Zoltán Gazdag

Prevention of intracellular oxidation in yeast: the role of vitamin E analogue, Trolox (6-hydroxy-2,5,7,8-tetramethylkroman-2-carboxyl acid)

Reactive oxygen species (ROS) are not only generated in conditions of cellular stress but are also constitutively produced in most cell types by specific metabolic processes. This research focused on a potential antioxidant Trolox (model compound for α‐tocopherol), with the aim to establish exact mechanisms of Trolox intracellular oxidation prevention on model organism Saccharomyces cerevisiae. Measuring intracellular oxidation of Trolox‐treated yeast cells revealed that Trolox decreased intracellular oxidation during normal metabolism. Trolox treatment decreased cyto‐ and geno‐toxicity of treated yeast cells in MES buffer, lowered intracellular oxidation, decreased intracellular peroxides formation, and increased H2O2 degradation and superoxide quenching yeast extract ability.

Chromate sensitivity in fission yeast is caused by increased glutathione reductase activity and peroxide overproduction

The Cr(VI)‐sensitive mutant chr‐51S of the Schizosaccharomyces pombe accumulated chromate (CrO42–) and reduced Cr(V) to much greater extent, than did its parental strain 6 chr+. Sublethal doses of K2Cr2O7 did not induce any adaptive stress response, while H2O2 or menadione pretreatment proved protective against the cell injuries caused by Cr(VI). The intracellular GSH concentration in chr‐51S cells was approximately half of that for the 6 chr+. Moreover, the glutathione disulfide reducing capacity of chr‐51S was characterized by significantly increased glutathione reductase (GR) and glucose‐6‐phosphate dehydrogenase activities. These data strongly suggested that, instead of GSH, the NADPH/GR system was the major one‐electron Cr(VI) reductant in vivo. The increased Cr(V) reduction in chr‐51S mutant was accompanied by high intracellular superoxide and peroxide concentrations, required for formation of the hydroxyl radical (•OH). The decreased intracellular GSH levels and the Cr(VI)‐sensitive phenotype of the chr‐51S cells indicates that GSH might act effectively against chromate by scavenging •OH.

In vivo direct patulin-induced fluidization of the plasma membrane of fission yeast Schizosaccharomyces pombe.

Patulin is a toxic metabolite produced by various species of Penicillium, Aspergillus and Byssochlamys. In the present study, its effects on the plasma membrane of fission yeast Schizosaccharomyces pombe were investigated. The phase-transition temperature (G) of untreated cells, measured by electron paramagnetic resonance spectrometry proved to be 14.1 degrees C. Treatment of cells for 20 min with 50, 500, or 1000 microM patulin resulted in a decrease of the G value of the plasma membrane to 13.9, 10.1 or 8.7 degrees C, respectively. This change in the transition temperature was accompanied by the loss of compounds absorbing light at 260 nm. Treatment of cells with 50, 500 or 1000 microM patulin for 20 min induced the efflux of 25%, 30.5% or 34%, respectively, of these compounds. Besides its cytotoxic effects an adaptation process was observed. This is the first study to describe the direct interaction of patulin with the plasma membrane, a process which could definitely contribute to the adverse toxic effects induced by patulin.

Regulation of patulin-induced oxidative stress processes in the fission yeast Schizosaccharomyces pombe.

Patulin (PAT), is one of the most widely disseminated mycotoxins found in agricultural products. In this study the PAT-induced accumulation of reactive oxygen species (ROS) and the regulation of the specific activities of antioxidant enzymes were investigated in the single cell eukaryotic organism Schizosaccharomyces pombe. In comparison with the untreated cells, 500 μM PAT treatment caused a 43% decrease in the concentration of the main intracellular antioxidant, glutathione (GSH); this depletion of GSH initiated a 2.44- and a 2.6-fold accumulation of superoxide anion and hydrogen peroxide, respectively, but did not increase the concentration of hydroxyl radicals; the reduction of ROS-induced adaptation processes via the activation of Pap1 transcription factor resulted in significantly increased specific activities of Cu/Zn superoxide dismutase, catalase and glutathione S-transferase to protect the cells against the ROS-induced unbalanced redox state. However, no change was measured in the activities of glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. It seems reasonable to assume that the temporary PAT-induced ROS accumulation plays a crucial role in adaptation processes. The adverse effects of PAT may be exerted mainly through the destruction of cellular membranes and protein/enzyme functions.

