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The role of glutathione in yeast dehydration tolerance
Cryobiology 47 (2003) 236–241 www.elsevier.com/locate/ycryo
The role of glutathione in yeast dehydration toleranceq Aline de Souza Espindola, Debora Silva Gomes, Anita Dolly Panek, and Elis Cristina Araujo Eleutherio* Departamento Bioquımica, Instituto de Quımica, UFRJ, Rio de Janeiro, Brazil Received 2 September 2003; accepted 15 October 2003
Abstract Among the factors that affect cell resistance against dehydration, oxidation is considered to be of great importance. In this work, we verified that both control and glutathione deficient mutant strains were much more oxidized after dehydration. Moreover, cells lacking glutathione showed a twofold higher increase in oxidation and lipid peroxidation than the control strain. While glucose 6-phosphate dehydrogenase and glutathione reductase activities did not change in response to dehydration in the control strain, the mutant strain gsh1 (glutathione deficient) showed a reduction of 50% in both activities, which could explain the high levels of oxidation shown by gsh1 cells. In conformity with these results, the mutant lacking GSH1 showed a high sensitivity to dehydration. Furthermore, the addition of glutathione to gsh1 cells restored survival rates to the levels of the control strain. We conclude that glutathione plays a significant role in the maintenance of intracellular redox balance during dehydration. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Glutathione; Oxidative stress; Dehydration; Saccharomyces cerevisiae
Water is usually thought to be required for the living state, but several organisms are capable of surviving complete dehydration. The phenomenon is widespread in the plant kingdom, but also occurs in prokaryotes, fungi, and small animals such as tardigrades, nematodes, and cysts of some crustacean embryos [12]. However, no vertebrate exhibits desiccation tolerance. With recent advances in tissue engineering, cell transplantation and genetic technology, successful q This work was supported by grants from FAPERJ and CNPq. * Corresponding author. Fax: +5521-2562-7735. E-mail address: [email protected] (E.C.A. Eleutherio).
long-term storage of living cells is of critical importance. Even common requirements, such as the storage of blood cells (platelets and erythrocytes) in blood banks, is still a major problem. The possibility of drying mammalian cells would greatly simplify the storage and transportation of cells and possibly of organs. Elucidation of the mechanisms that endow some organisms with the capacity to survive dehydration may lead to development of new methods for preserving biological materials that do not normally support drying. Anhydrobiosis (life without water) requires a coordinated series of events during dehydration that are associated with maintaining the native structure of biomolecules and with preventing
0011-2240/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2003.10.003
A.S. Espindola et al. / Cryobiology 47 (2003) 236–241
oxidative damage [14]. Recent advances in our understanding of the mechanisms of acquiring tolerance to the dry state include immobilization of the cytoplasm in a stable glassy state, which seems to prevent conformational changes in biomolecules [6]. Furthermore, in a glassy state, the rates of molecular diffusion and chemical reactions are greatly reduced [9]. Both conditions might protect cells against oxidation. Curtailing the production of reactive oxygen species (ROS) is also an important mechanism of survival against dehydration [15,18]. These species can damage proteins by causing modifications of amino acid side chains, formation of cross-links between proteins and fragmentation of the polypeptide backbone [3]. In addition, ROS can modify the bases and sugars in DNA, leading to DNA chain breaks [23], and cause lipid peroxidation in cell membranes [25]. Reduction of metabolism, noticeable as a reduction of respiration rate, seems to be essential in avoiding oxidative stress during acquisition of desiccation tolerance [5,13]. On the other hand, as ROS increase with drying, free-radical scavenging mechanisms are activated [20]. To protect against oxidative damage, cells possess defense mechanisms that include enzymes such as peroxidases and superoxide dismutases and antioxidants such as glutathione and vitamins C and E [11]. Perhaps the best-known example of a non-enzymatic defense system is glutathione (GSH: c-L glutamyl-L -cystinylglycine). GSH is present in high concentrations in most living cells from prokaryotes to eukaryotes [16]. Inside the cells, GSH assumes pivotal roles not only in bioreduction and protection against oxidative stress, but also in detoxification of xenobiotics or endogenous toxic metabolites, in transport, as well as in sulphur and nitrogen metabolisms [16]. The genes involved in GSH biosynthesis in Saccharomyces cerevisiae are GSH1 and GSH2, which encode c-glutamylcysteine synthetase and glutathione synthetase, respectively [2]. GSH acts as a radical scavenger with the redox active suphydryl group reacting with oxidants to produce oxidized glutathione (GSSG) [2]. Crucial to the role of glutathione as an antioxidant is the maintenance of a high reduced-oxidized ratio inside the cell. The enzyme glutathione reductase is primarily responsible for the reduction
237
of oxidized glutathione (GSSG), which is reduced by NADPH generated by glucose-6-phosphate dehydrogenase in the pentose phosphate pathway. Since very little is known about the damage caused by water loss due to oxidative stress, we have investigated the importance of some antioxidant defense systems in the dehydration tolerance of S. cerevisiae, a usual model in the study of stress response. Recently we demonstrated that both cytosolic and mitochondrial superoxide dismutases are capable of protecting dry cells against oxidation and that the presence of only one isoform is sufficient to support survival [17]. In this work, we analyze the role of glutathione in the ability of yeast cells to survive dehydration.
