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Oxidative stress during aging of stationary cultures of the yeast Saccharomyces cerevisiae
Free Radical Biology & Medicine, Vol. 28, No. 5, pp. 659 – 664, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter
PII S0891-5849(99)00266-X
Original Contribution OXIDATIVE STRESS DURING AGING OF STATIONARY CULTURES OF THE YEAST SACCHAROMYCES CEREVISIAE WITOLD JAKUBOWSKI,* TOMASZ BILIN´ SKI†
and
GRZEGORZ BARTOSZ*†
*Department of Molecular Biophysics, University of L 兾 o´dz´, L 兾 o´dz´, Poland and †Institute of Biology and Environmental Sciences, Pedagogical University of Rzeszo´w, Rzeszo´w, Poland (Received 7 June 1999; Revised 7 December 1999; Accepted 7 December 1999)
Abstract—Comparison of 5 d old stationary cultures of Saccharomyces cerevisiae and of cultures aged for 3 months revealed increased generation of reactive oxygen species assessed by 2⬘,7⬘-dichlorofluorescin oxidation, decreased activity of superoxide dismutase, decreased content of glutathione and increased protein carbonyl content during prolonged incubation of stationary yeast cultures. These results point to the occurrence of oxidative stress during aging of stationary cultures of the yeast. The magnitude of this stress was augmented in antioxidant-deficient strains, devoid of superoxide dismutases and catalases, and of decreased glutathione content. © 2000 Elsevier Science Inc. Keywords—Yeast, Saccharomyces cerevisiae, Aging, Oxidative stress, Stationary culture, Superoxide dismutase, Catalase, Glutathione, Protein carbonyl, Free radicals
INTRODUCTION
cerevisiae processes contributing to replicative aging take place. The free radical theory of aging postulates that free radical–mediated oxidative damage constitutes an important if not the main cause of cellular aging [10]; it can also be suspected to underlie the deleterious effects of incubation of stationary cultures on yeast longevity. The present study was aimed at examination whether oxidative stress occurs during prolonged incubation of stationary cultures of S. cerevisiae.
Individual cells of the yeast Saccharomyces cerevisiae are capable of a limited number of divisions and in this sense are subject to replicative aging. Thus, S. cerevisiae constitutes an interesting model for studies of the aging process, which makes possible experimentation not easily available in higher eukaryotes. A number of genes have been identified in S. cerevisiae, which affect the replicative life span of this organism [1– 6], and may be a starting point for search of homologous genes in other species. Stationary cultures of yeast seem to constitute another interesting model for the studies of aging. When yeast culture enters the stationary phase, cell division stops and cellular metabolic is slowed down. Cells are able to survive in this phase for weeks or months without nutrient supplementation [7]. Interestingly, the replicative life span of individual yeast cells decreases when yeast are stored for prolonged times in stationary cultures [8], a phenomenon reminiscent to that observed for human fibroblasts [9]. It suggests that during incubation of stationary cultures of S.
MATERIALS AND METHODS
Cell cultures The following Saccharomyces cerevisiae strains were obtained from the Department of Biochemistry, Institute of Agriculture at Zamos´c´, Agricultural University of Lublin: Sp4 (wild-type), phenotype Mat␣ leu1 arg4, A50 (catalase-deficient mutant), phenotype Mata leu1 arg4; ctt1 cta1; C4 (glutathione-poor mutant), phenotype Mat␣ leu1 arg4 als1* and DSCD6-6B (double mutant devoid of Cu,ZnSOD and MnSOD), phenotype Mat␣ ura3 sod1 sod2. The yeast were grown in liquid YPG medium 1% yeast extract (Difco), 1% bactopeptone (Difco, Detroit, MI, USA), and 2% glucose (POCh, Gliwice, Poland) at 28°C.
