Protection against oxidation during dehydration of yeast

Abstract

Based on the well-documented notion that oxygen affects the stability of dried cells, the role of the cytosolic and mitochondrial forms of superoxide dismutase (Sod) in the capacity of cells to resist dehydration was examined. Both enzymes are important for improving survival, and the absence of only 1 isoform did not impair tolerance against dehydration. In addition, sod strains showed the same Sod activity as the control strain, indicating that the deficiency in either cytoplasmic Cu/Zn or mitochondrial Mn was overcome by an increase in activity of the remaining Sod. To measure the level of intracellular oxidation produced by dehydration, a fluorescent probe, 2′,7′-dichlorofluorescein, was used. Dry cells exhibited a high increase in fluorescence: both control and sod mutant strains became almost 10-fold more oxidized after dehydration. Furthermore, the disaccharide trehalose was shown to protect dry cells against oxidation.

INTRODUCTION

Preservation of cells and biological materials by drying is of enormous practical importance in industry, clinical medicine, and agriculture. Water accounts for 50% to 95% of the weight of organisms, therefore the attraction of drying cells is clear: an increased shelf life added to a large reduction in weight and volume, which favors transport and storage of dry materials. Furthermore, organisms in anhydrobiosis are more resistant to adverse conditions (Potts 1994). The snag is that the process of dehydration causes a series of harmful structural and metabolic alterations, hindering the maintenance of viability.

Dehydration is known to cause severe damage to organisms at the membrane level as well as to their proteins (Crowe et al 1990). Nevertheless, many organisms, including yeast cells, are able to survive complete dehydration and rapidly resume their metabolic activities when they again come in contact with water (Crowe et al 1984). These organisms tolerate the lack of water because of their ability to synthesize large quantities of the disaccharide trehalose (Coutinho et al 1988). Although no vertebrate has been shown to exhibit dehydration tolerance, the cloning of trehalose genes in human fibroblasts allowed these cells to be maintained in the dry state for several days (Guo et al 1999).

The mechanism by which trehalose mediates desiccation tolerance has not been completely elucidated but seems to involve maintenance of the shape or conformation of biomolecules, as well as prevention against chemical changes. An already widely accepted hypothesis is that trehalose preserves macromolecules because its hydroxyl groups form hydrogen bonds, replacing water molecules that are normally bound to the surface of the macromolecule (Crowe et al 1984, 1998). Another important aspect of how trehalose protects cells is related to its ability to form glasses within cells (Burke 1986). Recently, it has been reported that the disaccharide plays a major role in protecting cellular proteins from oxidative damage (Benaroudj et al 2001). The question remains whether the protective effect of trehalose during dehydration would be due, at least in part, to the reduction of the oxidative stress caused during the process.

Very little is known about the damage caused by dehydration due to an increased oxidative state of yeast cells. Our knowledge goes little beyond the fact that when produced industrially, yeast cells are dried under vacuum to preserve the leavening capacity of the product (Chen et al 1966). Thus, it seems to be implied that dry cells are subjected to oxidative stress. Loss of water increases the ionic concentration (which can lead to the formation of reactive oxygen species [ROS]), and in the dry state, biomolecules become more susceptible to the attack of oxygen.

Oxidative stress arises when cellular defenses or repair mechanisms (or both) against oxidative damage are compromised or overwhelmed by excessive generation of oxidative species (known as ROS). 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 (Berlett and Stadtman 1997). In addition, ROS can modify bases and sugars in deoxyribonucleic acid (DNA), leading to DNA chain breaks (Storz et al 1987), and cause lipid peroxidation in cell membranes (Wolff et al 1986). Besides damage to cellular components, oxidative stress may contribute to the aging process and plays an important role in many diseases. In yeast and bacteria, oxidation also contributes to cell death on exposure to high temperatures (Davidson et al 1996), and this might also be the case in dehydration.

A number of biochemical systems have evolved to protect cells against ROS, including enzymes that remove and repair the products of oxidatively damaged components, as well as the nonenzymatic protective molecules glutathione and thioredoxin, which are scavengers of ROS (Jamieson 1998; Oliver et al 2001). From microorganisms to humans, a primary defense against oxygen toxicity involves one or the other form of the enzyme superoxide dismutase. This enzyme is involved in the conversion of superoxide anion to dioxygen and hydrogen peroxide, which is further degraded by catalase or peroxidases (Jamieson 1998). Null mutants of superoxide dismutase in Saccharomyces cerevisiae are associated with several biochemical defects, indicating that eukaryotic SOD genes may protect numerous metabolic enzymes against oxygen-induced damage (Longo et al 1996).