Effects of decreased specific glutathione reductase activity in a chromate-tolerant mutant of Schizosaccharomyces pombe

A chromate-tolerant mutant chr1-663T bearing a stable one-gene mutation and its parental strain 6chr+ were used to investigate the background of CrVI tolerance in the fission yeast Schizosaccharomyces pombe. The mutant chr1-663T displayed a significantly decreased specific glutathione reductase (GR) activity coded by the pgr1+ gene compared with its parental strain. Transformants of the mutant chr1-663T with a nonintegrative pUR18N vector expressing the pgr1+ gene exhibited the same CrVI sensitivity and specific GR activity as their parental strain, demonstrating the importance of the GR-NADPH system in CrVI tolerance. Transformants, nevertheless, exhibited an increased intracellular peroxide concentration, a decreased CrVI-reducing and HO•-producing ability, which suggested an unbalanced oxidoreduction state of cells and partial complementation of the GR function. No mutation was found in the sequences of the pgr1+ and the pap1+ (transcriptional regulatory gene of GR) genes of the CrVI-tolerant mutant by sequence analysis.

The abc1−/coq8− respiratory-deficient mutant of Schizosaccharomyces pombe suffers from glutathione underproduction and hyperaccumulates Cd2+

The abc1−/coq8− gene deletion respiratory-deficient mutant NBp17 of fission yeast Schizosaccharomyces pombe displayed a phenotypic fermentation pattern with enhanced production of glycerol and acetate, and also possessed oxidative stress-sensitive phenotypes to H2O2, menadione, tBuOOH, Cd2+, and chromate in comparison with its parental respiratory-competent strain HNT. As a consequence of internal stress-inducing mutation, adaptation processes to restore the redox homeostasis of mutant NBp17 cells were detected in minimal glucose medium. Mutant NBp17 produced significantly increased amounts of O2•− and H2O2 as a result of the decreased internal glutathione concentration and the only slightly increased glutathione reductase activity. The Cr(VI) reduction capacity and hence the •OH production ability were decreased. The mutant cells demonstrated increased specific activities of superoxide dismutases and glutathione reductase (but not catalase) to detoxify at least partially the overproduction of reactive oxygen species. All these features may be explained by the decreased redox capacity of the mutant cells. Most notably, mutant NBp17 hyperaccumulated yellow CdS.

Pro-oxidative versus antioxidative reactions between Trolox and Cr(VI): The role of H2O2

The effect of the Vitamin E model compound Trolox in reactions with Cr(VI) in the presence or absence of hydrogen peroxide was investigated. The aim of this study was to establish and discuss potential Trolox-mediated pro-oxidative reactions. The importance of the Trolox:Cr(VI) ratio in the Cr(VI) reduction process was determined from the EPR spectra and DNA cleavage reactions. In the absence of hydrogen peroxide, reduction of Cr(VI) occurred with concomitant oxidation of Trolox to the phenoxyl radical. In the presence of hydrogen peroxide, Cr(V), produced by the reduction of Cr(VI), reduced hydrogen peroxide to the hydroxyl radical. The latter was detected by spin-trapping the methyl radical following reaction with N-methyl sulfoxide. During Cr(VI) reduction with Trolox, DNA single- or double-strand breaks due to Trolox radical formation were not observed. Relaxed DNA appeared only when H(2)O(2) was added to Trolox/Cr(VI) mixtures most probably due to hydroxyl radical formation during the redox cycling of Cr(V/IV)-species. Fenton-like reactions do not play a significant role in the Trolox/Cr(VI) system in the absence of added H(2)O(2).

