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Targets of oxidative stress in yeast sod mutants
Biochimica et Biophysica Acta 1620 (2003) 245 – 251 www.bba-direct.com
Targets of oxidative stress in yeast sod mutants M.D. Pereira, R.S. Herdeiro, P.N. Fernandes, E.C.A. Eleutherio *, A.D. Panek Departamento de Bioquı´mica, Instituto de Quı´mica, UFRJ, 21949-900 Rio de Janeiro, Brazil Received 26 June 2002; received in revised form 9 December 2002; accepted 16 December 2002
Abstract Eukaryotic cells have developed mechanisms to rapidly respond towards the environment by changing the expression of a series of genes. There is increasing evidence that reactive oxygen species (ROS), besides causing damage, may also fulfill an important role as second messengers involved in signal transduction. Recently, we have demonstrated that deletion of SOD1 is beneficial for the acquisition of tolerance towards heat and ethanol stresses. The present report demonstrates that a sod1 mutant was the only one capable of acquiring tolerance against a subsequent stress produced by menadione, although this mutant strain had exhibited high sensitivity to oxidative stress. By measuring the level of intracellular oxidation, lipid peroxidation as well as glutathione metabolism, we have shown that in the SOD1deleted strain, an unbalance occurs in the cell redox status. These results indicated that the capacity of acquiring tolerance to oxidative stress is related to a signal given by one or all of the above factors. D 2003 Elsevier Science B.V. All rights reserved. Keywords: ROS; SOD; Oxidative stress; Saccharomyces cerevisiae
1. Introduction Organisms adapted to an aerobic way of life have to cope with the toxic effects of reactive oxygen species (ROS). These reactive molecules can be generated through normal cellular processes, such as respiratory metabolism and phagocytosis, or by the exposure of cells to stress conditions. Most of the cellular components (lipids, proteins, DNA and sugars) can become targets of ROS causing loss of their function and often leading to cell death. Paradoxically, while excess of ROS has been strongly related to a variety of diseases and to the process of aging [1– 3], small amounts of these reactive species have proven to be involved in important physiological functions such as signal transduction associated with the control of gene expression and cell proliferation [4]. Recently, increasing evidence has shown the importance of ROS in signal transduction pathways, through the regulation of the redox state. Several proteins involved in the initial, as well as the final step of signal cascades, are subjected to intracellular redox regulation influencing their biological activities [5 –7]. Moreover, with respect to tran-
* Corresponding author. Tel.: +55-21-2562-7824; fax: +55-21-25627735/7266. E-mail address: [email protected] (E.C.A. Eleutherio).
scription activators, ROS may influence DNA binding and transactivation [8]. In mammalian and yeast cells, this regulation is often mediated through disruption (reduction) or by formation (oxidation) of a disulfide bond within the cysteine residues present in these signaling proteins [8,9]. The yeast Saccharomyces cerevisiae, a well-known eukaryotic model for studies of oxidative stress, constantly senses and adapts to intracellular redox disturbances by the induction of genes or stimulons whose products act to maintain the cellular redox environment [10]. The cytoplasmic Cu/Zn-superoxide dismutase (SOD), which is coded by the SOD1 gene, appears to be a key enzyme involved in the regulation of intracellular levels of ROS and in protecting cells from the exogenous toxicity of oxidant agents [10,11]. On the other hand, overexpression of Cu/Zn-SOD has also been associated with various pathologies, such as Down’s syndrome and Alzheimer’s disease [12,13]. Cellular antioxidant defenses also include several other important factors, such as the mitochondrial Mn-SOD, coded by the SOD2 gene. It protects mitochondria from ROS generated during respiration and exposure to ethanol [14]. Furthermore, reduced glutathione (GSH) and the protein metallothionein (Cup1p) are molecules that act as radical and metal scavengers, being oxidized by ROS or metals and thereby removing oxidants from solution thus protecting cells against oxidation [15,16].
0304-4165/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4165(03)00003-5
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It has been proven that S. cerevisiae possesses two independent mechanisms for protection against peroxide and superoxide stresses [17]. These distinct responses include expression of proteins, which are induced either by hydrogen peroxide or by menadione, as a source of superoxide [18]. On the other hand, expression of some proteins in both oxidative conditions demonstrated that an overlap between these responses exists, probably due to the fact that dismutation of superoxide radicals leads to generation of hydrogen peroxide [18]. In addition, when sensitive first exponential cell cultures of S. cerevisiae growing on glucose are exposed to a sublethal stress condition, such as heat shock at 40 jC/60 min, they acquire tolerance against subsequent lethal oxidative stress conditions [19,20]. Although the phenomenon of acquisition of tolerance to oxidative stress is well documented in the literature, the mechanisms by which cells become more resistant are not yet elucidated. It has been recently reported that lack of cytosolic isoform of superoxide dismutase leads to an enhancement of intracellular oxidation after heat treatment, and a different pattern of Hsp104p and Hsp26p expression has also been observed [20]. These results suggest that the adaptive treatment produce an enhancement of intracellular oxidation, which might increase resistance to subsequent higher levels of oxidation. In order to characterize the sensors of oxidative stress, we have analyzed acquisition of tolerance in yeast cells with Sodp deficiencies, as well as intracellular oxidation during exposure to oxidative stress. To achieve this goal, menadione, a source of superoxide radicals, and tert-butylhydroperoxide, which causes membrane damage, were used and sensibility to these drugs was analyzed. We also monitored the levels of GSH/oxidized glutathione (GSSG) and their involvement with the maintenance of the cellular redox status. The hypothesis that these factors could be involved in the regulation of the expression of genes related to protective mechanisms, like CUP1, will also be discussed.