Address correspondence to: Witold Jakubowski, M. Sc., Department of Molecular Biophysics, University of L 兾 o´dz´, Banacha 12/16, 90-137 L 兾 o´dz´, Poland; Tel: ⫹48 (42) 6354476; Fax: ⫹48 (42) 6354473; E-Mail: [email protected]. 659
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W. JAKUBOWSKI et al. Table 1. Percent of Viable Yeast Cells After 3 Month Cultivation in Yeast Stationary Cultures Estimated Using the Live/Death Test (mean ⫾ SD) Strain Sp4 A50 C4 DSCD6-6B
Percentage of viable cells 92.1 ⫾ 5.1 89.4 ⫾ 6.3 83.2 ⫾ 5.6 86.4 ⫾ 6.5
Stationary cultures Yeast cultures obtained by growing cells from inoculum for 5 d were assumed as “young” stationary cultures. “Old” stationary cultures were obtained by incubating stationary cultures for 3 months at 28°C. During this time the cultures were supplied with fresh complete medium two times a week to prevent carbon source depletion. Each time the cells were allowed to sediment and 80 ml of the supernatant of a 110 ml culture was withdrawn and replaced by 80 ml of fresh medium. Cell density was about (5.1–5.5) ⫻ 108 cells/ml and did not change significantly during the incubation period. Cell viability was estimated using the LIVE/DEATH test (Molecular Probes, Eugene, OR, USA). The fraction of dead cells rose in course of time of experiment but did not exceed 17% of the population after 3 months (Table 1).
sodium carbonate buffer, pH ⫽ 10.2, suspended in the buffer at a concentration of 2 % (v/v) and broken with glass beads (4°C, 10 min). To a spectrophotometric cuvette containing 500 l of the carbonate buffer containing 200 M xanthine, 50 M NBT, and 200 M ethylenediaminetetraacetate (EDTA) was added xanthine oxidase and the buffer up to the volume of 1000 l. The amount of xanthine oxidase was adjusted so to obtain a rate of absorption increase at 560 nm of 0.0165 per min ( ⫽ 560 nm) at 25°C. Similarly cuvettes containing variable amounts of the yeast homogenate were measured. One unit of SOD activity was defined as the amount of the enzyme resulting in a decrease of the rate of absorbance increase by 50%. Catalase activity Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM sodium phosphate buffer, pH ⫽ 7.4, suspended in the buffer at a concentration of 2% (v/v) and broken by shaking the sediment with glass beads (4°C, 10 min). 333 l of aqueous solution of H2O2 (54 mM) were mixed with 567 l of buffer (pH 7.0) and quickly added with 100 l of cells homogenate and the rate of decrease of absorbance at 240 nm was measured. One unit of enzyme activity corresponded to decomposition of 1 mole substrate during 1 min.
2⬘,7⬘-Dichlorofluorescin oxidation
Total glutathione (GSH ⫹ GSSG)
Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM sodium phosphate buffer, pH ⫽ 7.4, suspended in the buffer at a concentration of 2% (v/v) and preincubated at 28°C for 15 min. H2DCFDA (carboxyfluorescin diacetate, Molecular Probes) was added as stock 1 mM ethanol solutions to final a concentration of 10 M [11,12]). After incubation (28°C, 20 min) cell suspensions were sedimented by centrifugation, suspended in the buffer and broken by shaking the sediment with glass beads (4°C, 10 min). The homogenates were clarified by centrifugation (3500 ⫻ g, 3 min) and fluorescence of the supernatant was measured in a Perkin-Elmer (Beaconfield, UK) LS-5B spectrofluorimeter (excitation and emission wavelengths of 488 and 520 nm, respectively).
Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed, suspended in 50 mM potassium phosphate buffer (pH 7.4) at a concentration of 10% (v/v) and added with an equal volume of cold 2 M HClO4 containing 4 mM EDTA and mixed thoroughly. After 15 min incubation the suspensions were centrifuged; 100 l aliquots of the supernatants of KOHneutralized perchloric-acid extracts were added to cuvettes containing 1000 l of 100 mM potassium phosphate buffer (pH 7.0) containing 1mM EDTA, 50 l 0.4% nicotinamide adenine dinucleotide phosphate (reduced form) in 0.5% NaHCO2, 20 l 0.15% 5,5⬘-dithiobis(2-nitrobenzoic acid) and 20 l of glutathione reductase solution (activity of 6 U/ml). The reaction rate measured as an increase in absorbance at 412 nm is proportional to the glutathione concentration [14].
