Features of the microalga Raphidocelis subcapitata: physiology and applications.

The microalga Raphidocelis subcapitata was isolated from the Nitelva River (Norway) and subsequently deposited in the collection of the Norwegian Institute of Water Research as "Selenastrum capricornutum Printz". This freshwater microalga, also known as Pseudokirchneriella subcapitata, acquired much of its notoriety due to its high sensitivity to different chemical species, which makes it recommended by different international organizations for the assessment of ecotoxicity. However, outside this scope, R. subcapitata continues to be little explored. This review aims to shed light on a microalga that, despite its popularity, continues to be an "illustrious" unknown in many ways. Therefore, R. subcapitata taxonomy, phylogeny, shape, size/biovolume, cell ultra-structure, and reproduction are reviewed. The nutritional and cultural conditions, chronological aging, and maintenance and preservation of the alga are summarized and critically discussed. Applications of R. subcapitata, such as its use in aquatic toxicology (ecotoxicity assessment and elucidation of adverse toxic outcome pathways) are presented. Furthermore, the latest advances in the use of this alga in biotechnology, namely in the bioremediation of effluents and the production of value-added biomolecules and biofuels, are highlighted. To end, a perspective regarding the future exploitation of R. subcapitata potentialities, in a modern concept of biorefinery, is outlined. KEY POINTS: • An overview of alga phylogeny and physiology is critically reviewed. • Advances in alga nutrition, cultural conditions, and chronological aging are presented. • Its use in aquatic toxicology and biotechnology is highlighted.

Perspective on the biotechnological production of bacterial siderophores and their use.

Iron (Fe) is an essential element in several fundamental cellular processes. Although present in high amounts in the Earth's crust, Fe can be a scarce element due to its low bioavailability. To mitigate Fe limitation, microorganism (bacteria and fungi) and grass plant biosynthesis and secret secondary metabolites, called siderophores, with capacity to chelate Fe(III) with high affinity and selectivity. This review focuses on the current state of knowledge concerning the production of siderophores by bacteria. The main siderophore types and corresponding siderophore-producing bacteria are summarized. A concise outline of siderophore biosynthesis, secretion and regulation is given. Important aspects to be taken into account in the selection of a siderophore-producing bacterium, such as biological safety, complexing properties of the siderophores and amount of siderophores produced are summarized and discussed. An overview containing recent scientific advances on culture medium formulation and cultural conditions that influence the production of siderophores by bacteria is critically presented. The recovery, purification and processing of siderophores are outlined. Potential applications of siderophores in different sectors including agriculture, environment, biosensors and the medical field are sketched. Finally, future trends regarding the production and use of siderophores are discussed. KEY POINTS : • An overview of siderophore production by bacteria is critically presented • Scientific advances on factors that influence siderophores production are discussed • Potential applications of siderophores, in different fields, are outlined.

Life and death of Pseudokirchneriella subcapitata: physiological changes during chronological aging.

The green alga Pseudokirchneriella subcapitata is widely used in ecotoxicity assays and has great biotechnological potential as feedstock. This work aims to characterize the physiology of this alga associated with the aging resulting from the incubation of cells for 21 days, in the OECD medium, with continuous agitation and light exposure, in a batch mode. After inoculation, cells grow exponentially during 3 days, and the culture presents a typical green color. In this phase, "young" algal cells present, predominantly, a lunate morphology with the chloroplast occupying a large part of the cell, maximum photosynthetic activity and pigments concentration, and produce starch as a reserve material. Between the 5th and the 12th days of incubation, cells are in the stationary phase. The culture becomes less green, and the cells stop dividing (≥ 99% have one nucleus) and start to age. "Old" algal cells present chloroplast shrinkage, an abrupt decline of chlorophylls content, and photosynthetic capacity (Fv/Fm and ɸPSII), accompanied by a degradation of starch and an increase of neutral lipids content. The onset of the death phase occurs after the 12th day and is characterized by the loss of cell membrane integrity of some algae (cell death). The culture stays, progressively, yellow, and the majority of the population (~93%) is composed of live cells, chronologically "old," with a significant drop in photosynthetic activity (decay > 75% of Fv/Fm and ɸPSII) and starch content. The information here achieved can be helpful when exploring the potential of this alga in toxicity studies or in biotechnological applications. KEY POINTS: • Physiological changes of P. subcapitata with chronological aging are shown • "Young" algae exhibit a semilunar shape, high photosynthetic activity, and accumulated starch • "Old"-live algae show reduced photosynthetic capacity and accumulated lipids.