Because there is so little information on the oxidative stress caused during drying, we decided to investigate the role of the isoforms of Sod, an antioxidant enzyme, in the acquisition of resistance to dehydration. S cerevisiae possesses 2 isoforms, the cytoplasmic Cu/Zn (Sod1p) and the mitochondrial Mn (Sod2p) (Jamieson 1998). In this study, we also analyzed the role of trehalose in protecting cells against oxidation during dehydration.

RESULTS AND DISCUSSION

The effect of isoforms of Sod during dehydration

To investigate whether oxidative stress contributes to the lethal effect of dehydration, we used mutant strains with deletions in either SOD1 or SOD2 genes. As represented in Figure 1A, when mutant strains deleted in SOD1 or SOD2 were subjected to drying, their survival capacity proved to be quite similar, indicating that the presence of only one isoform is sufficient to guarantee about 70% tolerance to dehydration. Considering the importance of Sod1 or Sod2 in protecting cells exposed to an oxidative stress, these results could be explained by the fact that a deficiency in either SOD1 or SOD2 in S cerevisiae cells is overcome by an increase in the remaining Sod activity when compared with the control strain (Costa et al 1993). There seems to be a compensation in regulating the expression of the Sod proteins because the total activity of the 2 mutants is very similar to that of the control strain (Table 1). Furthermore, total activity was not significantly changed in response to dehydration.

Fig 1.

Effect of dehydration on survival. Dehydration was done on a special scale at 37°C. After reaching constant weight, samples were left to equilibrate for 5 minutes. Final water content was 4–9%. Before and after dehydration, cells were diluted with 50 mM phosphate buffer, pH 6.0, and plated on yeast extract peptone dextrose (YPD) plates (containing 2.0% glucose, 2.0% peptone, 1.0% yeast extract, and 2.0% agar) to determine survival. Colonies were counted after incubation at 28°C for 72 hours. Plates were done in triplicate. Tolerance was measured as the percentage of viable cells that survive stress. The results represent the mean ± standard deviation of at least 3 independent experiments. (A) BY4741 (Matα his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) and its isogenic mutants sod1 and sod2, harboring the genes SOD1 and SOD2 interrupted by the gene KanMX4, were from Euroscarf, Frankfurt, Germany. Cells were grown up to the 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 volume/medium of 5:1. Cells were dehydrated in the presence or absence of 10% external trehalose. (B) The control strain Eg103 (Matα leu2; his3Δ1; trp1–289a; ura3–52; GAL⁺; mal) was transformed with a multicopy plasmid, and the isogenic mutant strains Wt1g (pYE-SOD1 LEU2) and Wt2n (pYE-SOD1/SOD2 LEU2) were obtained. These strains were a kind gift from Dr J. M. de Freitas, University of California, USA. The strains were grown on yeast nitrogen base (YNB) medium (2% glucose, 0.67% yeast nitrogen base without amino acids, and 0.01% appropriate auxothrophic requirements) under the conditions described previously. Eg103, Wt1g, and Wt2n strains were always dehydrated in the presence of 10% trehalose

Table 1.

Sod activity and level of intracellular trehalose

To determine whether yeast mutants carrying Sod deficiencies generate increased levels of ROS during dehydration, we used the 2′,7′-dichlorofluorescein diacetate probe. Deacetylation by esterases to dichlorofluorescein occurs within the cell, and thereafter, the probe is no longer able to leave the cell. Once inside the cell, this probe becomes susceptible to attack by ROS, producing a more fluorescent compound, which is measured spectrofluorimetrically (Davidson et al 1996). Fluorescence of cells, which reflects the level of intracellular oxidation, was determined before and after dehydration, and the results were expressed as a relation between the fluorescence of dried and fresh cells. As can be seen in Table 2, all strains showed more than 10-fold increase in oxidation after dehydration, confirming that the loss of water generates an oxidative stress. These results, in fact, may argue in favor of considering oxidation as playing a major role in the lethal effect of dehydration. We also observed that the increase in oxidation produced by dehydration was quite similar in the mutants, when compared with their parental strain, corroborating the interpretation that the absence of only 1 Sod isoform did not affect tolerance. It is interesting to note that although the Sodp2 enzyme is sequestered inside the mitochondria, it is able to fulfill completely the role of protecting cells against oxidative damage.

Table 2.