Superoxide dismutase (SOD) activity SOD activity was estimated with xanthine ⫹ xanthine oxidase and Nitro Blue Tetrazolium (NBT) [13]. Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM
Protein carbonyl groups Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM sodium phosphate buffer, pH ⫽ 7.4, suspended
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in this buffer and brought to a protein concentration of 10 mg/ml. Duplicate aliquots of the suspension (0.5 ml) were and added with 0.5 ml of 10% trichloroacetic acid. The samples were centrifuged and the precipitates were added with 1 ml of either 2M HCl or 0.2% 2,4-dinitrophenylhydrazine (DNPH) in 2M HCl and incubated at 37°C for 60 min. Then protein was dissolved by addition of 600 mg of guanidine hydrochloride, dinitrophenylhydrazine excess was removed by passing the samples through Sephadex G25 columns (Pharmacia, Upsala, Sweden), the eluates were brought to protein concentration of 1 mg/ml and absorbance of the DNPH-treated sample was read against the HCl-treated sample. The carbonyl content was calculated using millimolar absorption coefficient of the hydrazone of 21.0 mmol-1 cm-1 [15]. The results are mean ⫾ SD of at least four independent measurements. The values measured are expressed per milliliter cells (determined from cytocrit measurements of concentrated cell suspensions in a microhematocrit centrifuge) rather than per cell number due to small interstrain differences in cell size. For the wild-type strain (Sp4) cytocrit of 2% corresponds to 5.4 ⫻ 108 cells per milliliter. RESULTS
We compared biochemical changes in wild-type yeast and yeast strains deficient in various elements of the antioxidant defense during prolonged incubation (aging) of stationary cultures of S. cerevisiae. The content of glutathione decreased during aging of yeast stationary cultures. The magnitude of this decrease was different in different strains of S. cerevisiae and was higher for the catalase-deficient and, especially, for the SOD-deficient strain (Fig. 1). SOD activity decreased considerably during aging of yeast stationary cultures. The decrease was the highest for the wild-type strain; however, the initial levels of SOD activity were different in different strains and that in the glutathione-deficient strain was lowered already in young cultures (Fig. 2). Catalase activity was increased in aged stationary cultures, the increase was most pronounced in the wildtype strain and the lowest in the SOD-deficient strain (Fig. 3). The formation of reactive oxygen species measured by H2O2-dependent oxidation of H2DCF was significantly augmented in old stationary cultures of the yeast (Fig. 4). The level of protein carbonyl groups, a measure of the oxidative damage to cellular macromolecules, was increased in all strains. The increase was higher in the antioxidant-deficient strains than in the control strain, the
Fig. 1. Glutathione concentration in young (5 d) and old (3 month) stationary cultures of different strains of S. cerevisiae. Empty bars, young cultures; filled bars, old cultures. Top: absolute concentrations, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
most pronounced increase being noted for the glutathione-deficient yeast (Fig. 5). DISCUSSION
Our experimental system differs somewhat from the most commonly used one in which stationary cultures are incubated without nutritional additives and cells are subject to metabolic starvation. In order to avoid the latter effect we changed the medium periodically during long-term incubation of stationary cultures. This procedure did not lead to a detectable population growth during the period of experiment, apparently due to growth inhibition by the crowd-sensing effect and provided high viability of cells even after the 3 month period. The obtained results evidence the occurrence of oxidative stress and accumulation of oxidative damage during long-term incubation (“aging”) of stationary cultures of S. cerevisiae. The increased rate of H2DCF oxidation in old cultures of the yeast indicates an increased level of reactive oxygen species in these cultures. This phenomenon may be due to increased generation of reactive
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Fig. 2. Superoxide dismutase activity in young and old stationary cultures of different strains of S. cerevisiae. Empty bars ⫽ young cultures; filled bars ⫽ old cultures. Top: absolute activities, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
Fig. 3. Catalase activity in young and old stationary cultures off different strains of S. cerevisiae. Empty bars ⫽ young cultures; filled bars ⫽ old cultures. Top: absolute activities, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
oxygen species by cellular sources and/or to the decreased antioxidant defense, especially the lower level of glutathione. The increased content of protein carbonyl groups as well as decreased SOD activity also suggests an increased exposure of macromolecules to reactive oxygen species. SOD is known to be inactivated by exposure to hydrogen peroxide and other peroxides while catalase and glutathione peroxidase are inactivated by superoxide [16,17]. Therefore, decreased antioxidative defense in aged stationary cultures may lead to a self-perpetuating damage in spite of the increase in antioxidative defense on entering the stationary phase. However, an alternative explanation that the accumulation of protein carbonyls and decreased activity of SOD are due to decreased protein turnover in stationary cultures cannot be excluded. The oxidative stress in stationary cultures of yeast may be connected to the better oxygen access to the cells due to decreased metabolism. Metabolically active cells intensively consume oxygen under conditions of limited aeration which may lead to its low concentration within the cells. In stationary cultures the metabolism is considerably attenuated so the oxygen concentration within cells may be higher; one-electron reactions of autoxida-