Materials and methods Strains and culture conditions Control strain BY4741 (Mata; his3D1; leu2D0; met15D0; ura3D0) and its isogenic mutants gsh1 and gsh2, harboring the genes GSH1 and GSH2 interrupted by the gene KanMX4, were acquired from Euroscarf, Frankfurt, Germany. Cells were grown up to stationary phase (4.0 mg dry weight/ mL) in liquid YPD medium (1% yeast extract, 2% glucose, and 2% peptone), using an orbital shaker at 28 °C and 160 rpm, with the ratio of flask volume/medium of 5/1. Dehydration Dehydration was performed on a special balance at 37 °C. After reaching constant weight, samples were left to equilibrate for 5 min. Final water content was 4–9%. Additionally, gsh1 cells were treated with 5 mM glutathione monoethyl ester (GME) during 90 min before being dehydrated. Fluorescence assays Fluorescence was measured using a PTI (Photo Technology International) spectrofluorimeter, set at an excitation wavelength of 504 nm and an emission wavelength of 524 nm. Cells (50 mg) were
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resuspended in 12.5 mL of 50 mM phosphate buffer, pH 6.0 and 20 ,70 -dichlorofluorescein was added from a fresh 5 mM stock in ethanol to a final concentration of 10 lM. Incubation continued for 15 min at room temperature to permit uptake of the probe. After that, cells were harvested by centrifugation, washed twice with the same buffer and dehydrated at 37 °C. Cells were resuspended in 500 lL of the same buffer and 1.5 g of glass beads were added. Dry cells were lysed by three cycles of 1 min agitation on a vortex mixer followed by 1 min on ice. The supernatant solution was obtained after centrifugation at 25,000g for 5 min, diluted sixfold with water and then, fluorescence was measured [1]. As a control, fluorescence was measured in fresh cells. Lipid peroxidation Cells were resuspended in 500 lL of the same buffer, containing 10% trichloroacetic acid and 1.5 g of glass beads were added. The samples were lysed by three cycles of 1 min agitation on a vortex mixer followed by 1 min on ice. The supernatants obtained after centrifugation were mixed with 0.1 mL of 0.1 M EDTA and 0.6 mL 1% (w/v) thiobarbituric acid in 0.05 M NaOH. The reaction mixture was incubated in a boiling water bath for 15 min and after cooling, the absorbance at 532 nm was measured [21]. Determination of glutathione Reduced glutathione (GSH) concentrations were determined spectrophotometrically in neutralized trichloroacetic acid (TCA 10%) extracts by following the glyoxylase catalysed production of S-lactoyl-GSH at 240 nm [4]. Oxidized glutathione (GSSG) was determined in the same cuvette by addition of NADPH and glutathione reductase and then following the change in absorbance at 340 nm [4]. The redox ratio was expressed as the relation between GSSG and GSH content. Enzyme assays Extracts were obtained by disruption of 50 mg of cells with glass beads in 200 mM Tris/HCl
buffer, pH 7.2, on a vortex mixer [17]. Glucose-6phosphate dehydrogenase activity was determined based on NADPH production [10]. Glutathione reductase activity was measured using a kit from Calbiochem (Cat. No. 359962). Protein concentrations were determined by the method of Stickland [22]. Cell viability Before and following dehydration, cells were diluted with 50 mM phosphate buffer, pH 6.0 and plated on YPD plates (containing 2.0% glucose, 2.0% peptone, 1.0% yeast extract, and 2.0% agar) to determinate survival. Colonies were counted after incubation at 28 °C for 72 h. Plates were done in triplicate. Tolerance was measured as the percentage of viable cells that survived stress.