Adaptation to tert-butyl hydroperoxide at a plasma membrane level in the fission yeast Schizosaccharomyces pombe parental strain and its t-BuOOH-resistant mutant.

The one‐gene mutant hyd1‐190 of the fission yeast Schizosaccharomyces pombe displayed four‐fold resistance to tert‐butyl hydroperoxide (t‐BuOOH) in comparison with its parental strain hyd+. The cells of hyd1‐190 exhibited a quantitative alteration in the sterol content and hence in the fatty acid composition of the plasma membrane, reflected in a two‐fold amphotericin B sensitivity, increased rigidity of the plasma membrane, revealed by an elevated (Δ7.9 °C) phase‐transition temperature, measured by means of electron paramagnetic resonance spectroscopy, and a significantly decreased uptake of glycerol. Treatment of the strains with a subinhibitory concentration (0.2 mM) of t‐BuOOH induced adaptation via modification of the sterol and fatty acid compositions, resulting in increased (Δ3.95 °C) and decreased (Δ6.83 °C) phase‐transition temperatures of the hyd+ and hyd1‐190 strains, respectively, in order to defend the cells against the consequences of t‐BuOOH‐induced external oxidative stress. However, in contrast with hyd+, hyd1‐190 lacks the ability to adapt to t‐BuOOH at a cell level.

Oxidative stress induced by HIV-1 F34IVpr in Schizosaccharomyces pombe is one of its multiple functions

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during infection, including induction of cell cycle G(2) arrest and cell death in both human and the fission yeast Schizosaccharomyces pombe cells. In this study, a mutant derivative of Vpr (F34IVpr), which causes transient G2 arrest with little or no effect of cell killing, was used to study the molecular impact of Vpr on cellular oxidative stress responses in S. pombe. We demonstrated here that F34IVpr triggers low level of complex and atypical oxidative stress responses in comparison with its parental strain SP223 in early (14-h) and late (35-h) log phase cultures. Specifically, F34IVpr production in S. pombe causes significantly elevated levels of reactive oxygen species such as superoxide and peroxides; meanwhile, it also induces decreased levels of glutathione, hydroxyl radical concentrations and specific enzyme activities such as those of antioxidant enzymes including superoxide dismutases, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione transferase. These observations may provide functional insights into the significance of Vpr-induced oxidative stress as part of the multifaceted functions of Vpr, and contribute to the development of future new strategies aimed to reduce the adverse Vpr-mediated effects in HIV-infected patients.

Overexpression of the YAP1, PDE2, and STB3 genes enhances the tolerance of yeast to oxidative stress induced by 7‐chlorotetrazolo[5,1‐c]benzo[1,2,4]triazine

7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) is an antifungal agent that induces oxidative stress and enhances the activity of other antifungals with different modes of action. A genome-wide screening of Saccharomyces cerevisiae genomic library in the high-copy-number plasmid revealed three genes, YAP1, PDE2, and STB3, which increased the CTBT tolerance of the parental strain. The YAP1 gene is known to activate many genes in response to oxidants. The PDE2 and STB3 genes encode the high-affinity cAMP phosphodiesterase and the transcription factor recognizing the ribosomal RNA processing element in promoter sequences, respectively. The protective effects of their overexpression against CTBT toxicity was observed in the absence of certain proteins involved in stress responses, cell wall integrity signaling, and chromatin remodeling. The enhanced CTBT tolerance of the YAP1, PDE2, and STB3 transformants was a consequence of their high antioxidant enzyme activities at the beginning of CTBT treatment in comparison with that of the parental strain, for that they inactivated the CTBT-induced reactive oxygen species. These results point to the complex interplay among the oxidant sensing, cAMP-protein kinase A signaling, and transcription reprogramming of yeast cells, leading to their better adaptation to the stress imposed by CTBT.