tion-prone cellular components may produce superoxide and, in turn, other reactive oxygen species. Mitochondrial respiration have been suggested to be the major source of reactive oxygen species under these conditions, even or especially under conditions of low aeration. While under high aeration of long-term stationary cultures of S. cerevisiae yeast mutants devoid of Cu,ZnSOD were less viable than MnSOD mutants, this situation was reversed under low aeration when mutants devoid of mitochondrial MnSOD showed a more severe growth reduction [18]. An alternative source of oxygen free radicals, detectable on freezing and/or thawing of the yeast, has also been identified in the cytoplasm [19] and may contribute to the oxidative damage. Similar changes (accumulation of protein carbonyls and disulfide bridges) [20] accompanied by protein racemization [21] have been observed during incubation of stationary cultures of Escherichia coli. Oxidative stress may be a factor limiting the survival of microorganisms in long-term stationary culture. S. cerevisiae mutants devoid of either Cu,ZnSOD or MnSOD show lower survival in stationary cultures [18,22]. In Rhodobacter capsulatus, a similar decrease in stationary phase survival was noted for mutants lacking catalase-peroxidase
Aging of yeast cultures
Fig. 4. Oxidation of 2⬘7⬘-dichlorofluorescin in young and old stationary cultures of different strains of S. cerevisiae. Empty bars, young cultures; filled bars, old cultures. Top: relative fluorescence values read; bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
[23]. The increase in antioxidant content and increase in the levels of antioxidant enzymes including SOD [24] and glutathione reductase [25] on yeast entering into the stationary phase may, therefore, constitute an adaptive response to the enhanced oxidative danger. The increase in catalase activity may, however, represent a stress response; this enzyme is easily inducible in yeast by various types of stress. The transcription of CTT1 gene which codes catalase, is regulated by the cAMP level [26,27] and the deficiency of cAMP induces the biosynthesis of catalase. A drop of cAMP was observed on various types of stress, in particular in oxidative and heat stress [28,29]. It can be suggested that the accumulation of oxidative damage in stationary cultures of yeast may be reminiscent of processes taking place in nondividing mammalian cells in culture or postmitotic cells in a multicellular organism. Fibroblasts which remain proliferation-inhibited by confluency for up to 3 months show increased frequency of single-stranded sites in telomeres, measured as sensitivity to degradation by S1 nuclease, apparently due to oxidative damage [30]. This similarity is limited in some aspects. Most of the yeast cells can resume divisions on dilution. We were unable to demonstrate
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Fig. 5. The level of protein carbonyl groups in young and old stationary cultures of different strains of S. cerevisiae. Empty bars ⫽ young cultures; filled bars ⫽ old cultures. Top: absolute concentrations, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 5.
accumulation of lipofuscinlike material in old stationary cultures of the yeast (not shown). Nevertheless, as suggested by other authors [8,18,31,32], stationary yeast cultures may represent a model system, with respect to cells of higher eucaryotes, of oxidative damage to nondividing cells. Acknowledgement — This study was partially supported by Grant No. 512/P04/97/13 of the Polish Committee for Scientific Research.
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aspartyl residues is important in Escherichia coli stationary-phase survival and heat-shock resistance. Proc. Natl. Acad. Sci. USA 89:9885–9889; 1992. Longo, V. D.; Liou, L. L.; Valentine, J. S.; Gralla, E. B. Mitochondrial superoxide decreases yeast survival in stationary phase. Arch. Biochem. Biophys. 365:131–142; 1999. Hochman, A.; Figueredo, A.; Wall, J. D. Physiological functions of hydroperoxidases in Rhodobacter capsulatus. J. Bacteriol. 174:3386 –3391;1992. Flattery-O’Brien, J. A.; Grant, C. M.; Daews, I. W. Stationaryphase regulation of the Saccharomyces cerevisiae SOD2 gene is dependent on additive effects of HAP2/3/4/5- and STRE-binding elements. Mol. Microbiol. 23:303–312; 1997. Grant, C. M.; Maciver, F. H.; DAews, I. W. Stationary-phase induction of GLR1 expression is mediated by the yAP-1 transcriptional regulatory protein in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 22:739 –746; 1996. Lida, H. Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5⬘ coding region of the adenylate cyclase gene. Mol. Cell. Biol. 8:5555– 5560; 1988. Shin, D. Y.; Matsumoto, K.; Lida, H.; Uno, I.; Ishikawa, T. Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation. Mol. Cell Biol. 7:244 –250; 1987. Schu¨ller, C. H.; Brewster, J. L.; Alexander, M. R.; Gustin, M. C.; Ruis, H. The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene. EMBO J. 13:4382– 4389; 1994. Wieser, R.; Adam, G.; Wagner, A.; Schu¨ller, C. H.; Marchler, G.; Ruis, H.; Krawiec, Z.; Bilin´ski, T. Heat shock factor-independent heat control of transcription of the CTT1 gene encoding the cytosolic catalase T of Saccharomyces cerevisiae. J. Biol. Chem. 266:12406 –12411; 1991. Sitte, N.; Saretzki, G.; von Zglinicki, T. Accelerated telomere shortening in fibroblasts after extended periods of confluency. Free Radic. Biol. Med.24:885– 893; 1998. Jazwinski, S. M. An experimental system for the molecular analysis of the aging process: the budding yeast Saccharomyces cerevisiae. J. Gerontol. 45:B68 –B74; 1990. Kim, S.; Villeponteau, B.; Jazwinski, S. M. Effect of replicative age on transcriptional silencing near telomeres in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 219:370 –376; 1996.