2. Material and methods 2.1. Yeast strains, media and growth conditions S. cerevisiae strains used in this work are listed in Table 1. Stocks of all strains were maintained on solid YPD medium (1% yeast extract, 2% glucose, 2% peptone and 2% agar) in appropriate conditions to avoid selection of petites or supTable 1 Strains of the yeast S. cerevisiae used in this work Strains
Genotype
Eg103 Eg110 Eg118 Eg133
MATa, leu2, his3D1, trp1-289a, ura3-52, GAL+, mal like Eg103 except sod2::TRP1 like Eg103 except sod1::URA3 like Eg103 except sod2::TRP1, sod1::URA3
pressors. For all experiments, cells were grown up to the middle of exponential phase (1.0 mg dry weight/ml) in liquid YPD medium, using an orbital shaker at 28 jC and 160 rpm, with the ratio of flask volume/medium of 5:1. To confirm that we were working with first exponential cells, the presence of glucose in the medium was analyzed by the glucose oxidase/ peroxidase assay (kit from Merck—cat. no. 3395). 2.2. Stress conditions Oxidative stress was performed by exposure of 10 ml of the cell culture to 30 mM menadione or 2 mM tertbutylhydroperoxide, during 60 min at 28 jC/160 rpm. In all experimental conditions, the cultures were divided into two parts: one was immediately exposed to stress conditions, while the other was previously submitted to a heat treatment at 40 jC/60 min and then exposed to menadione or tert-butylhydroperoxide. 2.3. Viability determinations Cell viability was analyzed by plating, in triplicate, on solidified YPD medium, after proper dilution. The plates were incubated at 28 jC for 72 h and the colonies counted. Viability was determined before and after stress conditions, using cells adapted or not at 40 jC/60 min. Tolerance was expressed as percentage of survival. 2.4. Fluorescence assays The oxidation of the sensitive probe, 2V,7V-dichlorofluorescein was used to measure the enhancement of intracellular oxidation levels caused by 30 mM menadione or 2 mM tert-butylhydroperoxide in cells previously treated or not at 40 jC/60 min. Fluorescence was measured using a Photo Technology International (PTI) spectrofluorimeter set at an excitation wavelength of 504 nm and an emission wavelength of 524 nm [21]. A fresh 5 mM stock solution of dichlorofluorescein dissolved in ethanol was added to the culture (to a final concentration of 10 AM) and incubation at 28 jC continued for 15 min to allow uptake of the probe. Half of the culture was heated at 40 jC and the other was maintained at 28 jC. After 60 min, cells were exposed to menadione or tert-butylhydroperoxide stress. After the severe oxidative stress, 50 mg of cells (adapted or not at 40 jC) were harvested by centrifugation and washed twice with water. The pellets were resuspended in 500 Al of water 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 supernatant solutions were obtained after centrifugation at 25,000 g for 5 min, diluted 6-fold with water, and then fluorescence was measured. As control, fluorescence was measured in cells not exposed to oxidative stress. The results were expressed as the relation between the fluorescence of stressed and non-stressed cells.
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2.5. Determination of glutathione levels
3. Results and discussion
GSH and GSSG concentrations were determined spectrophotometrically, in neutralized trichloroacetic acid (10% TCA) extracts before and after heat treatment at 40 jC/60 min, according to Bernt and Bergmeyer [22].
3.1. Menadione and tert-butylhydroperoxide tolerance in sod mutant cells
2.6. Enzyme assays Total protein extracts were obtained by disruption of 50 mg cells with glass beads in 200 mM Tris/HCl buffer, pH 7.2 on a vortex. Glutathione reductase (GR) activity was determined using a kit from Calbiochem (cat. no. 359962), which is based on NADPH oxidation. The oxidation of NADPH to NADP+ is followed by a decrease in absorbance at 340 nm. h-Galactosidase activity was assayed by measuring o-nitrophenyl-h-D-galactopyranoside hydrolysis as reported by Miller [23]. Protein concentrations were determined by the method of Stickland [24]. 2.7. Detection of lipid peroxidation For measuring lipid peroxidation, 50 mg of cells submitted directly or after heat treatment (40 jC/60 min) to an oxidative stress, were cooled on ice, harvested by centrifugation and washed twice with 20 mM Tris/HCl buffer, pH 7.4. The pellets were resuspended in 500 Al of the same buffer 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 extracts were used for detection of lipid peroxidation using an assay based on the condensation of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) with a chromogenic reagent, which yields a stable chromophore with maximal absorbance at 586 nm (kit from Calbiochem—cat. no. 437634). 2.8. Construction of yeast transformants The pYI plasmid, containing the Cup1 5Vupstream promoter joined in frame with the LacZ reporter gene [16], was used to transform yeast cells by the lithium acetate method [25]. This plasmid was a kind gift from Dr. D.J. Thiele (Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606). Transformed cells were selected by plating on solid YNB medium (2% glucose, 0.67% yeast nitrogen base without amino acids, 0.01% appropriate auxothrophic requirements and 2% agar). 2.9. Expression of metallothionein For determination of metallothionein expression, transformants collected at the middle of the exponential phase in liquid YNB medium, containing or not 50 AM CuSO4, were harvested before and after heat shock and assayed for h-galactosidase activity as described in enzyme assays.