Results and discussion Water loss induces an oxidative stress, which seems to affect the longevity of anhydrobiotes [9,14]. In this work, oxidative stress produced by dehydration was monitored by measuring changes in fluorescence resulting from oxidation of an intracellular probe. The probe 20 ,70 -dichlorofluorescein is a molecule that can permeate cell membranes by passive diffusion [24]. Once inside the cell, it becomes susceptible to attack by freeradical species, producing a more fluorescent compound [24]. The intensity of fluorescence can be measured and it is the basis of a commonly used assay for oxidative stress. According to the results (Table 1), control and glutathione deficient strains showed a meaningful Table 1 Enhancement of intracellular oxidation measured as fold increase in fluorescence Strains
Increase of fluorescence
BY4741 gsh1 gsh2
2.0 0.1 4.0 0 2.4 0.3
The results represent a relation between the fluorescence of dry and fresh cells and are expressed as means SD of three independent experiments.
A.S. Espindola et al. / Cryobiology 47 (2003) 236–241
increase in oxidation after dehydration, confirming that oxidative stress plays a major role in the lethal effect of dehydration. However, cells deleted in GSH1 presented an increase of oxidation twofold higher than the control strain. Strain gsh2, which is deficient in the enzyme necessary to join glycine to the dipeptide c-glutamyl cysteine, showed the same level of oxidation as control cells. Glutathione synthetase (Gsh2) is dispensable for growth under oxidative stress conditions due to an accumulation of c-glutamyl cysteine [8]. This data supports our results, which showed that the dipeptide could substitute, at least in part, for the essential function of glutathione as a cellular redox buffer during dehydration. A crucial strategy for survival in dried anhydrobiotes is the control of membrane dynamics [9,12]. Under dehydration, cell membranes become fluidized and perturbed, as well as being more susceptible to ROS attack [7]. ROS often cause an extensive peroxidation and de-esterification of membrane lipids at intermediate ranges of water loss [19]. Given that the properties of glutathione include peroxide reduction and removal of many lipophilic xenobiotics from the cytosol [16], it seems likely that GSH is important in protecting cell membranes under anhydrous conditions. There are several ways to detect lipid peroxidation processes; we used the method of TBARS (thiobarbituric acid reactive species), which detects malondialdehyde (MDA). Dehydration produced a high increase in the levels of lipid peroxidation in both control strain (BY4741) and gsh mutants (Table 2). However, corroborating the idea that GSH might protect membrane against oxidation, the levels of lipid peroxidation in the glutathione deficient mutants, even in fresh cells, were two- to four-fold higher than in the control strain BY4741.
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Similar to oxidation during dehydration, gsh1 cells showed the highest levels of MDA. As expected, after dehydration, there was a significant enhancement of the intracellular GSSG content in the control strain (Table 2), confirming once again the key role played by glutathione in maintaining the redox homeostasis of dried cells. In accordance to this result, fresh and dried cells of the BY4741 strain showed similar glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GLR) activities (Figs. 1A and B). In seeds and desiccation-resistant plants, intracellular glasses reduce the rates of chemical reaction and, therefore of metabolic activity [9]. Therefore, it should be taken into consideration that, although ROS increase with drying, the enzymatic antioxidant systems can be activated only under conditions of sufficient water and, in the dried state, only molecular antioxidants (e.g., glutathione and sugars) can alleviate oxidative stress. The mutant strain gsh1 (glutathione deficient) showed a reduction of 50% in G6PDH activity in response to dehydration (Fig. 1A), which could explain the high levels of oxidation shown by gsh1 cells. In this mutant strain, a low G6PDH activity would impair NADPH production, affecting other sulphydryl groups involved with antioxidant protection, like glutaredoxins and thioredoxins. Interestingly, both gsh1 and gsh2 strains did loose approximately 50% of their GLR activities (Fig. 1B), as compared to the control strain, suggesting that glutathione might regulate GLR activity. Although gsh2 cells had shown similar G6PDH activity to that of the control strain, a reduced GLR activity could imply on reduced GSH-turnover, which might be associated with the high levels of lipid peroxidation shown by this mutant (Table 2).
Table 2 Lipid peroxidation and GSSG/GSH ratio Strains
BY4741 gsh1 gsh2
Lipid peroxidation (pmol MDA/mg cell)
GSSG/GSH ratio
Fresh cells
Dried cells
Fresh cells
Dried cells
48 2 191 7 189 5
97 4 421 12 231 9
2.0 0.5 ND ND
8.6 0.2 ND ND
The results represent means SD of three independent experiments. * Not determined.