Harmful effects of metal(loid) oxide nanoparticles.

The 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. KEY POINTS: • The potential hazardous effects of MOx NPs are critically reviewed. • An overview of the main mechanisms associated with MOx NPs toxicity is presented. • Scientific advances about yeast cell responses to MOx NPs are updated and discussed.

Exposure of the alga Pseudokirchneriella subcapitata to environmentally relevant concentrations of the herbicide metolachlor: Impact on the redox homeostasis.

This study investigated the effect of the herbicide metolachlor (MET) on the redox homeostasis of the freshwater green alga Pseudokirchneriella subcapitata. At low MET concentrations (≤40 µg L-1), no effects on algal cells were detected. The exposure of P. subcapitata to 45-235 µg L-1 MET induced a significant increase of reactive oxygen species (ROS). The intracellular levels of ROS were particularly increased at high (115 and 235 µg L-1) but environmentally relevant MET concentrations. The exposure of algal cells to 115 and 235 µg L-1 MET originated a decrease in the levels of antioxidants molecules (reduced glutathione and carotenoids) as well as a reduction of the activity of scavenging enzymes (superoxide dismutase and catalase). These results suggest that antioxidant (non-enzymatic and enzymatic) defenses were affected by the excess of MET. As consequence of this imbalance (ROS overproduction and decline of the antioxidant system), ROS inflicted oxidative injury with lipid peroxidation and damage of cell membrane integrity. The results provide further insights about the toxic modes of action of MET on a non-target organism and emphasize the relevance of toxicological studies in the assessment of the impact of herbicides in freshwater environments.

Nickel Oxide Nanoparticles Trigger Caspase- and Mitochondria-Dependent Apoptosis in the Yeast Saccharomyces cerevisiae.

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.

Metal(loid) oxide (Al2O3, Mn3O4, SiO2 and SnO2) nanoparticles cause cytotoxicity in yeast via intracellular generation of reactive oxygen species.

In this work, the physicochemical characterization of five (Al2O3, In2O3, Mn3O4, SiO2 and SnO2) nanoparticles (NPs) was carried out. In addition, the evaluation of the possible toxic impacts of these NPs and the respective modes of action were performed using the yeast Saccharomyces cerevisiae. In general, in aqueous suspension, metal(loid) oxide (MOx) NPs displayed an overall negative charge and agglomerated; these NPs were practically insoluble (dissolution < 8%) and did not generate detectable amounts of reactive oxygen species (ROS) under abiotic conditions. Except In2O3 NPs, which did not induce an obvious toxic effect on yeast cells (up to 100 mg/L), the other NPs induced a loss of cell viability in a dose-dependent manner. The comparative analysis of the loss of cell viability induced by the NPs with the ions released by NPs (NPs supernatant) suggested that SiO2 toxicity was mainly caused by the NPs themselves, Al2O3 and SnO2 toxic effects could be attributed to both the NPs and the respective released ions and Mn3O4 harmfulness could be mainly due to the released ions. Al2O3, Mn3O4, SiO2 and SnO2 NPs induced the loss of metabolic activity and the generation of intracellular ROS without permeabilization of plasma membrane. The co-incubation of yeast cells with MOx NPs and a free radical scavenger (ascorbic acid) quenched intracellular ROS and significantly restored cell viability and metabolic activity. These results evidenced that the intracellular generation of ROS constituted the main cause of the cytotoxicity exhibited by yeasts treated with the MOx NPs. This study highlights the importance of a ROS-mediated mechanism in the toxicity induced by MOx NPs.