PII S0891-5849(99)00266-X
Original Contribution OXIDATIVE STRESS DURING AGING OF STATIONARY CULTURES OF THE YEAST SACCHAROMYCES CEREVISIAE WITOLD JAKUBOWSKI,* TOMASZ BILIN´ SKI†
and
GRZEGORZ BARTOSZ*†
*Department of Molecular Biophysics, University of L 兾 o´dz´, L 兾 o´dz´, Poland and †Institute of Biology and Environmental Sciences, Pedagogical University of Rzeszo´w, Rzeszo´w, Poland (Received 7 June 1999; Revised 7 December 1999; Accepted 7 December 1999)
Abstract—Comparison of 5 d old stationary cultures of Saccharomyces cerevisiae and of cultures aged for 3 months revealed increased generation of reactive oxygen species assessed by 2⬘,7⬘-dichlorofluorescin oxidation, decreased activity of superoxide dismutase, decreased content of glutathione and increased protein carbonyl content during prolonged incubation of stationary yeast cultures. These results point to the occurrence of oxidative stress during aging of stationary cultures of the yeast. The magnitude of this stress was augmented in antioxidant-deficient strains, devoid of superoxide dismutases and catalases, and of decreased glutathione content. © 2000 Elsevier Science Inc. Keywords—Yeast, Saccharomyces cerevisiae, Aging, Oxidative stress, Stationary culture, Superoxide dismutase, Catalase, Glutathione, Protein carbonyl, Free radicals
INTRODUCTION
cerevisiae processes contributing to replicative aging take place. The free radical theory of aging postulates that free radical–mediated oxidative damage constitutes an important if not the main cause of cellular aging [10]; it can also be suspected to underlie the deleterious effects of incubation of stationary cultures on yeast longevity. The present study was aimed at examination whether oxidative stress occurs during prolonged incubation of stationary cultures of S. cerevisiae.
Individual cells of the yeast Saccharomyces cerevisiae are capable of a limited number of divisions and in this sense are subject to replicative aging. Thus, S. cerevisiae constitutes an interesting model for studies of the aging process, which makes possible experimentation not easily available in higher eukaryotes. A number of genes have been identified in S. cerevisiae, which affect the replicative life span of this organism [1– 6], and may be a starting point for search of homologous genes in other species. Stationary cultures of yeast seem to constitute another interesting model for the studies of aging. When yeast culture enters the stationary phase, cell division stops and cellular metabolic is slowed down. Cells are able to survive in this phase for weeks or months without nutrient supplementation [7]. Interestingly, the replicative life span of individual yeast cells decreases when yeast are stored for prolonged times in stationary cultures [8], a phenomenon reminiscent to that observed for human fibroblasts [9]. It suggests that during incubation of stationary cultures of S.
MATERIALS AND METHODS
Cell cultures The following Saccharomyces cerevisiae strains were obtained from the Department of Biochemistry, Institute of Agriculture at Zamos´c´, Agricultural University of Lublin: Sp4 (wild-type), phenotype Mat␣ leu1 arg4, A50 (catalase-deficient mutant), phenotype Mata leu1 arg4; ctt1 cta1; C4 (glutathione-poor mutant), phenotype Mat␣ leu1 arg4 als1* and DSCD6-6B (double mutant devoid of Cu,ZnSOD and MnSOD), phenotype Mat␣ ura3 sod1 sod2. The yeast were grown in liquid YPG medium 1% yeast extract (Difco), 1% bactopeptone (Difco, Detroit, MI, USA), and 2% glucose (POCh, Gliwice, Poland) at 28°C.