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Fig. 1. Glucose-6-phosphate dehydrogenase (A) and glutathione reductase (B) activities. Cell-free extracts were prepared for enzyme determinations before and after dehydration. One unit of G6PDH activity is defined as the amount of enzyme that catalyses the production of 1 lmol NADPH in 1 min under the assay conditions. One unit of GLR activity is defined as the amount of enzyme that catalyses the conversion of 1 lmol NADPH in 1 min under the assay conditions. The results represent means SD of three independent experiments.
As seen in Fig. 2, survival correlated well with capacity to synthesize glutathione. Mutants lacking glutathione, especially the gsh1 strain, showed a higher sensitivity to dehydration than the control
Fig. 2. Effect of dehydration on survival. Tolerance was measured as percentage of viable cells that survive stress. The mutant strain gsh1 was also dehydrated in the presence of 5 mM glutathione momoethyl ester (GME). The results represent means SD of at least three independent experiments.
strain. The importance of GSH in desiccation tolerance was confirmed by the addition of glutathione monoethyl ester (GME) to gsh1 cells, which restored the levels of survival to those of the control strain. The drug GME is a cell-permeable derivative of GSH that undergoes hydrolysis by intra-cellular esterases, thereby, increasing intracellular GSH concentration [2]. We conclude, therefore, that glutathione plays a remarkable role in protecting cell membranes and in maintaining redox homeostasis under water deficiency, favoring tolerance to the dry state. However, the study of the ability to survive dehydration indicates that other aspects are important for conferring tolerance and long-term stability. It has often been found that one specific mechanism does not endow resistance on its own; interplay of several defense systems is necessary. On the other hand, cell protection can be achieved in different ways so as to guarantee optimal survival.
Acknowledgments We thank Prof. Ricardo Chaloub and Prof. Marcoaurelio Almenara (Dep. Bioquımica–I.Q./ UFRJ, Brazil) for the use of the spectrofluorimeter.
References [1] P.D.B. Adamis, A.D. Panek, S.G.F. Leite, E.C.A. Eleutherio, Factors involved with cadmium absorption by a wild-type strain of Saccharomyces cerevisiae, Braz. J. Microbiol. 34 (2003) 55–60. [2] M.E. Anderson, Glutathione: an overview of biosynthesis and modulation, Chem. Biol. Interact. 111–112 (1998) 1–14. [3] B.S. Berlett, E.R. Stadtman, Protein oxidation in aging, disease, and oxidative stress, J. Biol. Chem. 272 (1997) 20313–20316. [4] E. Bernt, H.U. Bergmeyer, Methods Enzymatic Anal. 4 (1974) 1643–1647. [5] J.S. Clegg, The physical properties and metabolic status of Artemia cysts at low water contents: The water replacement hypothesis, in: C.A. Leopold (Ed.), Membrane, Metabolism and Dry Organisms, Cornell University Press, Ithaca, NY, 1986, pp. 169–187.
A.S. Espindola et al. / Cryobiology 47 (2003) 236–241 [6] J.H. Crowe, J.F. Carpenter, L.M. Crowe, The role of vitrification in anhydrobiosis, Annu. Rev. Physiol. 60 (1998) 73–103. [7] J.H. Crowe, B.D. McKersie, L.M. Crowe, Effects of free fatty acids and transition temperature on the stability of dry liposomes, Biochim. Biophys. Acta 979 (1989) 7–10. [8] C.M. Grant, F.H. Maciver, I.W. Dawes, Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gammaglutamylcysteine, Mol. Biol. Cell 8 (1997) 1699–1707. [9] F.A. Hoekstra, E.A. Golovina, J. Buitink, Mechanisms of plant desiccation tolerance, Trends Plant Sci. 6 (2001) 431– 438. [10] S. Izawa, K. Maeda, T. Miki, J. Mano, Y. Inoue, A. Kimura, Importance of glucose-6-phosphate dehydrogenase in adaptative response to hydrogen peroxide in Saccharomyces cerevisiae, Biochem. J. 330 (1998) 811–817. [11] D.J. Jamieson, Oxidative stress responses of the yeast Saccharomyces cerevisiae, Yeast 14 (1998) 1511–1527. [12] A.C. Leopold, Membranes, Metabolism and Dry Organisms, Cornell University Press, Ithaca, 1986. [13] O. Leprince, N.M. Atherton, R. Deltour, G.A.F. Hendry, The involvement of respiration in free radical processes during loss of desiccation tolerance in germinating Zea mays, L. Plant Physiol. 104 (1994) 1333–1339. [14] A.E. Oliver, O. Leprince, W.F. Wolkers, D.K. Hincha, A.G. Heyer, J.H. Crowe, Non-disaccharide-based mechanisms of protection during drying, Cryobiology 43 (2001) 151–167. [15] N.W. Pammenter, P. Berjak, A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms, Seed Sci. Res. 9 (1999) 13–37.