Nickel Oxide (NiO) Nanoparticles Induce Loss of Cell Viability in Yeast Mediated by Oxidative Stress.

The present work aimed to elucidate whether the toxic effects of nickel oxide (NiO) nanoparticles (NPs) on the yeast Saccharomyces cerevisiae were associated with oxidative stress (OS) and what mechanisms may have contributed to this OS. Cells exposed to NiO NPs accumulated superoxide anions and hydrogen peroxide, which were intracellularly generated. Yeast cells coexposed to NiO NPs and antioxidants (l-ascorbic acid and N- tert-butyl-α-phenylnitrone) showed quenching of reactive oxygen species (ROS) and increased resistance to NiO NPs, indicating that the loss of cell viability was associated with ROS accumulation. Mutants lacking mitochondrial DNA (ρ0) displayed reduced levels of ROS and increased resistance to NiO NPs, which suggested the involvement of the mitochondrial respiratory chain in ROS production. Yeast cells exposed to NiO NPs presented decreased levels of reduced glutathione (GSH). Mutants deficient in GSH1 ( gsh1Δ) or GSH2 ( gsh2Δ) genes displayed increased levels of ROS and increased sensitivity to NiO NPs, which underline the central role of GSH against NiO NPs-induced OS. This work suggests that the increased levels of intracellular ROS (probably due to the perturbation of the electron transfer chain in mitochondria) combined with the depletion of GSH pool constitute important mechanisms of NiO NPs-induced loss of cell viability in the yeast S. cerevisiae.

Nickel oxide (NiO) nanoparticles disturb physiology and induce cell death in the yeast Saccharomyces cerevisiae.

The increasing use of nanoparticles (NPs) has spurred concerns about their toxic effects. This work aimed to assess the potential hazards of nickel oxide (NiO) NPs using the yeast Saccharomyces cerevisiae as a cell model. Yeast cells exposed for 6 h to 100 mg/L NiO NPs presented reduced metabolic activity (esterase activity and FUN-1 dye processing) and enhanced accumulation of reactive oxygen species. NiO NPs induced the loss of cell viability in a dose-dependent manner. Study of the dissolution of NiO NPs in aqueous media, together with the toxicological data, suggests that the nickel released by the NPs cannot explain all the toxic effects observed in S. cerevisiae caused by the NPs. Transmission electron microscopy observations revealed that NiO NPs were adsorbed onto cell surface but did not enter into yeast cells. Isogenic mutants (cwp1∆ and cwp2∆) with increased cell wall porosity did not display enhanced susceptibility to NiO NPs compared to the wild type strain. Our results suggest that NiO NPs exert their toxic effect by an indirect mechanism. This work contributes to knowledge of the potential hazards of NiO NPs and to the elucidation of their mechanisms of toxic action.

Toxicity Induced by a Metal Mixture (Cd, Pb and Zn) in the Yeast Pichia kudriavzevii: The Role of Oxidative Stress.

The present work aims to contribute for the elucidation of the role of oxidative stress in the toxicity associated with the exposure of Pichia kudriavzevii to multi-metals (Cd, Pb and Zn). Cells of the non-conventional yeast P. kudriavzevii exposed for 6 h to the action of multi-metals accumulated intracellular reactive oxygen species (ROS), evaluated through the oxidation of the probe 2',7'-dichlorodihydrofluorescein diacetate. A progressive loss of membrane integrity (monitored using propidium iodide) was observed in multi-metal-treated cells. The triggering of intracellular ROS accumulation preceded the loss of membrane integrity. These results suggest that the disruption of membrane integrity can be attributed to the oxidative stress. The exposure of yeast cells to single metal showed that, under the concentrations tested, Pb was the metal responsible for the induction of the oxidative stress. Yeast cells coexposed to an antioxidant (ascorbic acid) and multi-metals did not accumulate intracellular ROS, but loss proliferation capacity. Together, the data obtained indicated that intracellular ROS accumulation contributed to metal toxicity, namely for the disruption of membrane integrity of the yeast P. kudriavzevii. It was proposed that Pb toxicity (the metal responsible for the toxic symptoms under the conditions tested) result from the combination of an ionic mechanism and the intracellular ROS accumulation.