Viability of S. cerevisiae strains, exposed to oxidative conditions was analyzed in cells, either in first exponential phase in the presence of glucose, which promotes a strong repression of various antioxidant genes [26], or harvested at that stage and submitted to an adaptive treatment at 40 jC/ 60 min. This non-lethal treatment causes a set of metabolic changes, which are related to the acquisition of tolerance against many severe stress conditions, including oxidative stress [17,19,26]. Although a previous report showed sensitivity of sod mutants to superoxide and peroxide [18], the process of acquisition of tolerance in these mutant cells has not been analyzed. As expected, the role of Sod1p seems extremely important for protection against oxidative stress caused by exogenous menadione, since strains deficient in Sod1p were unable to survive when directly exposed to this condition (Fig. 1A). Results obtained with the double mutant, Eg133, confirmed the importance of Sod1p to confer high tolerance to cells under the same conditions. It is interesting to note that, after exposure of cells to a sublethal heat shock at 40 jC/60 min, only cells of the single sod1 mutant strain acquired tolerance. The double mutant continued to be hypersensitive to the superoxide stress produced by menadione. This result suggests that the acquisition of tolerance, after heat treatment, observed only in the Sod1p-deficient strain, is due to induction of Sod2p, which is absent in the double mutant strain. During this adaptive treatment, the activity of Sod2p is induced, indicating that the deficiency in Sod1p is overcome by an enhancement in the mitochondrial isoform activity [14,27]. In the case of the control and Sod2p-deficient strains, we did not observe any change in tolerance after heat treatment, probably due to the fact that the Sod1p present in these cells is sufficient to protect them against this stress (Fig. 1A). With respect to the second oxidative stress generator used in this study, tert-butylhydroperoxide, a different pattern of action was observed. Although, it has been suggested that exposure to peroxide leads to lipid peroxidation, our results demonstrated that the presence of Sod1p is also important to detoxify ROS production caused by tertbutylhydroperoxide (Fig. 1B). This result suggests that besides being involved in superoxide dismutation, Sod1p might be involved in another mechanism for ROS elimination. On the other hand, the Sod2p isoform might not be required for protection against this stress, since only strains carrying sod1 mutation exhibited a significant reduction in survival. The heat treatment, which often leads to an acquisition of tolerance against different kinds of adverse conditions including oxidative stress, caused a decrease in tolerance to peroxide in all strains tested. It has been documented that during such a heat treatment, an alteration
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M.D. Pereira et al. / Biochimica et Biophysica Acta 1620 (2003) 245–251 Table 2 Enhancement of intracellular oxidation measured as fold increase in fluorescence Strains
Control sod1
Menadione
tert-butylhydroperoxide
Non-adapted
Adapted
Non-adapted
Adapted
1.60 F 0 6.90 F 0.21
1.60 F 0.50 2.40 F 0
0.90 F 0.13 0.90 F 0.07
1.00 F 0.40 2.00 F 0.21
Relation between fluorescence of stressed and non-stressed cells during exposure to oxidative stress. Cells were directly exposed to stress (nonadapted) or previously treated at 40 jC/60 min (adapted). The results represent the mean F standard deviation of three independent experiments.
Fig. 1. Effect of 30 mM menadione (A) and 2 mM tert-butylhydroperoxide (B) on cell viability. Control and sod mutant cells, harvested in first exponential phase, were directly stressed (black bars) or were previously submitted to a mild heat shock at 40 jC/60 min before being exposed to oxidative stress conditions (white bars). The results represent the mean F standard deviation of three independent experiments.
of the lipid composition occurs and the plasma membrane becomes more unsaturated [28,29], which might contribute to enhance the toxicity of peroxide, producing more lipid peroxides and ROS. 3.2. Analysis of cell redox status Many aerobic organisms show adaptive responses to oxidative stress by increasing the levels of antioxidant enzymes. A part of this adaptive response is regulated at the transcriptional level. The activities of some of these transcription factors, like mammalian NF-nB and yeast Yap1, are reversibly controlled through the redox status and modulated by thiol-disulfide oxidoreductases [8,30,31]. Thus in order to address the question why only sod1 mutant cells were capable of acquiring tolerance to a superoxide stress (Fig. 1A), the cell redox status was monitored. To determine whether yeast cells carrying a Sod1p deficiency generate increased levels of ROS during oxidative stress conditions caused by menadione and tert-butylhydroperoxide, the fluorescent probe, 2V,7V-dichlorofluorescein diacetate (DCF) was used. This probe is absorbed and trapped
inside the cells after cleavage of the diacetates by an intracellular esterase, and thereafter, is no longer able to leave the cell [32]. Once inside the cell, it becomes susceptible to attack by ROS, producing a more fluorescent compound [32]. According to Table 2, after direct exposure of cells to a menadione stress, the mean of DCF fluorescence in sod1 cells was significantly increased (almost 7-fold), while the tolerant control strain showed less than a 2-fold increase. These results suggest that an abrupt enhancement of intracellular ROS levels could be responsible for conferring hypersensitivity to oxidative stress conditions in the sod1 mutant strain. On the other hand, after heat treatment, we observed a strong reduction in the fluorescence rates of the Sod1p-deficient strain, demonstrating that the heat treatment promoted an adaptation of the cells to a subsequent menadione lethal exposure. These results corroborate the survival experiments (Fig. 1A), which showed that sod1 cells were extremely sensitive to menadione when directly exposed. Tolerance was acquired only after a heat treatment. With respect to the oxidation of the fluorescent probe during exposure to tert-butylhydroperoxide, no significant differences between the isogenic strains were observed (Table 2). The peroxide stress did not increase intracellular oxidation, suggesting that this kind of stress did not represent great toxicity through intracellular ROS generation. However, when sod1 mutant strains were previously heattreated, a 2-fold increase in fluorescence was observed. Since the target of tert-butylhydroperoxide seems to be the membrane, we measured the level of lipid peroxidation in cells submitted to this oxidative stress. As can be seen in Table 3, the sod1 mutant strain, when exposed to stress, produced higher levels of lipid peroxidation as compared to Table 3 Determination of lipid peroxidation in control and sod1 mutant strains grown to first exponential phase Strains
Control sod1
Levels of lipid peroxidation (pmol/mg cell) Non-stressed
Adapted (40 jC/ 1 h)
Directly stressed
Adapted at 40 jC and stressed
50 F 7 70 F 30
54 F 20 86 F 7
70 F 20 130 F 20
100 F 3 140 F 14
Cells were submitted or not to an adaptive heat stress (40 jC/60 min), before being exposed to 2 mM tert-butylhydroperoxide (stressed cells). The results represent the mean F standard deviation of three independent experiments.