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[16] M.J. Penninckx, An overview on glutathione in Saccharomyces versus non-conventional yeasts, FEM Yeast Res. 2 (2002) 295–305. [17] E.J. Pereira, A.D. Panek, E.C.A. Eleutherio, Protection against oxidation during dehydration of yeast, Cell Stress Chaperon. 8 (2003) 120–124. [18] M. Potts, Desiccation tolerance in prokaryotes, Microbiol. Rev. 58 (1994) 755–805. [19] T. Senaratna, B.D. McKersie, A. Borochov, Desiccation and free radical mediated changes in plant membranes, J. Exp. Bot. 38 (1987) 2005–2014. [20] H.W. Sherwin, J.M. Farrant, Protection mechanisms against excess light in the ressuction plants Craterostigma wilmsii and Xerophyta viscosa, Plant Growth Regul. 24 (1998) 203–210. [21] E.L. Steels, R.P. Learmonth, K. Watson, Stress tolerance and membrane lipid unsaturation in Saccharomyces cerevisiae grown aerobically or anaerobically, Microbiology 140 (1994) 569–576. [22] L.H. Stickland, The determination of small quantities of bacteria by means of the biuret reaction, J. Gen. Microbiol. 5 (1951) 698–703. [23] G. Storz, M.F. Christman, H. Sies, B.N. Ames, Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium, Proc. Natl. Acad. Sci. USA 84 (1987) 8917–8921. [24] M. Tsuchiya, M. Suematsu, In vivo visualization of oxygen radical-dependent photoemission, Methods Enzymol. (1994) 128–140. [25] S.P. Wolff, A. Garner, R.T. Dean, Free radicals, lipids and protein degradation, Trends Biochem. Sci. 11 (1986) 27–31.
The role of glutathione in yeast dehydration toleranceq Aline de Souza Espindola, Debora Silva Gomes, Anita Dolly Panek, and Elis Cristina Araujo Eleutherio* Departamento Bioquımica, Instituto de Quımica, UFRJ, Rio de Janeiro, Brazil Received 2 September 2003; accepted 15 October 2003
Abstract Among the factors that affect cell resistance against dehydration, oxidation is considered to be of great importance. In this work, we verified that both control and glutathione deficient mutant strains were much more oxidized after dehydration. Moreover, cells lacking glutathione showed a twofold higher increase in oxidation and lipid peroxidation than the control strain. While glucose 6-phosphate dehydrogenase and glutathione reductase activities did not change in response to dehydration in the control strain, the mutant strain gsh1 (glutathione deficient) showed a reduction of 50% in both activities, which could explain the high levels of oxidation shown by gsh1 cells. In conformity with these results, the mutant lacking GSH1 showed a high sensitivity to dehydration. Furthermore, the addition of glutathione to gsh1 cells restored survival rates to the levels of the control strain. We conclude that glutathione plays a significant role in the maintenance of intracellular redox balance during dehydration. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Glutathione; Oxidative stress; Dehydration; Saccharomyces cerevisiae
Water is usually thought to be required for the living state, but several organisms are capable of surviving complete dehydration. The phenomenon is widespread in the plant kingdom, but also occurs in prokaryotes, fungi, and small animals such as tardigrades, nematodes, and cysts of some crustacean embryos [12]. However, no vertebrate exhibits desiccation tolerance. With recent advances in tissue engineering, cell transplantation and genetic technology, successful q This work was supported by grants from FAPERJ and CNPq. * Corresponding author. Fax: +5521-2562-7735. E-mail address: [email protected] (E.C.A. Eleutherio).