Influence of the metabolic state on the tolerance of Pichia kudriavzevii to heavy metals.

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 Ca2+ 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.

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 µmol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 (.-)) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ρ(0) strain, lacking mitochondrial DNA, was used. The ρ(0) strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 (.-) precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.

Saccharomyces cerevisiae mutants affected in vacuole assembly or vacuolar H+-ATPase are hypersensitive to lead (Pb) toxicity.

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H(+)-ATPase (V-ATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1Δ or vma2Δ) or membrane domain (vph1Δ or vma3Δ) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.

Alternative chelating agents: evaluation of the ready biodegradability and complexation properties.

The 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.

Cleanup of industrial effluents containing heavy metals: a new opportunity of valorising the biomass produced by brewing industry.

Heavy metal pollution is a matter of concern in industrialised countries. Contrary to organic pollutants, heavy metals are not metabolically degraded. This fact has two main consequences: its bioremediation requires another strategy and heavy metals can be indefinitely recycled. Yeast cells of Saccharomyces cerevisiae are produced at high amounts as a by-product of brewing industry constituting a cheap raw material. In the present work, the possibility of valorising this type of biomass in the bioremediation of real industrial effluents containing heavy metals is reviewed. Given the auto-aggregation capacity (flocculation) of brewing yeast cells, a fast and off-cost yeast separation is achieved after the treatment of metal-laden effluent, which reduces the costs associated with the process. This is a critical issue when we are looking for an effective, eco-friendly, and low-cost technology. The possibility of the bioremediation of industrial effluents linked with the selective recovery of metals, in a strategy of simultaneous minimisation of environmental hazard of industrial wastes with financial benefits from reselling or recycling the metals, is discussed.

Evaluation of the role of glutathione in the lead-induced toxicity in Saccharomyces cerevisiae.

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.

Palmelloid-like phenotype in the alga Raphidocelis subcapitata exposed to pollutants: A generalized adaptive strategy to stress or a specific cellular response?

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.

Flocculation in ale brewing strains of Saccharomyces cerevisiae: re-evaluation of the role of cell surface charge and hydrophobicity.

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.

Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata.

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 EC(10) and EC(50) 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 high-throughput screening of toxics or environmental samples using the alga P. subcapitata.

Lead induces oxidative stress and phenotypic markers of apoptosis in Saccharomyces cerevisiae.

In the present work, the mode of cell death induced by Pb in Saccharomyces cerevisiae was studied. Yeast cells Pb-exposed, up to 6 h, loss progressively the capacity to proliferate and maintained the membrane integrity evaluated by the fluorescent probes bis(1,3-dibutylbarbituric acid trimethine oxonol) and propidium iodide. Pb-induced death is an active process, requiring the participation of cellular metabolism, since the simultaneous addition of cycloheximide attenuated the loss of cell proliferation capacity. Cells exposed to Pb accumulated intracellularly reactive oxygen species (ROS), evaluated by 2',7'-dichlorodihydrofluorescein diacetate. The addition of ascorbic acid (a ROS scavenger) strongly reduced the oxidative stress and impaired the loss of proliferation capacity in Pb-treated cells. Pb-exposed cells displayed nuclear morphological alterations, like chromatin fragmentation, as revealed by diaminophenylindole staining. Together, the data obtained indicate that yeast cells exposition to 1 mmol/l Pb results in severe oxidative stress which can be the trigger of programmed cell death by apoptosis.