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Fig. 2. Intracellular levels of reduced (A) and oxidized (B) glutathione in cells growing exponentially on glucose subjected or not to a heat treatment at 40 jC for 60 min. The results represent the mean F standard deviation of three independent experiments.
its parental strain, confirming that the Sod1p deficiency increases the level of intracellular oxidation. This phenotype exhibited by the sod1 mutant might be explained by the role of Sod1p in the elimination of ROS generated by peroxide. It has been reported that at higher temperatures an increase in lipid unsaturation levels occurs [28], constituting an optimum target for tert-butylhydroperoxide, which could explain the lower survival rates after heat treatment. According to Table 3, the control strain showed a significant increase in lipid peroxidation when previously treated at 40 jC (cells directly stressed presented a lower level of lipid peroxidation than adapted and stressed cells). These results are in agreement with tolerance to tert-butylhydroperoxide, since cells of control strain pre-heated and then exposed to this drug showed a lower survival rate than cells directly stressed (Fig 1B). On the other hand, the resistance of the sod1 mutant did not change with the same intensity when cells were adapted at 40 jC. In accordance with these results, the levels of lipid peroxidation produced by tert-
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butylhydroperoxide in sod1 cells (Table 3), although higher than those of control strain, did not differ after the heat treatment. Finally, intracellular GSH and GSSG contents were analyzed. It has recently been reported that oxidation of GSH to GSSG is intimately related to the regulation of expression of genes involved in cellular protection [29]. As seen in Fig. 2A, no differences between the sod1 mutant and the control strain were observed in relation to intracellular GSH levels during growth of cells harvested at the middle of the exponential phase. However, after heat treatment at 40 jC, we observed that there was a significant decline in intracellular GSH levels in the Sod1p-deficient strain, while in the control strain, there was no alteration in either GSSG or GSH contents before and after the heat treatment (Fig. 2B). This observation suggests that the concentration of this oxidized molecule is controlled by an efficient turnover of GSH catalyzed by GR. It should be emphasized that in exponentially growing cells of the sod1 mutant, the level of GSSG was much higher than in the control strain, demonstrating that these cells are oxidized even during normal cellular growth, without any additional treatment. The reduced GSH turnover in the Sod1p-deficient strain is probably due the fact that the enzyme glutathione reductase, involved in this process, has lost approximately 50% of its activity, as compared to the control strain (Fig. 3). These results confirm that the intracellular environment of the sod1 mutant strain is always more oxidized than in the control due to the incapacity of GSH, in this strain, of maintaining the cellular redox status. It was previously demonstrated that intracellular oxidation induced by the adaptive treatment at 40 jC is increased 2-fold in sod1 mutant cells as compared to their control strain [20]. A slightly more
Fig. 3. Glutathione reductase activity during adaptive heat. Cell-free extracts were prepared for enzyme determinations immediately before and after mild heat treatment at 40 jC for 60 min. One unit of enzyme activity is defined as the amount of enzyme that catalyses the conversions of 1 Amol NADPH in 1 min under the assay conditions. The results represent the mean F standard deviation of three independent experiments.
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oxidized cellular environment might generate a signal that triggers the transcriptional induction of genes, whose products could be beneficial for acquisition of tolerance. 3.3. Analysis of metallothionein expression Since a more oxidized intracellular environment produced by heat treatment seemed to be involved with a better response to a menadione stress in the sod1 mutant strain, the expression of the CUP1 gene was studied using the reporter gene LacZ. In mammalian and yeast, metallothionein serves to protect cells during exposure to oxidative stress, by removing the excess of metals, such as copper, involved with the production of ROS. CUP1 gene is also inducible following exposure to oxidants, demonstrating the importance of conferring tolerance against oxidative stress [16,33]. As seen in Fig. 4A, heat treatment at 40 jC/60 min did not induce the expression of CUP1 in either strain. However, these experiments were carried out in YNB minimal medium, a more oxidized environment than the YPD standard medium (results not shown), which may explain the absence of a change in expression. On the other hand, during normal growth, as well as after heat treatment, the sod1 mutant already showed a 4-fold increase in the expression of CUP1 (Fig. 4A). Due to the low levels of h-galactosidase activities observed, we added 50 AM CuSO4 to the culture media, which is known to induce the expression of metallothionein, with no injury to cell survival. The results observed in Fig. 4B demonstrate that in the sod1 mutant, in the presence of CuSO4, the h-galactosidase activity was also three to four times higher than that observed in the control strain, confirming that this increase in CUP1 expression is, in fact, related to the Sod1p deficiency. Under those conditions, the heat treatment was also inefficient in inducing the expression of metallothionein. The more oxidized environment of sod1 cells seems to contribute to the activation of stress responses at the transcriptional level, since a higher level of expression of CUP1 was found in this mutant strain. It has been well documented that heat and oxidative stresses (both conditions involved with ROS generation) lead to the activation of the Hsf transcription factor, resulting in increased expression of a diverse range of genes encoding proteins that protect the cell against stress-induced damages [33 –35]. It has been verified that Yap1p was constitutively activated and localized in the nucleus of a thiorredoxin mutant strain, which seemed to be related to the intracellular redox status [8,31]. Further experiments for elucidating Hsfp activation and Yap1p nuclear localization in this sod1 mutant are necessary to determine which regulatory pathway is triggering the sod1 mutant response of acquiring tolerance after an adaptive heat treatment. Finally, the results presented here represent strong evidence for correlating the cell capacity of acquiring tolerance with the level of intracellular oxidation. During non-lethal
Fig. 4. Metallothionein expression. Free extracts from transformed cells with CUP1-LacZ grown on minimal media were prepared for hgalactosidase activity determinations immediately before and after mild heat treatment at 40 jC for 60 min (A). The same experiment was conducted with cells growing in minimal medium added of 50 AM CuSO4 (B). One unit of enzyme activity is defined as the amount of enzyme that catalyses the production of 1 Amol ONP (o-nitrophenyl) in 1 min under the assay conditions. The results represent the mean F standard deviation of three independent experiments.
oxidative stress, as occurs during heat treatment, the level of oxidation could signal for activation of the expression of certain genes, here exemplified by CUP1, leading to a response intimately associated with the increase of tolerance. However, when the level of ROS exceeds the detoxification capacity of the antioxidant defense system, the fatal consequence is the loss of viability and death—under these circumstances Sod1p plays an important role for cell protection.
Acknowledgements We would like to thank Dr. E.B. Gralla and Dr. J.S. Valentine for providing the SOD strains as well as Prof. Ricardo Choloub and Dr. Marcoaurelio Almenara for the
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use of PTI spectrofluorimeter. This work was supported by grants from CNPq, FAPERJ and FAPESP.
