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 ρ0 strain, lacking mitochondrial DNA, was used. The ρ0 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.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013

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

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.The authors thank the Fundacao para a Ciencia e a Tecnologia (FCT) through the Portuguese Government for their financial support of this work through the grant PEST-OE/EQB/LA0023/2011 to IBB

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals toxic effects observed.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes" (NORTE-07-0124-FEDER-000028), Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010). Vanessa A. Mesquita gratefully acknowledges the grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES). The authors also thank to Doctor Rosane Freitas Schwan to offer the yeast strain and to Doctor Helena M.V.M. Soares, from the Faculty of Engineering of Porto University, for the use of analytical facilities (AAS with flame atomization and AAS with electrothermal atomization)

Effects of zinc on leaf decomposition by fungi in streams : studies in microcosms

The effect of zinc on leaf decomposition by aquatic fungi was studied in microcosms. Alder leaf disks were precolonized for 15 days at the source of the Este River, and exposed to different zinc concentrations during 25 days. Leaf mass loss, fungal biomass (based on ergosterol concentration), fungal production (rates of [1-14C]acetate incorporation into ergosterol), sporulation rates and species richness of aquatic hyphomycetes were determined. At the source of the Este River decomposition of alder leaves was fast and 50% of the initial mass was lost in 25 days. A total of 18 aquatic hyphomycete species were recorded during 42 days of leaf immersion. Articulospora tetracladia was the dominant species, followed by Lunulospora curvula and two unidentified species with sigmoid conidia. Cluster analysis suggested that zinc concentration and exposure time affected the structure of aquatic hyphomycete assemblages, even though richness had not been severely affected. Both zinc concentration and exposure time significantly affected leaf mass loss, fungal production and sporulation, but not fungal biomass. Zinc exposure reduced leaf mass loss, inhibited fungal production and affected fungal reproduction by either stimulating or inhibiting sporulation rates. The results of this work suggested zinc pollution might depress leaf decomposition in streams due to changes in the structure and activity of aquatic fungi.Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI) - POCTI/34024/BSE/2000

Intraspecific traits change biodiversity effects on ecosystem functioning under metal stress

The online version of this article (doi:10.1007/s00442-011-1930-3) contains supplementary material, which is available to authorized users.Studies investigating the impacts of biodiversity loss on ecosystem processes have often reached different conclusions, probably because insufficient attention has been paid to some aspects including (1) which biodiversity measure (e.g., species number, species identity or trait) better explains ecosystem functioning, (2) the mechanisms underpinning biodiversity effects, and (3) how can environmental context modulates biodiversity effects. Here, we investigated how species number (one to three species) and traits of aquatic fungal decomposers (by replacement of a functional type from an unpolluted site by another from a metal-polluted site) affect fungal production (biomass acumulation) and plant litter decomposition in the presence and absence of metal stress. To examine the putative mechanisms that explain biodiversity effects, we determined the contribution of each fungal species to the total biomass produced in multicultures by real-time PCR. In the absence of metal, positive diversity effects were observed for fungal production and leaf decomposition as a result of species complementarity. Metal stress decreased diversity effects on leaf decomposition in assemblages containing the functional type from the unpolluted site, probably due to competitive interactions between fungi. However, dominance effect maintained positive diversity effects under metal stress in assemblages containing the functional type from the metal-polluted site. These findings emphasize the importance of intraspecific diversity in modulating diversity effects under metal stress, providing evidence that trait-based diversity measures should be incorporated when examining biodiversity effects.The Portuguese Foundation for Science and Technology supported I. Fernandes (SFRH/BD/42215/2007

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 intracelullarly 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