growth promoting bacteria (PGPB), is presented as a very promising alternative to enhance yield of crops and performance. However, the use of microorganisms is not a risk-free solution and the potential biohazards associated with the utilization of bacteria in agriculture should be considered. The present work gives a current overview of the main mechanisms associated with the use of bacteria in the promotion of plant growth. The potentiality of several bacterial genera (Azotobacter, Azospirillum, Bacillus, Pantoea, Pseudomonas and Rhizobium) regarding to siderophore production capacity and other plant growth-promoting properties are presented. In addition, the field performance of these bacteria genera as well as the biosafety aspects related with their use for agricultural proposes are reviewed and discussed.
- Evaluation of the Efficacy of Two New Biotechnological-Based Freeze-Dried Fertilizers for Sustainable Fe Deficiency Correction of Soybean Plants Grown in Calcareous SoilsPublication . Ferreira, Carlos M. H.; López-Rayo, Sandra; Lucena, Juan J.; Soares, Eduardo V.; Soares, Helena M. V. M.Currently, fertilization with synthetic chelates is the most effective agricultural practice to prevent iron (Fe) deficiencies in crops, especially in calcareous soils. Because these compounds are not biodegradable, they are persistent in the environment, and so, there is the risk of metal leaching from the soils. Thus, new, more environment-friendly efficient solutions are needed to solve iron-deficiency-induced chlorosis (IDIC) in crops grown in calcareous soils. Therefore, the central aim of this work was to prepare new freeze-dried Fe products, using a biotechnological-based process, from two siderophores bacterial (Azotobacter vinelandii and Bacillus subtilis) cultures (which previously evidenced high Fe complexation ability at pH 9) and test their capacity for amending IDIC of soybean grown in calcareous soils. Results have shown that A. vinelandii iron fertilizer was more stable and interacted less with calcareous soils and its components than B. subtilis one. This behavior was noticeable in pot experiments where chlorotic soybean plants were treated with both fertilizer products. Plants treated with A. vinelandii fertilizer responded more significantly than those treated with B. subtilis one, when evaluated by their growth (20% more dry mass than negative control) and chlorophyll development (30% higher chlorophyll index than negative control) and in most parameters similar to the positive control, ethylenediamine-di(o-hydroxyphenylacetic acid). On average, Fe content was also higher in A. vinelandii-treated plants than on B. subtilis-treated ones. Results suggest that this new siderophore-based formulation product, prepared from A. vinelandii culture, can be regarded as a possible viable alternative for replacing the current nongreen Fe-chelating fertilizers and may envisage a sustainable and environment-friendly mending IDIC of soybean plants grown in calcareous soils.
- Harmful effects of metal(loid) oxide nanoparticlesPublication . Soares, Eduardo V.; Soares, HMVMThe incorporation of nanomaterials (NMs), including metal(loid) oxide (MOx) nanoparticles (NPs), in the most diversified consumer products, has grown enormously in recent decades. Consequently, the contact between humans and these materials increased, as well as their presence in the environment. This fact has raised concerns and uncertainties about the possible risks of NMs to human health and the adverse effects on the environment. These concerns underline the need and importance of assessing its nanosecurity. The present review focuses on the main mechanisms underlying the MOx NPs toxicity, illustrated with different biological models: release of toxic ions, cellular uptake of NPs, oxidative stress, shading effect on photosynthetic microorganisms, physical restrain and damage of cell wall. Additionally, the biological models used to evaluate the potential hazardous of nanomaterials are briefly presented, with particular emphasis on the yeast Saccharomyces cerevisiae, as an alternative model in nanotoxicology. An overview containing recent scientific advances on cellular responses (toxic symptoms exhibited by yeasts) resulting from the interaction with MOx NPs (inhibition of cell proliferation, cell wall damage, alteration of function and morphology of organelles, presence of oxidative stress bio-indicators, gene expression changes, genotoxicity and cell dead) is critically presented. The elucidation of the toxic modes of action of MOx NPs in yeast cells can be very useful in providing additional clues about the impact of NPs on the physiology and metabolism of the eukaryotic cell. Current and future trends of MOx NPs toxicity, regarding their possible impacts on the environment and human health, are discussed.
- 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.
- Chronic exposure of the freshwater alga Pseudokirchneriella subcapitata to five oxide nanoparticles: Hazard assessment and cytotoxicity mechanismsPublication . Sousa, Cátia A.; Soares, Helena M.V.M.; Soares, Eduardo V.The increasing use of nanoparticles (NPs) unavoidably enhances their unintended introduction into the aquatic systems, raising concerns about their nanosafety. This work aims to assess the toxicity of five oxide NPs (Al2O3, Mn3O4, In2O3, SiO2 and SnO2) using the freshwater alga Pseudokirchneriella subcapitata as a primary producer of ecological relevance. These NPs, in OECD medium, were poorly soluble and unstable (displayed low zeta potential values and presented the tendency to agglomerate). Using the algal growth inhibition assay and taking into account the respective 72 h-EC50 values, it was possible to categorize the NPs as: toxic (Al2O3 and SnO2); harmful (Mn3O4 and SiO2) and non-toxic/non-classified (In2O3). The toxic effects were mainly due to the NPs, except for SnO2 which toxicity can mainly be attributed to the Sn ions leached from the NPs. A mechanistic study was undertaken using different physiological endpoints (cell membrane integrity, metabolic activity, photosynthetic efficiency and intracellular ROS accumulation). It was observed that Al2O3, Mn3O4 and SiO2 induced an algistatic effect (growth inhibition without loss of membrane integrity) most likely as a consequence of the cumulative effect of adverse outcomes: i) reduction of the photosynthetic efficiency of the photosystem II (ФPSII); ii) intracellular ROS accumulation and iii) loss of metabolic activity. SnO2 NPs also provoked an algistatic effect probably as a consequence of the reduction of ФPSII since no modification of intracellular ROS levels and metabolic activity were observed. Altogether, the results here presented allowed to categorize the toxicity of the five NPs and shed light on the mechanisms behind NPs toxicity in the green alga P. subcapitata.
- (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a reviewPublication . Ferreira, Carlos M. H.; Pinto, Isabel S. S.; Soares, Eduardo V.; Soares, Helena M. V. M.The use of buffers to maintain the pH within a desired range is a very common practice in chemical, biochemical and biological studies. Among them, zwitterionic N-substituted aminosulfonic acids, usually known as Good’s buffers, although widely used, can complex metals and interact with biological systems. The present work reviews, discusses and updates the metal complexation characteristics of thirty one commercially available buffers. In addition, their impact on biological systems is also presented. The influences of these buffers on the results obtained in biological, biochemical and environmental studies, with special focus on their interaction with metal ions, are highlighted and critically reviewed. Using chemical speciation simulations, based on the current knowledge of the metal–buffer stability constants, a proposal of the most adequate buffer to employ for a given metal ion is presented.