Enhancement of intracellular oxidation measured as fold increase in fluorescence

To further investigate the role of Sod isoforms in protecting yeast cells against the increase in oxidation occurring under dry conditions, strains overexpressing SOD1 and SOD2 were used. Corroborating what has been shown for overexpression of dismutases in transgenic alfalfa (McKersie et al 1999), the overexpression of SOD1 in yeast cells led to a 5-fold increase in tolerance, whereas in the double mutant Wt2n, which overexpresses both enzymes, survival was 8-fold greater than that shown by the parental strain (Fig 1B).

The above results led us to conclude that the dismutases play a crucial role in protecting yeast cells from oxidative damage caused by dehydration; the presence of one of them, however, is sufficient to endow cells with resistance. This observation supports the fact that dried yeast is industrially produced under vacuum.

The role of trehalose in oxidative stress

The control strain Eg103, as reported in a previous article (Pereira et al 2001), does not accumulate endogenous trehalose under any circumstances; therefore, it was unable to survive dehydration. Trehalose has been shown to endow yeast cells with the capacity to survive dehydration (Eleutherio et al 1993). Endogenously accumulated trehalose is translocated from the cytosol to the outer membrane of the cell by a high-affinity trehalose H⁺-symport system (Stambuk et al 1998). In the absence of the 2 conditions, ie, accumulation and transport, survival is greatly reduced (Eleutherio et al 1993). Addition of exogenous trehalose to mutants that lack one of the above requirements enhances tolerance to dehydration.

On the basis of our previous experience, we decided to dry cells in the presence of 10% trehalose. The question we wished to address was whether this sugar would be able to reduce oxidative damage caused by dehydration and, if so, whether the membrane would be the main target.

The presence of external trehalose endowed the control strain Eg103, as well as its isogenic mutant strains Wt1g and Wt2n, with the capacity to tolerate dehydration, in spite of the absence of endogenous accumulation of this disaccharide (Fig 1B). The control strain BY4741 and its mutants sod1 and sod2 were able to accumulate trehalose (Table 1), which might be the cause for the high tolerance shown by all strains. Furthermore, addition of external trehalose increased dehydration resistance, especially in the control and sod2 strains (Fig 1A).

As seen in Table 2, the ratio of fluorescence between dried and fresh cells, which is indicative of the state of cell oxidation, was significantly reduced when cells were dried in the presence of 10% trehalose. Although in the control strain BY4741 reduction was only 23%, in the mutant strains the effect of trehalose was 56% and 83% for the SOD1 and SOD2 deletions, respectively. These results confirm the importance of this disaccharide in protection against water deficiency.

Because one of the targets of dehydration seems to be the membrane, we measured the level of lipid peroxidation in cells subjected to water stress. There are several ways to detect the process of lipid peroxidation; we used the method of thiobarbituric acid reactive species, which detects malondialdehyde (MDA). As seen in Table 3, dehydration produced a high increase in the levels of lipid peroxidation in control (BY4741) and sod1 and sod2 strains. The levels of lipid peroxidation, before and after dehydration, were similar in the control and sod2 strains. Interestingly enough, the sod1 mutant strain, which accumulated the highest quantity of trehalose, showed low levels of lipid peroxidation. Moreover, by measuring the levels of MDA produced after dehydration in the control strain Eg103, which is unable to accumulate trehalose, we verified that they were almost 50% higher than in the control strain BY4741, which showed normal levels of the endogenous disaccharide. As shown for oxidation during dehydration, external trehalose was able to reduce the levels of peroxidation in all strains. As seen with the fluorescence determinations, the level of lipid peroxidation was reduced by the addition of external trehalose, suggesting that this disaccharide in dry cells might not only stabilize membranes but also play a role in protecting cellular constituents from oxidative damage.

Table 3.

Lipid peroxidation^a

Recently, it has been demonstrated that trehalose is also capable of reducing oxidant-induced modifications of proteins during exposure of yeast cells to H₂O₂ (Benaroudj et al 2001). Besides dehydration, trehalose has been correlated with tolerance to other stresses, such as heat and oxidative stresses (Eleutherio et al 1995; Benaroudj et al 2001); in all of them an increase in the level of intracellular oxidation occurs (Davidson et al 1996). Under these adverse conditions, trehalose accumulation might reduce oxidative damage in cells by scavenging free radicals. Moreover, the ability of trehalose to reduce chemical modifications of lipids under oxidative conditions suggests an additional property for this sugar, which could help explain the mechanism by which trehalose stabilizes biomolecules. In the case of dehydration, a stress that also causes a great increase in oxidation, not only ROS scavenger molecules but also antioxidant enzymes seem to play an important role in protecting cells against damage.