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Targets of oxidative stress in yeast sod mutants M.D. Pereira, R.S. Herdeiro, P.N. Fernandes, E.C.A. Eleutherio *, A.D. Panek Departamento de Bioquı´mica, Instituto de Quı´mica, UFRJ, 21949-900 Rio de Janeiro, Brazil Received 26 June 2002; received in revised form 9 December 2002; accepted 16 December 2002
Abstract Eukaryotic cells have developed mechanisms to rapidly respond towards the environment by changing the expression of a series of genes. There is increasing evidence that reactive oxygen species (ROS), besides causing damage, may also fulfill an important role as second messengers involved in signal transduction. Recently, we have demonstrated that deletion of SOD1 is beneficial for the acquisition of tolerance towards heat and ethanol stresses. The present report demonstrates that a sod1 mutant was the only one capable of acquiring tolerance against a subsequent stress produced by menadione, although this mutant strain had exhibited high sensitivity to oxidative stress. By measuring the level of intracellular oxidation, lipid peroxidation as well as glutathione metabolism, we have shown that in the SOD1deleted strain, an unbalance occurs in the cell redox status. These results indicated that the capacity of acquiring tolerance to oxidative stress is related to a signal given by one or all of the above factors. D 2003 Elsevier Science B.V. All rights reserved. Keywords: ROS; SOD; Oxidative stress; Saccharomyces cerevisiae
1. Introduction Organisms adapted to an aerobic way of life have to cope with the toxic effects of reactive oxygen species (ROS). These reactive molecules can be generated through normal cellular processes, such as respiratory metabolism and phagocytosis, or by the exposure of cells to stress conditions. Most of the cellular components (lipids, proteins, DNA and sugars) can become targets of ROS causing loss of their function and often leading to cell death. Paradoxically, while excess of ROS has been strongly related to a variety of diseases and to the process of aging [1– 3], small amounts of these reactive species have proven to be involved in important physiological functions such as signal transduction associated with the control of gene expression and cell proliferation [4]. Recently, increasing evidence has shown the importance of ROS in signal transduction pathways, through the regulation of the redox state. Several proteins involved in the initial, as well as the final step of signal cascades, are subjected to intracellular redox regulation influencing their biological activities [5 –7]. Moreover, with respect to tran-
* Corresponding author. Tel.: +55-21-2562-7824; fax: +55-21-25627735/7266. E-mail address: [email protected] (E.C.A. Eleutherio).
scription activators, ROS may influence DNA binding and transactivation [8]. In mammalian and yeast cells, this regulation is often mediated through disruption (reduction) or by formation (oxidation) of a disulfide bond within the cysteine residues present in these signaling proteins [8,9]. The yeast Saccharomyces cerevisiae, a well-known eukaryotic model for studies of oxidative stress, constantly senses and adapts to intracellular redox disturbances by the induction of genes or stimulons whose products act to maintain the cellular redox environment [10]. The cytoplasmic Cu/Zn-superoxide dismutase (SOD), which is coded by the SOD1 gene, appears to be a key enzyme involved in the regulation of intracellular levels of ROS and in protecting cells from the exogenous toxicity of oxidant agents [10,11]. On the other hand, overexpression of Cu/Zn-SOD has also been associated with various pathologies, such as Down’s syndrome and Alzheimer’s disease [12,13]. Cellular antioxidant defenses also include several other important factors, such as the mitochondrial Mn-SOD, coded by the SOD2 gene. It protects mitochondria from ROS generated during respiration and exposure to ethanol [14]. Furthermore, reduced glutathione (GSH) and the protein metallothionein (Cup1p) are molecules that act as radical and metal scavengers, being oxidized by ROS or metals and thereby removing oxidants from solution thus protecting cells against oxidation [15,16].
0304-4165/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4165(03)00003-5
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It has been proven that S. cerevisiae possesses two independent mechanisms for protection against peroxide and superoxide stresses [17]. These distinct responses include expression of proteins, which are induced either by hydrogen peroxide or by menadione, as a source of superoxide [18]. On the other hand, expression of some proteins in both oxidative conditions demonstrated that an overlap between these responses exists, probably due to the fact that dismutation of superoxide radicals leads to generation of hydrogen peroxide [18]. In addition, when sensitive first exponential cell cultures of S. cerevisiae growing on glucose are exposed to a sublethal stress condition, such as heat shock at 40 jC/60 min, they acquire tolerance against subsequent lethal oxidative stress conditions [19,20]. Although the phenomenon of acquisition of tolerance to oxidative stress is well documented in the literature, the mechanisms by which cells become more resistant are not yet elucidated. It has been recently reported that lack of cytosolic isoform of superoxide dismutase leads to an enhancement of intracellular oxidation after heat treatment, and a different pattern of Hsp104p and Hsp26p expression has also been observed [20]. These results suggest that the adaptive treatment produce an enhancement of intracellular oxidation, which might increase resistance to subsequent higher levels of oxidation. In order to characterize the sensors of oxidative stress, we have analyzed acquisition of tolerance in yeast cells with Sodp deficiencies, as well as intracellular oxidation during exposure to oxidative stress. To achieve this goal, menadione, a source of superoxide radicals, and tert-butylhydroperoxide, which causes membrane damage, were used and sensibility to these drugs was analyzed. We also monitored the levels of GSH/oxidized glutathione (GSSG) and their involvement with the maintenance of the cellular redox status. The hypothesis that these factors could be involved in the regulation of the expression of genes related to protective mechanisms, like CUP1, will also be discussed.