long-term storage of living cells is of critical importance. Even common requirements, such as the storage of blood cells (platelets and erythrocytes) in blood banks, is still a major problem. The possibility of drying mammalian cells would greatly simplify the storage and transportation of cells and possibly of organs. Elucidation of the mechanisms that endow some organisms with the capacity to survive dehydration may lead to development of new methods for preserving biological materials that do not normally support drying. Anhydrobiosis (life without water) requires a coordinated series of events during dehydration that are associated with maintaining the native structure of biomolecules and with preventing
0011-2240/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2003.10.003
A.S. Espindola et al. / Cryobiology 47 (2003) 236–241
oxidative damage [14]. Recent advances in our understanding of the mechanisms of acquiring tolerance to the dry state include immobilization of the cytoplasm in a stable glassy state, which seems to prevent conformational changes in biomolecules [6]. Furthermore, in a glassy state, the rates of molecular diffusion and chemical reactions are greatly reduced [9]. Both conditions might protect cells against oxidation. Curtailing the production of reactive oxygen species (ROS) is also an important mechanism of survival against dehydration [15,18]. These species can damage proteins by causing modifications of amino acid side chains, formation of cross-links between proteins and fragmentation of the polypeptide backbone [3]. In addition, ROS can modify the bases and sugars in DNA, leading to DNA chain breaks [23], and cause lipid peroxidation in cell membranes [25]. Reduction of metabolism, noticeable as a reduction of respiration rate, seems to be essential in avoiding oxidative stress during acquisition of desiccation tolerance [5,13]. On the other hand, as ROS increase with drying, free-radical scavenging mechanisms are activated [20]. To protect against oxidative damage, cells possess defense mechanisms that include enzymes such as peroxidases and superoxide dismutases and antioxidants such as glutathione and vitamins C and E [11]. Perhaps the best-known example of a non-enzymatic defense system is glutathione (GSH: c-L glutamyl-L -cystinylglycine). GSH is present in high concentrations in most living cells from prokaryotes to eukaryotes [16]. Inside the cells, GSH assumes pivotal roles not only in bioreduction and protection against oxidative stress, but also in detoxification of xenobiotics or endogenous toxic metabolites, in transport, as well as in sulphur and nitrogen metabolisms [16]. The genes involved in GSH biosynthesis in Saccharomyces cerevisiae are GSH1 and GSH2, which encode c-glutamylcysteine synthetase and glutathione synthetase, respectively [2]. GSH acts as a radical scavenger with the redox active suphydryl group reacting with oxidants to produce oxidized glutathione (GSSG) [2]. Crucial to the role of glutathione as an antioxidant is the maintenance of a high reduced-oxidized ratio inside the cell. The enzyme glutathione reductase is primarily responsible for the reduction
237
of oxidized glutathione (GSSG), which is reduced by NADPH generated by glucose-6-phosphate dehydrogenase in the pentose phosphate pathway. Since very little is known about the damage caused by water loss due to oxidative stress, we have investigated the importance of some antioxidant defense systems in the dehydration tolerance of S. cerevisiae, a usual model in the study of stress response. Recently we demonstrated that both cytosolic and mitochondrial superoxide dismutases are capable of protecting dry cells against oxidation and that the presence of only one isoform is sufficient to support survival [17]. In this work, we analyze the role of glutathione in the ability of yeast cells to survive dehydration.
