Role of thioredoxin peroxidase in aging of stationary cultures of Saccharomyces cerevisiae

A soluble protein from Saccharomyces cerevisiae acts as a peroxidase but requires a NADPH-dependent thioredoxin system and was named thioredoxin peroxidase (TPx). The role of TPx in aging of stationary cultures of S. cerevisiae was investigated in a wild-type strain and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination. The occurrence of oxidative stress during aging of stationary cultures of the yeast has been proposed. Comparison of 5-day-old (young) stationary cultures of S. cerevisiae and of cultures aged for 3 months (old) revealed decreased viability, increased generation of reactive oxygen species, modulation of cellular redox status, and increased cellular oxidative damage reflected by increased protein carbonyl content and lipid peroxidation. The magnitude of this stress was augmented in yeast mutant lacking TPx. These results suggest that TPx may play a direct role in cellular defense against aging of stationary cultures presumably, functioning as an antioxidant enzyme.     

Oxidative stress during aging of the yeast in a stationary culture and its attenuation by antioxidants

Oxidative stress during aging of Saccharomyces cerevisiae in stationary culture was documented by demonstration of progressive increase in the formation of superoxide, decrease in the content of acid‐soluble thiols and of acid‐soluble antioxidant capacity of cell extracts, and accumulation of aldehydes and protein carbonyl groups in two yeast strains and decreases in activities of antioxidant enzymes. Cells of a CuZn‐SOD (superoxide dismutase)‐1‐deficient strain showed a higher loss of viability than cells of an isogenic wild‐type strain. Cell survival was augmented, and changes in biochemical parameters were ameliorated, by addition of exogenous antioxidants (ascorbic acid, glutathione and melatonin) in both strains.     

Ascorbate and thiol antioxidants abolish sensitivity of yeast Saccharomyces cerevisiae to disulfiram

Sensitivity of baker’s yeast to disulfiram (DSF) and hypersensitivity of a mutant devoid of Cu, Zn-superoxide dismutase to this compound is reported, demonstrating that yeast may be a simple convenient eukaryotic model to study the mechanism of DSF toxicity. DSF was found to induce oxidative stress in yeast cells demonstrated by increased superoxide production and decrease of cellular glutathione content. Anoxic atmosphere and hydrophilic antioxidants (ascorbate, glutathione, dithiothreitol, cysteine, and N-acetylcysteine) ameliorated DSF toxicity to yeast indicating that oxidative stress plays a critical role in the cellular action of DSF.     

Oxidative stress and antioxidant content in Chlorella vulgaris after exposure to ultraviolet-B radiation

Growth of Chlorella vulgaris was measured in cultures irradiated with 0, 0.8, 2.0 and 4.4 kJ m² UV-B. Growth expressed as chlorophyll content, declined significantly with increased UV-B dose. Ultraviolet-B irradiated cultures in log phase of growth showed a 284% increase in oxygen radical generation and a 145% increase in lipid peroxidation compared with unirradiated cultures, whereas cultures in the stationary growth phase showed no significant changes in these parameters. The activities of superoxide dismutase and catalase increased by 40 and 500%, respectively, after exposure to a UV-B dose of 4.4 kJ m⁻². Contents of the lipophilic antioxidants α-tocopherol and β-carotene increased by 180 and 63 amol cell⁻¹ respectively, between log and stationary phases in unirradiated cultures; but in UV-B-irradiated cultures these increases were significantly depressed. Photoreducing capacities of chloroplasts were decreased following UV-B irradiation of both isolated chloroplasts and those isolated from irradiated algae. Cells exposed to UV-B exhibited increased size and starch accumulation. These results suggest that oxidative stress conditions related to UV-B exposure trigger an antioxidant response that includes an increase in the activity of the antioxidant enzymes (superoxide dismutase and catalase).     