2. Material and methods 2.1. Yeast strains, media and growth conditions S. cerevisiae strains used in this work are listed in Table 1. Stocks of all strains were maintained on solid YPD medium (1% yeast extract, 2% glucose, 2% peptone and 2% agar) in appropriate conditions to avoid selection of petites or supTable 1 Strains of the yeast S. cerevisiae used in this work Strains
Genotype
Eg103 Eg110 Eg118 Eg133
MATa, leu2, his3D1, trp1-289a, ura3-52, GAL+, mal like Eg103 except sod2::TRP1 like Eg103 except sod1::URA3 like Eg103 except sod2::TRP1, sod1::URA3
pressors. For all experiments, cells were grown up to the middle of exponential phase (1.0 mg dry weight/ml) in liquid YPD medium, using an orbital shaker at 28 jC and 160 rpm, with the ratio of flask volume/medium of 5:1. To confirm that we were working with first exponential cells, the presence of glucose in the medium was analyzed by the glucose oxidase/ peroxidase assay (kit from Merck—cat. no. 3395). 2.2. Stress conditions Oxidative stress was performed by exposure of 10 ml of the cell culture to 30 mM menadione or 2 mM tertbutylhydroperoxide, during 60 min at 28 jC/160 rpm. In all experimental conditions, the cultures were divided into two parts: one was immediately exposed to stress conditions, while the other was previously submitted to a heat treatment at 40 jC/60 min and then exposed to menadione or tert-butylhydroperoxide. 2.3. Viability determinations Cell viability was analyzed by plating, in triplicate, on solidified YPD medium, after proper dilution. The plates were incubated at 28 jC for 72 h and the colonies counted. Viability was determined before and after stress conditions, using cells adapted or not at 40 jC/60 min. Tolerance was expressed as percentage of survival. 2.4. Fluorescence assays The oxidation of the sensitive probe, 2V,7V-dichlorofluorescein was used to measure the enhancement of intracellular oxidation levels caused by 30 mM menadione or 2 mM tert-butylhydroperoxide in cells previously treated or not at 40 jC/60 min. Fluorescence was measured using a Photo Technology International (PTI) spectrofluorimeter set at an excitation wavelength of 504 nm and an emission wavelength of 524 nm [21]. A fresh 5 mM stock solution of dichlorofluorescein dissolved in ethanol was added to the culture (to a final concentration of 10 AM) and incubation at 28 jC continued for 15 min to allow uptake of the probe. Half of the culture was heated at 40 jC and the other was maintained at 28 jC. After 60 min, cells were exposed to menadione or tert-butylhydroperoxide stress. After the severe oxidative stress, 50 mg of cells (adapted or not at 40 jC) were harvested by centrifugation and washed twice with water. The pellets were resuspended in 500 Al of water 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 supernatant solutions were obtained after centrifugation at 25,000 g for 5 min, diluted 6-fold with water, and then fluorescence was measured. As control, fluorescence was measured in cells not exposed to oxidative stress. The results were expressed as the relation between the fluorescence of stressed and non-stressed cells.
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2.5. Determination of glutathione levels
3. Results and discussion
GSH and GSSG concentrations were determined spectrophotometrically, in neutralized trichloroacetic acid (10% TCA) extracts before and after heat treatment at 40 jC/60 min, according to Bernt and Bergmeyer [22].
3.1. Menadione and tert-butylhydroperoxide tolerance in sod mutant cells
2.6. Enzyme assays Total protein extracts were obtained by disruption of 50 mg cells with glass beads in 200 mM Tris/HCl buffer, pH 7.2 on a vortex. Glutathione reductase (GR) activity was determined using a kit from Calbiochem (cat. no. 359962), which is based on NADPH oxidation. The oxidation of NADPH to NADP+ is followed by a decrease in absorbance at 340 nm. h-Galactosidase activity was assayed by measuring o-nitrophenyl-h-D-galactopyranoside hydrolysis as reported by Miller [23]. Protein concentrations were determined by the method of Stickland [24]. 2.7. Detection of lipid peroxidation For measuring lipid peroxidation, 50 mg of cells submitted directly or after heat treatment (40 jC/60 min) to an oxidative stress, were cooled on ice, harvested by centrifugation and washed twice with 20 mM Tris/HCl buffer, pH 7.4. The pellets were resuspended in 500 Al of the same buffer 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 extracts were used for detection of lipid peroxidation using an assay based on the condensation of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) with a chromogenic reagent, which yields a stable chromophore with maximal absorbance at 586 nm (kit from Calbiochem—cat. no. 437634). 2.8. Construction of yeast transformants The pYI plasmid, containing the Cup1 5Vupstream promoter joined in frame with the LacZ reporter gene [16], was used to transform yeast cells by the lithium acetate method [25]. This plasmid was a kind gift from Dr. D.J. Thiele (Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606). Transformed cells were selected by plating on solid YNB medium (2% glucose, 0.67% yeast nitrogen base without amino acids, 0.01% appropriate auxothrophic requirements and 2% agar). 2.9. Expression of metallothionein For determination of metallothionein expression, transformants collected at the middle of the exponential phase in liquid YNB medium, containing or not 50 AM CuSO4, were harvested before and after heat shock and assayed for h-galactosidase activity as described in enzyme assays.