Materials and methods Strains and culture conditions Control strain BY4741 (Mata; his3D1; leu2D0; met15D0; ura3D0) and its isogenic mutants gsh1 and gsh2, harboring the genes GSH1 and GSH2 interrupted by the gene KanMX4, were acquired from Euroscarf, Frankfurt, Germany. Cells were grown up to stationary phase (4.0 mg dry weight/ mL) in liquid YPD medium (1% yeast extract, 2% glucose, and 2% peptone), using an orbital shaker at 28 °C and 160 rpm, with the ratio of flask volume/medium of 5/1. Dehydration Dehydration was performed on a special balance at 37 °C. After reaching constant weight, samples were left to equilibrate for 5 min. Final water content was 4–9%. Additionally, gsh1 cells were treated with 5 mM glutathione monoethyl ester (GME) during 90 min before being dehydrated. Fluorescence assays Fluorescence was measured using a PTI (Photo Technology International) spectrofluorimeter, set at an excitation wavelength of 504 nm and an emission wavelength of 524 nm. Cells (50 mg) were
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resuspended in 12.5 mL of 50 mM phosphate buffer, pH 6.0 and 20 ,70 -dichlorofluorescein was added from a fresh 5 mM stock in ethanol to a final concentration of 10 lM. Incubation continued for 15 min at room temperature to permit uptake of the probe. After that, cells were harvested by centrifugation, washed twice with the same buffer and dehydrated at 37 °C. Cells were resuspended in 500 lL of the same buffer and 1.5 g of glass beads were added. Dry cells were lysed by three cycles of 1 min agitation on a vortex mixer followed by 1 min on ice. The supernatant solution was obtained after centrifugation at 25,000g for 5 min, diluted sixfold with water and then, fluorescence was measured [1]. As a control, fluorescence was measured in fresh cells. Lipid peroxidation Cells were resuspended in 500 lL of the same buffer, containing 10% trichloroacetic acid and 1.5 g of glass beads were added. The samples were lysed by three cycles of 1 min agitation on a vortex mixer followed by 1 min on ice. The supernatants obtained after centrifugation were mixed with 0.1 mL of 0.1 M EDTA and 0.6 mL 1% (w/v) thiobarbituric acid in 0.05 M NaOH. The reaction mixture was incubated in a boiling water bath for 15 min and after cooling, the absorbance at 532 nm was measured [21]. Determination of glutathione Reduced glutathione (GSH) concentrations were determined spectrophotometrically in neutralized trichloroacetic acid (TCA 10%) extracts by following the glyoxylase catalysed production of S-lactoyl-GSH at 240 nm [4]. Oxidized glutathione (GSSG) was determined in the same cuvette by addition of NADPH and glutathione reductase and then following the change in absorbance at 340 nm [4]. The redox ratio was expressed as the relation between GSSG and GSH content. Enzyme assays Extracts were obtained by disruption of 50 mg of cells with glass beads in 200 mM Tris/HCl
buffer, pH 7.2, on a vortex mixer [17]. Glucose-6phosphate dehydrogenase activity was determined based on NADPH production [10]. Glutathione reductase activity was measured using a kit from Calbiochem (Cat. No. 359962). Protein concentrations were determined by the method of Stickland [22]. Cell viability Before and following dehydration, cells were diluted with 50 mM phosphate buffer, pH 6.0 and plated on YPD plates (containing 2.0% glucose, 2.0% peptone, 1.0% yeast extract, and 2.0% agar) to determinate survival. Colonies were counted after incubation at 28 °C for 72 h. Plates were done in triplicate. Tolerance was measured as the percentage of viable cells that survived stress.
Results and discussion Water loss induces an oxidative stress, which seems to affect the longevity of anhydrobiotes [9,14]. In this work, oxidative stress produced by dehydration was monitored by measuring changes in fluorescence resulting from oxidation of an intracellular probe. The probe 20 ,70 -dichlorofluorescein is a molecule that can permeate cell membranes by passive diffusion [24]. Once inside the cell, it becomes susceptible to attack by freeradical species, producing a more fluorescent compound [24]. The intensity of fluorescence can be measured and it is the basis of a commonly used assay for oxidative stress. According to the results (Table 1), control and glutathione deficient strains showed a meaningful Table 1 Enhancement of intracellular oxidation measured as fold increase in fluorescence Strains
Increase of fluorescence
BY4741 gsh1 gsh2
2.0 0.1 4.0 0 2.4 0.3
The results represent a relation between the fluorescence of dry and fresh cells and are expressed as means SD of three independent experiments.
A.S. Espindola et al. / Cryobiology 47 (2003) 236–241
increase in oxidation after dehydration, confirming that oxidative stress plays a major role in the lethal effect of dehydration. However, cells deleted in GSH1 presented an increase of oxidation twofold higher than the control strain. Strain gsh2, which is deficient in the enzyme necessary to join glycine to the dipeptide c-glutamyl cysteine, showed the same level of oxidation as control cells. Glutathione synthetase (Gsh2) is dispensable for growth under oxidative stress conditions due to an accumulation of c-glutamyl cysteine [8]. This data supports our results, which showed that the dipeptide could substitute, at least in part, for the essential function of glutathione as a cellular redox buffer during dehydration. A crucial strategy for survival in dried anhydrobiotes is the control of membrane dynamics [9,12]. Under dehydration, cell membranes become fluidized and perturbed, as well as being more susceptible to ROS attack [7]. ROS often cause an extensive peroxidation and de-esterification of membrane lipids at intermediate ranges of water loss [19]. Given that the properties of glutathione include peroxide reduction and removal of many lipophilic xenobiotics from the cytosol [16], it seems likely that GSH is important in protecting cell membranes under anhydrous conditions. There are several ways to detect lipid peroxidation processes; we used the method of TBARS (thiobarbituric acid reactive species), which detects malondialdehyde (MDA). Dehydration produced a high increase in the levels of lipid peroxidation in both control strain (BY4741) and gsh mutants (Table 2). However, corroborating the idea that GSH might protect membrane against oxidation, the levels of lipid peroxidation in the glutathione deficient mutants, even in fresh cells, were two- to four-fold higher than in the control strain BY4741.