Stress response of yeast candida intermedia to Cr(VI)

Stress response of yeast Candida intermedia ZIM 156 exposed to chromium(VI) was investigated. Yeast cells were treated with Cr(VI) in concentrations of 50, 100, 300 and 500 microM in the mid-exponential growth phase. Monitoring of some bioprocess parameters during growth, specifically pO(2), showed that Cr(VI) addition, specifically in concentration of 100 and partially 50 micromol/L, increased metabolism intensity, which is connected to induced stress responses. Furthermore, oxidation of 2',7'-dichlorofluorescin indicated increased intracellular oxidant level, specifically at 100 microM Cr(VI) concentration. Antioxidant defense systems were further investigated. Catalase and superoxide dismutase activity was not increased in the cells exposed to the both Cr(VI) concentrations, which indicate that catalase and superoxide dismutase do not participate in cell defense systems. In contrast intracellular glutathione content in reduced form increased significantly in the cells exposed to 100 micromol Cr(VI)/L. Therefore, we demonstrated that glutathione plays an important role in the stress response of C. intermedia to Cr(VI).     

Ammonia production by IMR-90 fibroblast cultures: effects of ammonia on glutathione, gamma-glutamyl transpeptidase, lysosomal enzymes, and cell division

gamma-Glutamyl transpeptidase has multi-catalytic activities. It degrades glutathione and can produce ammonia from glutamine. The present study was designed to examine whether the decreased cell proliferation, cellular glutathione content and concurrent increase in ammonia production in senescent cells in culture are the result of increased gamma-glutamyl transpeptidase activity. We used IMR-90 fibroblast and 3T3 LI preadipocyte cultures. The cellular glutathione content depended upon cell proliferation and cell density. The glutathione content was higher in cells at logarithmic growth, and lower at stationary growth or post confluency; dead cells had no detectable glutathione by the method currently used. The glutathione content was minimal in "old" IMR-90 cells, regardless of cell density. On the other hand, an increase occurred in the unit number of molecules of bound 5-iodoacetoamidofluorescein, an active-site directed stoichiometric inhibitor of transpeptidase. That result corresponded favorably with the increased enzyme activity, suggesting that the number of enzyme molecules per cell was increased. The inhibition of ammonia production of the cultures by inhibition of gamma-glutamyl transpeptidase by 5-iodoacetoamidofluorescein and reversible inhibition of ammonia production by a serine-borate mixture were consistent with our postulate. Addition of NH4Cl (0.1 mM) to IMR-90 cultures caused increased activities of transpeptidase and some of the lysosomal enzymes; concurrently, the amount of cellular glutathione and the number of cell divisions decreased. This suggests that the increased ammonia production presumably resulting from glutaminase activity of the observed increase of transpeptidase may profoundly affect certain cellular functions.     

Enhanced heme oxygenase activity increases the antioxidant defense capacity of guinea pig liver upon acute cobalt chloride loading: comparison with rat liver

Changes in the activity of so-called oxidative stress defensive enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and heme oxygenase, as well as changes in lipid peroxidation and reduced glutathione levels, were measured in guinea pig and rat liver after acute cobalt loading. Cobalt chloride administration produced a much higher degree of lipid peroxidation in guinea pig than in rat liver compared with the control animals. The intrahepatic reduced glutathione content in control guinea pig was higher than that in rat, but was equally decreased in both species after cobalt administration. The enzymatic scavengers of free radicals, superoxide dismutase, catalase and glutathione peroxidase, were significantly decreased in rat liver after acute cobalt loading, and as a compensatory reaction, the heme oxygenase activity was increased (seven-fold). In guinea pig liver, only superoxide dismutase activity was depleted in response to cobalt-induced oxidative stress, while catalase and glutathione peroxidase were highly activated and the heme oxygenase activity was dramatically increased (13-fold). It is assumed that enhanced heme oxygenase activity may have important antioxidant significance by increasing the liver oxidative-stress defense capacity.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Accumulation of hydrogen peroxide and functioning of defense system in overwatered barley seedlings