Viability of S. cerevisiae strains, exposed to oxidative conditions was analyzed in cells, either in first exponential phase in the presence of glucose, which promotes a strong repression of various antioxidant genes [26], or harvested at that stage and submitted to an adaptive treatment at 40 jC/ 60 min. This non-lethal treatment causes a set of metabolic changes, which are related to the acquisition of tolerance against many severe stress conditions, including oxidative stress [17,19,26]. Although a previous report showed sensitivity of sod mutants to superoxide and peroxide [18], the process of acquisition of tolerance in these mutant cells has not been analyzed. As expected, the role of Sod1p seems extremely important for protection against oxidative stress caused by exogenous menadione, since strains deficient in Sod1p were unable to survive when directly exposed to this condition (Fig. 1A). Results obtained with the double mutant, Eg133, confirmed the importance of Sod1p to confer high tolerance to cells under the same conditions. It is interesting to note that, after exposure of cells to a sublethal heat shock at 40 jC/60 min, only cells of the single sod1 mutant strain acquired tolerance. The double mutant continued to be hypersensitive to the superoxide stress produced by menadione. This result suggests that the acquisition of tolerance, after heat treatment, observed only in the Sod1p-deficient strain, is due to induction of Sod2p, which is absent in the double mutant strain. During this adaptive treatment, the activity of Sod2p is induced, indicating that the deficiency in Sod1p is overcome by an enhancement in the mitochondrial isoform activity [14,27]. In the case of the control and Sod2p-deficient strains, we did not observe any change in tolerance after heat treatment, probably due to the fact that the Sod1p present in these cells is sufficient to protect them against this stress (Fig. 1A). With respect to the second oxidative stress generator used in this study, tert-butylhydroperoxide, a different pattern of action was observed. Although, it has been suggested that exposure to peroxide leads to lipid peroxidation, our results demonstrated that the presence of Sod1p is also important to detoxify ROS production caused by tertbutylhydroperoxide (Fig. 1B). This result suggests that besides being involved in superoxide dismutation, Sod1p might be involved in another mechanism for ROS elimination. On the other hand, the Sod2p isoform might not be required for protection against this stress, since only strains carrying sod1 mutation exhibited a significant reduction in survival. The heat treatment, which often leads to an acquisition of tolerance against different kinds of adverse conditions including oxidative stress, caused a decrease in tolerance to peroxide in all strains tested. It has been documented that during such a heat treatment, an alteration
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M.D. Pereira et al. / Biochimica et Biophysica Acta 1620 (2003) 245–251 Table 2 Enhancement of intracellular oxidation measured as fold increase in fluorescence Strains
Control sod1
Menadione
tert-butylhydroperoxide
Non-adapted
Adapted
Non-adapted
Adapted
1.60 F 0 6.90 F 0.21
1.60 F 0.50 2.40 F 0
0.90 F 0.13 0.90 F 0.07
1.00 F 0.40 2.00 F 0.21
Relation between fluorescence of stressed and non-stressed cells during exposure to oxidative stress. Cells were directly exposed to stress (nonadapted) or previously treated at 40 jC/60 min (adapted). The results represent the mean F standard deviation of three independent experiments.
Fig. 1. Effect of 30 mM menadione (A) and 2 mM tert-butylhydroperoxide (B) on cell viability. Control and sod mutant cells, harvested in first exponential phase, were directly stressed (black bars) or were previously submitted to a mild heat shock at 40 jC/60 min before being exposed to oxidative stress conditions (white bars). The results represent the mean F standard deviation of three independent experiments.
of the lipid composition occurs and the plasma membrane becomes more unsaturated [28,29], which might contribute to enhance the toxicity of peroxide, producing more lipid peroxides and ROS. 3.2. Analysis of cell redox status Many aerobic organisms show adaptive responses to oxidative stress by increasing the levels of antioxidant enzymes. A part of this adaptive response is regulated at the transcriptional level. The activities of some of these transcription factors, like mammalian NF-nB and yeast Yap1, are reversibly controlled through the redox status and modulated by thiol-disulfide oxidoreductases [8,30,31]. Thus in order to address the question why only sod1 mutant cells were capable of acquiring tolerance to a superoxide stress (Fig. 1A), the cell redox status was monitored. To determine whether yeast cells carrying a Sod1p deficiency generate increased levels of ROS during oxidative stress conditions caused by menadione and tert-butylhydroperoxide, the fluorescent probe, 2V,7V-dichlorofluorescein diacetate (DCF) was used. This probe is absorbed and trapped
inside the cells after cleavage of the diacetates by an intracellular esterase, and thereafter, is no longer able to leave the cell [32]. Once inside the cell, it becomes susceptible to attack by ROS, producing a more fluorescent compound [32]. According to Table 2, after direct exposure of cells to a menadione stress, the mean of DCF fluorescence in sod1 cells was significantly increased (almost 7-fold), while the tolerant control strain showed less than a 2-fold increase. These results suggest that an abrupt enhancement of intracellular ROS levels could be responsible for conferring hypersensitivity to oxidative stress conditions in the sod1 mutant strain. On the other hand, after heat treatment, we observed a strong reduction in the fluorescence rates of the Sod1p-deficient strain, demonstrating that the heat treatment promoted an adaptation of the cells to a subsequent menadione lethal exposure. These results corroborate the survival experiments (Fig. 1A), which showed that sod1 cells were extremely sensitive to menadione when directly exposed. Tolerance was acquired only after a heat treatment. With respect to the oxidation of the fluorescent probe during exposure to tert-butylhydroperoxide, no significant differences between the isogenic strains were observed (Table 2). The peroxide stress did not increase intracellular oxidation, suggesting that this kind of stress did not represent great toxicity through intracellular ROS generation. However, when sod1 mutant strains were previously heattreated, a 2-fold increase in fluorescence was observed. Since the target of tert-butylhydroperoxide seems to be the membrane, we measured the level of lipid peroxidation in cells submitted to this oxidative stress. As can be seen in Table 3, the sod1 mutant strain, when exposed to stress, produced higher levels of lipid peroxidation as compared to Table 3 Determination of lipid peroxidation in control and sod1 mutant strains grown to first exponential phase Strains
Control sod1
Levels of lipid peroxidation (pmol/mg cell) Non-stressed
Adapted (40 jC/ 1 h)
Directly stressed
Adapted at 40 jC and stressed
50 F 7 70 F 30
54 F 20 86 F 7
70 F 20 130 F 20
100 F 3 140 F 14
Cells were submitted or not to an adaptive heat stress (40 jC/60 min), before being exposed to 2 mM tert-butylhydroperoxide (stressed cells). The results represent the mean F standard deviation of three independent experiments.