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Similar to oxidation during dehydration, gsh1 cells showed the highest levels of MDA. As expected, after dehydration, there was a significant enhancement of the intracellular GSSG content in the control strain (Table 2), confirming once again the key role played by glutathione in maintaining the redox homeostasis of dried cells. In accordance to this result, fresh and dried cells of the BY4741 strain showed similar glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GLR) activities (Figs. 1A and B). In seeds and desiccation-resistant plants, intracellular glasses reduce the rates of chemical reaction and, therefore of metabolic activity [9]. Therefore, it should be taken into consideration that, although ROS increase with drying, the enzymatic antioxidant systems can be activated only under conditions of sufficient water and, in the dried state, only molecular antioxidants (e.g., glutathione and sugars) can alleviate oxidative stress. The mutant strain gsh1 (glutathione deficient) showed a reduction of 50% in G6PDH activity in response to dehydration (Fig. 1A), which could explain the high levels of oxidation shown by gsh1 cells. In this mutant strain, a low G6PDH activity would impair NADPH production, affecting other sulphydryl groups involved with antioxidant protection, like glutaredoxins and thioredoxins. Interestingly, both gsh1 and gsh2 strains did loose approximately 50% of their GLR activities (Fig. 1B), as compared to the control strain, suggesting that glutathione might regulate GLR activity. Although gsh2 cells had shown similar G6PDH activity to that of the control strain, a reduced GLR activity could imply on reduced GSH-turnover, which might be associated with the high levels of lipid peroxidation shown by this mutant (Table 2).
Table 2 Lipid peroxidation and GSSG/GSH ratio Strains
BY4741 gsh1 gsh2
Lipid peroxidation (pmol MDA/mg cell)
GSSG/GSH ratio
Fresh cells
Dried cells
Fresh cells
Dried cells
48 2 191 7 189 5
97 4 421 12 231 9
2.0 0.5 ND ND
8.6 0.2 ND ND
The results represent means SD of three independent experiments. * Not determined.
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Fig. 1. Glucose-6-phosphate dehydrogenase (A) and glutathione reductase (B) activities. Cell-free extracts were prepared for enzyme determinations before and after dehydration. One unit of G6PDH activity is defined as the amount of enzyme that catalyses the production of 1 lmol NADPH in 1 min under the assay conditions. One unit of GLR activity is defined as the amount of enzyme that catalyses the conversion of 1 lmol NADPH in 1 min under the assay conditions. The results represent means SD of three independent experiments.
As seen in Fig. 2, survival correlated well with capacity to synthesize glutathione. Mutants lacking glutathione, especially the gsh1 strain, showed a higher sensitivity to dehydration than the control
Fig. 2. Effect of dehydration on survival. Tolerance was measured as percentage of viable cells that survive stress. The mutant strain gsh1 was also dehydrated in the presence of 5 mM glutathione momoethyl ester (GME). The results represent means SD of at least three independent experiments.
strain. The importance of GSH in desiccation tolerance was confirmed by the addition of glutathione monoethyl ester (GME) to gsh1 cells, which restored the levels of survival to those of the control strain. The drug GME is a cell-permeable derivative of GSH that undergoes hydrolysis by intra-cellular esterases, thereby, increasing intracellular GSH concentration [2]. We conclude, therefore, that glutathione plays a remarkable role in protecting cell membranes and in maintaining redox homeostasis under water deficiency, favoring tolerance to the dry state. However, the study of the ability to survive dehydration indicates that other aspects are important for conferring tolerance and long-term stability. It has often been found that one specific mechanism does not endow resistance on its own; interplay of several defense systems is necessary. On the other hand, cell protection can be achieved in different ways so as to guarantee optimal survival.
Acknowledgments We thank Prof. Ricardo Chaloub and Prof. Marcoaurelio Almenara (Dep. Bioquımica–I.Q./ UFRJ, Brazil) for the use of the spectrofluorimeter.
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