The effects of overwatering (flooding) on the oxidative potential, the level of low-molecular-weight antioxidants, the content of stress proteins, and activities of antioxidant enzymes in green barley (Hordeum vulgare L.) seedlings were studied. Overwatering retarded barley seedling growth and induced hydrogen peroxide accumulation, a decrease in the total ascorbate content and an increase in the content of reduced glutathione (GSH), but it did not affect the content of oxidized glutathione (GSSG). After the cessation of stress factor action (post-stress period), the content of hydrogen peroxide declined to the initial level, the content of ascorbate reduced still stronger, whereas the content of GSH continued to rise. Under flooding conditions, activities of glutathione reductase (GR) and superoxide dismutase (SOD) increased. After the cessation of stress factor action, activities of these enzymes decreased but remained at rather high levels as compared with control. Activity of catalase (CAT) reduced during stress, whereas activity of ascorbate peroxidase (APX) was not essentially changed. In the post-stress period, CAT activity remained to be low; in contrast, APX activity increased. Barley seedling flooding induced the synthesis of stress proteins, HSP70 and dehydrins (DH). In the post-stress period, the content of stress proteins decreased; however, the content of DH in experimental leaves remained rather high. The results obtained indicate that barley defense system manifested a complex response to overwatering, which may be related to the oxygen shortage under stress conditions and sharp metabolism activation at re-aeration in the post-stress period.     

Synthesis and localization of L-lactate oxidase in yeasts Yarrowia lipolytica

Conditions for L-lactate oxidase synthesis by the yeast Yarrowia lipolytica were investigated. The enzyme was found to be synthesized during growth on L-lactate in the exponential growth phase. L-lactate oxidase synthesis was also observed on glucose after adaptation to stress conditions (oxidative or thermal stress) during the stationary growth phase after glucose consumption. The cells grown on L-lactate exhibited high levels of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase), which exceeded those of glucose-grown cells. Ultrastructurally, L-lactate-grown cells and the cells grown on glucose and adapted to various stress conditions were also found to be similar, with increased mitochondria, elevated number and size of peroxisomes, and formation of lipid and polyphosphate inclusions. In order to determine the intracellular localization of L-lactate oxidase, the cells were disintegrated by the lytic enzyme complex from Helix pomatia. Centrifugation of the homogenate in Percoll gradient resulted in the isolation of purified fractions of the native mitochondria and peroxisomes. L-lactate oxidase was shown to be localized in peroxisomes.     

Ethanol-induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and gamma-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, gamma-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration.     

Monitoring <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> NCYC 921 batch fermentations growing under carbon and nitrogen limitation by flow cytometry

The yeast Rhodosporidium toruloides NCYC 921 was grown on carbon or nitrogen limited batch cultures. The fermentations were monitored using traditional techniques and multi-parameter flow cytometry. The lipid content was assessed by flow cytometry in association with the fluorocrome Nile Red which emits yellow gold fluorescence when dissolved in neutral lipids and red fluorescence when dissolved in polar lipids. In this way, it was possible to at-line monitor the yeast lipid composition in terms of polarity classes throughout the batch growths. It was found that the neutral lipids decreased during the carbon-limited stationary phase, and increased during the nitrogen-limited batch growth. The maximum lipid content was obtained for the nitrogen-limited yeast culture (24% w/w lipids). The yeast cells with permeabilised membranes profile remained almost unchanged during the time course of both fermentations. The scatter light measurements (forward and side scatter signals) provided information on the yeast growth phase. The multi-parameter flow cytometric approach here reported represents a better control system based on measurements made at the single cell level for optimization of the yeast lipid production bioprocess performance.     

A possible competition between 5'-monodeiodination of thyroxine and the respiratory burst in human granulocytes

Thyroxine (T4) pretreatment of A 23187-stimulated human granulocytes in 10(-5)-10(-6) M concentration range inhibited the superoxide anion production of these cells. T4 increased the level of oxidized form of glutathione, whereas the intracellular level of the reduced form decreased. A similar alteration in the ratio of the oxidized to reduced forms of glutathione was detected in granulocytes during yeast cell phagocytosis. In addition, conversion of T4 to triiodothyronine (T3) was also inhibited during phagocytosis. A possible competition between 5'-monodeiodination of T4 and the oxidative burst of human granulocytes is discussed.     