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Fig. 2. Intracellular levels of reduced (A) and oxidized (B) glutathione in cells growing exponentially on glucose subjected or not to a heat treatment at 40 jC for 60 min. The results represent the mean F standard deviation of three independent experiments.
its parental strain, confirming that the Sod1p deficiency increases the level of intracellular oxidation. This phenotype exhibited by the sod1 mutant might be explained by the role of Sod1p in the elimination of ROS generated by peroxide. It has been reported that at higher temperatures an increase in lipid unsaturation levels occurs [28], constituting an optimum target for tert-butylhydroperoxide, which could explain the lower survival rates after heat treatment. According to Table 3, the control strain showed a significant increase in lipid peroxidation when previously treated at 40 jC (cells directly stressed presented a lower level of lipid peroxidation than adapted and stressed cells). These results are in agreement with tolerance to tert-butylhydroperoxide, since cells of control strain pre-heated and then exposed to this drug showed a lower survival rate than cells directly stressed (Fig 1B). On the other hand, the resistance of the sod1 mutant did not change with the same intensity when cells were adapted at 40 jC. In accordance with these results, the levels of lipid peroxidation produced by tert-
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butylhydroperoxide in sod1 cells (Table 3), although higher than those of control strain, did not differ after the heat treatment. Finally, intracellular GSH and GSSG contents were analyzed. It has recently been reported that oxidation of GSH to GSSG is intimately related to the regulation of expression of genes involved in cellular protection [29]. As seen in Fig. 2A, no differences between the sod1 mutant and the control strain were observed in relation to intracellular GSH levels during growth of cells harvested at the middle of the exponential phase. However, after heat treatment at 40 jC, we observed that there was a significant decline in intracellular GSH levels in the Sod1p-deficient strain, while in the control strain, there was no alteration in either GSSG or GSH contents before and after the heat treatment (Fig. 2B). This observation suggests that the concentration of this oxidized molecule is controlled by an efficient turnover of GSH catalyzed by GR. It should be emphasized that in exponentially growing cells of the sod1 mutant, the level of GSSG was much higher than in the control strain, demonstrating that these cells are oxidized even during normal cellular growth, without any additional treatment. The reduced GSH turnover in the Sod1p-deficient strain is probably due the fact that the enzyme glutathione reductase, involved in this process, has lost approximately 50% of its activity, as compared to the control strain (Fig. 3). These results confirm that the intracellular environment of the sod1 mutant strain is always more oxidized than in the control due to the incapacity of GSH, in this strain, of maintaining the cellular redox status. It was previously demonstrated that intracellular oxidation induced by the adaptive treatment at 40 jC is increased 2-fold in sod1 mutant cells as compared to their control strain [20]. A slightly more
Fig. 3. Glutathione reductase activity during adaptive heat. Cell-free extracts were prepared for enzyme determinations immediately before and after mild heat treatment at 40 jC for 60 min. One unit of enzyme activity is defined as the amount of enzyme that catalyses the conversions of 1 Amol NADPH in 1 min under the assay conditions. The results represent the mean F standard deviation of three independent experiments.
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oxidized cellular environment might generate a signal that triggers the transcriptional induction of genes, whose products could be beneficial for acquisition of tolerance. 3.3. Analysis of metallothionein expression Since a more oxidized intracellular environment produced by heat treatment seemed to be involved with a better response to a menadione stress in the sod1 mutant strain, the expression of the CUP1 gene was studied using the reporter gene LacZ. In mammalian and yeast, metallothionein serves to protect cells during exposure to oxidative stress, by removing the excess of metals, such as copper, involved with the production of ROS. CUP1 gene is also inducible following exposure to oxidants, demonstrating the importance of conferring tolerance against oxidative stress [16,33]. As seen in Fig. 4A, heat treatment at 40 jC/60 min did not induce the expression of CUP1 in either strain. However, these experiments were carried out in YNB minimal medium, a more oxidized environment than the YPD standard medium (results not shown), which may explain the absence of a change in expression. On the other hand, during normal growth, as well as after heat treatment, the sod1 mutant already showed a 4-fold increase in the expression of CUP1 (Fig. 4A). Due to the low levels of h-galactosidase activities observed, we added 50 AM CuSO4 to the culture media, which is known to induce the expression of metallothionein, with no injury to cell survival. The results observed in Fig. 4B demonstrate that in the sod1 mutant, in the presence of CuSO4, the h-galactosidase activity was also three to four times higher than that observed in the control strain, confirming that this increase in CUP1 expression is, in fact, related to the Sod1p deficiency. Under those conditions, the heat treatment was also inefficient in inducing the expression of metallothionein. The more oxidized environment of sod1 cells seems to contribute to the activation of stress responses at the transcriptional level, since a higher level of expression of CUP1 was found in this mutant strain. It has been well documented that heat and oxidative stresses (both conditions involved with ROS generation) lead to the activation of the Hsf transcription factor, resulting in increased expression of a diverse range of genes encoding proteins that protect the cell against stress-induced damages [33 –35]. It has been verified that Yap1p was constitutively activated and localized in the nucleus of a thiorredoxin mutant strain, which seemed to be related to the intracellular redox status [8,31]. Further experiments for elucidating Hsfp activation and Yap1p nuclear localization in this sod1 mutant are necessary to determine which regulatory pathway is triggering the sod1 mutant response of acquiring tolerance after an adaptive heat treatment. Finally, the results presented here represent strong evidence for correlating the cell capacity of acquiring tolerance with the level of intracellular oxidation. During non-lethal
Fig. 4. Metallothionein expression. Free extracts from transformed cells with CUP1-LacZ grown on minimal media were prepared for hgalactosidase activity determinations immediately before and after mild heat treatment at 40 jC for 60 min (A). The same experiment was conducted with cells growing in minimal medium added of 50 AM CuSO4 (B). One unit of enzyme activity is defined as the amount of enzyme that catalyses the production of 1 Amol ONP (o-nitrophenyl) in 1 min under the assay conditions. The results represent the mean F standard deviation of three independent experiments.
oxidative stress, as occurs during heat treatment, the level of oxidation could signal for activation of the expression of certain genes, here exemplified by CUP1, leading to a response intimately associated with the increase of tolerance. However, when the level of ROS exceeds the detoxification capacity of the antioxidant defense system, the fatal consequence is the loss of viability and death—under these circumstances Sod1p plays an important role for cell protection.
Acknowledgements We would like to thank Dr. E.B. Gralla and Dr. J.S. Valentine for providing the SOD strains as well as Prof. Ricardo Choloub and Dr. Marcoaurelio Almenara for the
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use of PTI spectrofluorimeter. This work was supported by grants from CNPq, FAPERJ and FAPESP.
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