Address correspondence to: Witold Jakubowski, M. Sc., Department of Molecular Biophysics, University of L 兾 o´dz´, Banacha 12/16, 90-137 L 兾 o´dz´, Poland; Tel: ⫹48 (42) 6354476; Fax: ⫹48 (42) 6354473; E-Mail: [email protected]. 659
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W. JAKUBOWSKI et al. Table 1. Percent of Viable Yeast Cells After 3 Month Cultivation in Yeast Stationary Cultures Estimated Using the Live/Death Test (mean ⫾ SD) Strain Sp4 A50 C4 DSCD6-6B
Percentage of viable cells 92.1 ⫾ 5.1 89.4 ⫾ 6.3 83.2 ⫾ 5.6 86.4 ⫾ 6.5
Stationary cultures Yeast cultures obtained by growing cells from inoculum for 5 d were assumed as “young” stationary cultures. “Old” stationary cultures were obtained by incubating stationary cultures for 3 months at 28°C. During this time the cultures were supplied with fresh complete medium two times a week to prevent carbon source depletion. Each time the cells were allowed to sediment and 80 ml of the supernatant of a 110 ml culture was withdrawn and replaced by 80 ml of fresh medium. Cell density was about (5.1–5.5) ⫻ 108 cells/ml and did not change significantly during the incubation period. Cell viability was estimated using the LIVE/DEATH test (Molecular Probes, Eugene, OR, USA). The fraction of dead cells rose in course of time of experiment but did not exceed 17% of the population after 3 months (Table 1).
sodium carbonate buffer, pH ⫽ 10.2, suspended in the buffer at a concentration of 2 % (v/v) and broken with glass beads (4°C, 10 min). To a spectrophotometric cuvette containing 500 l of the carbonate buffer containing 200 M xanthine, 50 M NBT, and 200 M ethylenediaminetetraacetate (EDTA) was added xanthine oxidase and the buffer up to the volume of 1000 l. The amount of xanthine oxidase was adjusted so to obtain a rate of absorption increase at 560 nm of 0.0165 per min ( ⫽ 560 nm) at 25°C. Similarly cuvettes containing variable amounts of the yeast homogenate were measured. One unit of SOD activity was defined as the amount of the enzyme resulting in a decrease of the rate of absorbance increase by 50%. Catalase activity Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM sodium phosphate buffer, pH ⫽ 7.4, suspended in the buffer at a concentration of 2% (v/v) and broken by shaking the sediment with glass beads (4°C, 10 min). 333 l of aqueous solution of H2O2 (54 mM) were mixed with 567 l of buffer (pH 7.0) and quickly added with 100 l of cells homogenate and the rate of decrease of absorbance at 240 nm was measured. One unit of enzyme activity corresponded to decomposition of 1 mole substrate during 1 min.
2⬘,7⬘-Dichlorofluorescin oxidation
Total glutathione (GSH ⫹ GSSG)
Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM sodium phosphate buffer, pH ⫽ 7.4, suspended in the buffer at a concentration of 2% (v/v) and preincubated at 28°C for 15 min. H2DCFDA (carboxyfluorescin diacetate, Molecular Probes) was added as stock 1 mM ethanol solutions to final a concentration of 10 M [11,12]). After incubation (28°C, 20 min) cell suspensions were sedimented by centrifugation, suspended in the buffer and broken by shaking the sediment with glass beads (4°C, 10 min). The homogenates were clarified by centrifugation (3500 ⫻ g, 3 min) and fluorescence of the supernatant was measured in a Perkin-Elmer (Beaconfield, UK) LS-5B spectrofluorimeter (excitation and emission wavelengths of 488 and 520 nm, respectively).
Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed, suspended in 50 mM potassium phosphate buffer (pH 7.4) at a concentration of 10% (v/v) and added with an equal volume of cold 2 M HClO4 containing 4 mM EDTA and mixed thoroughly. After 15 min incubation the suspensions were centrifuged; 100 l aliquots of the supernatants of KOHneutralized perchloric-acid extracts were added to cuvettes containing 1000 l of 100 mM potassium phosphate buffer (pH 7.0) containing 1mM EDTA, 50 l 0.4% nicotinamide adenine dinucleotide phosphate (reduced form) in 0.5% NaHCO2, 20 l 0.15% 5,5⬘-dithiobis(2-nitrobenzoic acid) and 20 l of glutathione reductase solution (activity of 6 U/ml). The reaction rate measured as an increase in absorbance at 412 nm is proportional to the glutathione concentration [14].
Superoxide dismutase (SOD) activity SOD activity was estimated with xanthine ⫹ xanthine oxidase and Nitro Blue Tetrazolium (NBT) [13]. Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM
Protein carbonyl groups Cells were sedimented by centrifugation from 2 ml aliquots of the cultures, washed three times with 2 ml of 50 mM sodium phosphate buffer, pH ⫽ 7.4, suspended
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in this buffer and brought to a protein concentration of 10 mg/ml. Duplicate aliquots of the suspension (0.5 ml) were and added with 0.5 ml of 10% trichloroacetic acid. The samples were centrifuged and the precipitates were added with 1 ml of either 2M HCl or 0.2% 2,4-dinitrophenylhydrazine (DNPH) in 2M HCl and incubated at 37°C for 60 min. Then protein was dissolved by addition of 600 mg of guanidine hydrochloride, dinitrophenylhydrazine excess was removed by passing the samples through Sephadex G25 columns (Pharmacia, Upsala, Sweden), the eluates were brought to protein concentration of 1 mg/ml and absorbance of the DNPH-treated sample was read against the HCl-treated sample. The carbonyl content was calculated using millimolar absorption coefficient of the hydrazone of 21.0 mmol-1 cm-1 [15]. The results are mean ⫾ SD of at least four independent measurements. The values measured are expressed per milliliter cells (determined from cytocrit measurements of concentrated cell suspensions in a microhematocrit centrifuge) rather than per cell number due to small interstrain differences in cell size. For the wild-type strain (Sp4) cytocrit of 2% corresponds to 5.4 ⫻ 108 cells per milliliter. RESULTS
We compared biochemical changes in wild-type yeast and yeast strains deficient in various elements of the antioxidant defense during prolonged incubation (aging) of stationary cultures of S. cerevisiae. The content of glutathione decreased during aging of yeast stationary cultures. The magnitude of this decrease was different in different strains of S. cerevisiae and was higher for the catalase-deficient and, especially, for the SOD-deficient strain (Fig. 1). SOD activity decreased considerably during aging of yeast stationary cultures. The decrease was the highest for the wild-type strain; however, the initial levels of SOD activity were different in different strains and that in the glutathione-deficient strain was lowered already in young cultures (Fig. 2). Catalase activity was increased in aged stationary cultures, the increase was most pronounced in the wildtype strain and the lowest in the SOD-deficient strain (Fig. 3). The formation of reactive oxygen species measured by H2O2-dependent oxidation of H2DCF was significantly augmented in old stationary cultures of the yeast (Fig. 4). The level of protein carbonyl groups, a measure of the oxidative damage to cellular macromolecules, was increased in all strains. The increase was higher in the antioxidant-deficient strains than in the control strain, the
Fig. 1. Glutathione concentration in young (5 d) and old (3 month) stationary cultures of different strains of S. cerevisiae. Empty bars, young cultures; filled bars, old cultures. Top: absolute concentrations, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
most pronounced increase being noted for the glutathione-deficient yeast (Fig. 5). DISCUSSION
Our experimental system differs somewhat from the most commonly used one in which stationary cultures are incubated without nutritional additives and cells are subject to metabolic starvation. In order to avoid the latter effect we changed the medium periodically during long-term incubation of stationary cultures. This procedure did not lead to a detectable population growth during the period of experiment, apparently due to growth inhibition by the crowd-sensing effect and provided high viability of cells even after the 3 month period. The obtained results evidence the occurrence of oxidative stress and accumulation of oxidative damage during long-term incubation (“aging”) of stationary cultures of S. cerevisiae. The increased rate of H2DCF oxidation in old cultures of the yeast indicates an increased level of reactive oxygen species in these cultures. This phenomenon may be due to increased generation of reactive
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Fig. 2. Superoxide dismutase activity in young and old stationary cultures of different strains of S. cerevisiae. Empty bars ⫽ young cultures; filled bars ⫽ old cultures. Top: absolute activities, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