Effect of cellular glutathione content on the induction of DNA double strand breaks by 25 MeV electrons

The effect of endogenous glutathione (GSH) on the induction of DNA double strand breaks (dsb) by 25 MeV electrons was investigated using stationary haploid yeast cells defective in gamma-glutamyl-cysteine-synthetase (gsh 1) containing less than 5 per cent of the normal GSH content. In gsh 1 cells the induction of dsb is increased by a factor of 1.5 under oxic and 1.8 under anoxic irradiation conditions: whereas the oxygen enhancement ratio was only slightly decreased (1.9) compared to wild-type cells (2.4).     

Antioxidant system and photosynthetic characteristics responses to short-term PEG-induced drought stress in cucumber seedling leaves

The effect of short-term drought stress on the water content, antioxidant system and photosynthetic characteristics was investigated using cucumber (Cucumis sativus L.) seedlings. The results indicated that polyethylene glycol induced water stress reduced water content in shoots of cucumber seedling after treatment of 36 hours, and caused obvious reductions in net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration and transpiration of leaves. In addition, water stress significantly reduced the photosynthetic pigment content and inhibited photochemical activity, including actual photochemical efficiency, maximal quantum yield of photosystem II photochemistry and coefficient for photochemical quenching. Meanwhile non-photochemical quenching increased. As responses to drought stress, significant increases in electrolyte leakage, malondialdehyde, superoxide anion and hydrogen peroxide levels were detected in leaves. The superoxide dismutases, catalase, glutathione reductase and dehydroascorbate reductase activities, protein, ascorbate and glutathione content, all decreased and peroxidases activity increased, while ascorbate peroxidase and monodehydroascorbate reductase activities exhibited different trend under different degree of water stress. Therefore, it can be concluded that water stress strongly disrupted the normal metabolism of leaves and restrained water absorption.     

Influences of magnesium deficiency and cerium on antioxidant system of spinach chloroplasts

Magnesium-deficiency conditions applied to spinach cultures caused an oxidative stress status in spinach chloroplast monitored by an increase in reactive oxygen species (ROS) accumulation. The enhancement of lipids peroxide of spinach chloroplast grown in magnesium-deficiency media suggested an oxidative attack that was activated by a reduction of antioxidative defense mechanism measured by analysing the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase, as well as antioxidants such as carotenoids and glutathione content. As the antioxidative response of chloroplast was reduced in spinach grown in magnesium-deficiency media, it caused a significant reduction of spinach plant weight, old leaves turning chlorosis. However, cerium treatment grown in magnesium-deficiency conditions decreased the malondialdehyde and ROS, and increased activities of the antioxidative defense system, and improved spinach growth. Together, the experimental study implied that cerium could partly substitute for magnesium and increase the oxidative stress-resistance of spinach chloroplast grown in magnesium-deficiency conditions, but the mechanisms need further study.     

Effects of Glutathione Modulation on Oxidative Stress and Enzymatic Antioxidant Defence in Yeast Pachysolen tannophilus

The aim of this study was to explore the relationship of intracellular glutathione with various oxidative stress markers and the stress protectant marker trehalose. In the first group of yeast cells, diethyl maleate was used for depletion of glutathione. A second group of yeast cells were incubated with amino acids constituting glutathione (GIu, Cys, Gly) to increase glutathione level. Increased level of oxidative stress marker like ROS, protein carbonyl formation and lipid peroxidation and decreased viability in glutathione-depleted cells were observed in the present study. The increased activity of antioxidant enzymes SOD and CAT in the glutathione depleted group suggests the interaction of different antioxidant defence system in Pachysolen tannophilus. Furthermore, the increased levels of trehalose in glutathione-depleted group shows that trehalose acts as a stress reducer in glutathione depleted Pachysolen tannophilus.