Fig. 3. Catalase activity in young and old stationary cultures off different strains of S. cerevisiae. Empty bars ⫽ young cultures; filled bars ⫽ old cultures. Top: absolute activities, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
oxygen species by cellular sources and/or to the decreased antioxidant defense, especially the lower level of glutathione. The increased content of protein carbonyl groups as well as decreased SOD activity also suggests an increased exposure of macromolecules to reactive oxygen species. SOD is known to be inactivated by exposure to hydrogen peroxide and other peroxides while catalase and glutathione peroxidase are inactivated by superoxide [16,17]. Therefore, decreased antioxidative defense in aged stationary cultures may lead to a self-perpetuating damage in spite of the increase in antioxidative defense on entering the stationary phase. However, an alternative explanation that the accumulation of protein carbonyls and decreased activity of SOD are due to decreased protein turnover in stationary cultures cannot be excluded. The oxidative stress in stationary cultures of yeast may be connected to the better oxygen access to the cells due to decreased metabolism. Metabolically active cells intensively consume oxygen under conditions of limited aeration which may lead to its low concentration within the cells. In stationary cultures the metabolism is considerably attenuated so the oxygen concentration within cells may be higher; one-electron reactions of autoxida-
tion-prone cellular components may produce superoxide and, in turn, other reactive oxygen species. Mitochondrial respiration have been suggested to be the major source of reactive oxygen species under these conditions, even or especially under conditions of low aeration. While under high aeration of long-term stationary cultures of S. cerevisiae yeast mutants devoid of Cu,ZnSOD were less viable than MnSOD mutants, this situation was reversed under low aeration when mutants devoid of mitochondrial MnSOD showed a more severe growth reduction [18]. An alternative source of oxygen free radicals, detectable on freezing and/or thawing of the yeast, has also been identified in the cytoplasm [19] and may contribute to the oxidative damage. Similar changes (accumulation of protein carbonyls and disulfide bridges) [20] accompanied by protein racemization [21] have been observed during incubation of stationary cultures of Escherichia coli. Oxidative stress may be a factor limiting the survival of microorganisms in long-term stationary culture. S. cerevisiae mutants devoid of either Cu,ZnSOD or MnSOD show lower survival in stationary cultures [18,22]. In Rhodobacter capsulatus, a similar decrease in stationary phase survival was noted for mutants lacking catalase-peroxidase
Aging of yeast cultures
Fig. 4. Oxidation of 2⬘7⬘-dichlorofluorescin in young and old stationary cultures of different strains of S. cerevisiae. Empty bars, young cultures; filled bars, old cultures. Top: relative fluorescence values read; bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 4.
[23]. The increase in antioxidant content and increase in the levels of antioxidant enzymes including SOD [24] and glutathione reductase [25] on yeast entering into the stationary phase may, therefore, constitute an adaptive response to the enhanced oxidative danger. The increase in catalase activity may, however, represent a stress response; this enzyme is easily inducible in yeast by various types of stress. The transcription of CTT1 gene which codes catalase, is regulated by the cAMP level [26,27] and the deficiency of cAMP induces the biosynthesis of catalase. A drop of cAMP was observed on various types of stress, in particular in oxidative and heat stress [28,29]. It can be suggested that the accumulation of oxidative damage in stationary cultures of yeast may be reminiscent of processes taking place in nondividing mammalian cells in culture or postmitotic cells in a multicellular organism. Fibroblasts which remain proliferation-inhibited by confluency for up to 3 months show increased frequency of single-stranded sites in telomeres, measured as sensitivity to degradation by S1 nuclease, apparently due to oxidative damage [30]. This similarity is limited in some aspects. Most of the yeast cells can resume divisions on dilution. We were unable to demonstrate
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Fig. 5. The level of protein carbonyl groups in young and old stationary cultures of different strains of S. cerevisiae. Empty bars ⫽ young cultures; filled bars ⫽ old cultures. Top: absolute concentrations, bottom bars: relative values with respect to young cultures assumed as 100% for each strain, respectively. Mean ⫾ SD, n ⫽ 5.
accumulation of lipofuscinlike material in old stationary cultures of the yeast (not shown). Nevertheless, as suggested by other authors [8,18,31,32], stationary yeast cultures may represent a model system, with respect to cells of higher eucaryotes, of oxidative damage to nondividing cells. Acknowledgement — This study was partially supported by Grant No. 512/P04/97/13 of the Polish Committee for Scientific Research.
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