Saccharomyces cerevisiae

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Research article

Sodium nitroprusside induces mild oxidative stress in Saccharomyces cerevisiae Oleh V. Lushchak, Volodymyr I. Lushchak Department of Biochemistry, Vassyl Stefanyk Precarpathian National University, Ivano-Frankivsk, Ukraine

Nitric oxide is known to be a messenger in animals and plants. It may act either as a pro-oxidant or antioxidant. In the present work, the yeast Saccharomyces cerevisiae was treated under aerobic conditions with the nitric oxide donor, sodium nitroprusside (SNP), at concentrations of 1, 5 and 10 mM. The activities of antioxidant enzymes as well as concentrations of protein carbonyls and cellular thiols were measured. Yeast incubation with SNP increased the activities of catalase and superoxide dismutase. Cycloheximide, an inhibitor of translation, blocked SNP-induced catalase activation, but not SOD activation. Incubation with SNP increased the activity of peroxisomal catalase, whereas cytosolic catalase was not affected. SNP treatment inactivated aconitase in a dose-dependent manner. Surprisingly, in cells incubated with 1 mM SNP, the levels of lowmolecular weight thiols were significantly higher, whereas the concentrations of protein carbonyl groups were lower than those in untreated cells. The incubation of yeast cells either with decomposed SNP or with SNP under anaerobic conditions did not result in SOD and catalase activation. It is suggested, that under aerobic conditions, the SNP effects are connected with induction of mild oxidative/nitrosative stress. Keywords: yeast, nitrosative stress, sodium nitroprusside, antioxidant enzymes, aconitase

The important physiological function of nitric oxide (•NO) as a vasodilator and neurotransmitter was discovered over 20 years ago1 and, since that time, has received much attention. To study the effects of •NO in biological systems as well as to examine its possible pathophysiology, many •NO-donors have been used including sodium nitroprusside (SNP). SNP can act as a nitrosating agent at neutral pH and, being relatively non-toxic to mammalian cells, it is used clinically as a vasodilator.2,3

In mammalian and avian cells, •NO is involved in many physiological and pathophysiological processes. As an activator of soluble guanylate cyclase, it regulates the important second messenger cGMP concentrations and has other putative roles in several signal transduction pathways including, for example, regulation of apoptosis.3–6 In vertebrates, nitric oxide is generated by a family of specific enzymes, called • NO-synthases (NOS).1,2 However, •NO is not only a regulator of processes, but is also a powerful weapon against micro-organisms. In this case, the activation of white blood cells stimulates an inducible NOS (iNOS),

Correspondence to: Volodymyr I. Lushchak, Department of Biochemistry, Vassyl Stefanyk Precarpathian National University, 57 Shevchenko Str., Ivano-Frankivsk 76025, Ukraine. Tel/Fax: +38(0342)714683; E-mail: [email protected] Received 10 December 2007, revised manuscript accepted 29 Feb. 2008

Abbreviations: CP, protein carbonyl groups; G6PDH, NADP+-dependent glucose-6-phosphate dehydrogenase; GR, glutathione reductase; IDH, NAD+-dependent isocitrate dehydrogenase; LOOH, lipid peroxides; L-SH, low-molecular weight thiols; MDH, NAD+-dependent malate dehydrogenase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SNP, sodium nitroprusside; SOD, superoxide dismutase

Introduction

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leading to increased •NO generation.7 These cells also simultaneously generate superoxide anion (O2•–), hydrogen peroxide (H2O2) and hydroxyl radical (•OH) collectively named reactive oxygen species (ROS).7 The combination of •NO with O2•– gives a powerful oxidizing agent, peroxynitrite, that also possesses strong antimicrobial activity. Microbes protect themselves against ROS and reactive nitrogen species (RNS) using different mechanisms. For example, the enterobacteria Escherichia coli increases the expression of certain antioxidant enzymes grouped into two regulons, SoxRS and OxyR.9,10 In E. coli, •NO particularly activates the SoxRS pathway.10 Microbes also have special systems, which convert •NO to less toxic molecules. Flavohemoglobin, operating as an •NOdioxygenase under aerobic conditions and as an •NOreductase under anaerobic conditions, was found in yeast and bacteria.11–15 Although there are some data on the effects of RNS on yeast (as reviewed elsewhere16–19 and see references cited therein), in fact nothing is known about the possible response of the antioxidant system to RNS in yeast. Therefore, this study was designed to evaluate the effect of the •NO donor sodium nitroprusside on the activities of antioxidant and associated enzymes, some Krebs’ cycle enzymes as well as markers of oxidative stress – the levels of protein carbonyls, lipid peroxides and low-molecular weight thiols in Saccharomyces cerevisiae.

Materials and methods Materials Phenylmethylsulphonyl fluoride (PMSF), isocitrate, malate, glucose-6-phosphate, oxidized glutathione, 2,4-dinitrophenylhydrazine (DNPH), and 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich Chemie GmbH (Germany); sodium nitroprusside, NADPH, NADP, NAD, N,N,N′,N′tetramethyl ethylenediamine (TEMED) and quercetin were from Reanal (Hungary); and guanidine-HCl was from Fluka (Germany). All other chemicals were of analytical grade. Strains and growth condition The S. cerevisiae strains used in this study were: parental strain YPH250 (MATa trp1-∆1 his3-∆200 lys2-801 leu2∆1 ade2-101 ura3-52) and its derivative strains containing coding region insertions URA3 in YTT7 (ctt1∆::URA3), and TRP1 in YIT2 (cta1∆::TRP1). The strains were kindly provided by Dr Yoshiharu Inoue (Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto, Japan). Cells were grown to stationary phase

(72 h) at 28°C on an orbital shaker (175 rpm) in a liquid YPD medium (1% w/v yeast extract, 2% w/v Bacto-peptone, 2% w/v glucose). For experiments, cells were prepared from overnight cultures grown in YPD medium. The resulting cultures were counted and cells were inoculated into the main culture to a concentration of about 0.3 x 106 cells/ml. To create anaerobic conditions, the samples were placed in closed tubes, pre-incubated for 30 min, and further incubated with SNP for 1 h. Survival measurement Yeast cell survival was performed by counting of colony forming units (CFUs). Cell suspensions treated with sodium nitroprusside for 1 h were diluted in sterile distilled water and the resultant suspensions plated onto YPD-agar in Petri dishes (diameter 10 cm). Preparation of cell extracts and enzyme activity assays Cell extracts were prepared by vortexing with glass beads (0.5 mm) as described earlier20 and kept on ice for immediate use. The activity of SOD was assayed at 406 nm as the inhibition of quercetin oxidation by superoxide anion as described previously.20 One unit of SOD activity was defined as the amount of soluble supernatant protein that inhibited the maximal rate of quercetin oxidation by 50%. Measurement of glutathione reductase (GR), glucose-6phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) activities have been described elsewhere.20 NAD or NADP reduction and NADH or NADPH oxidation by respective enzymes were recorded at 340 nm and an extinction coefficient for reduced forms of these co-enzymes of 6.22 mM–1cm–1 was used. Dismutation of hydrogen peroxide by catalase was assayed as described earlier.20 Hydrogen peroxide consumption was measured at 240 nm using an extinction coefficient for hydrogen peroxide of 39.4 M–1cm–1. Aconitase activity was measured in a reaction medium containing 50 mM potassium phosphate (KPi) buffer pH 7.5, and 1 mM of isocitric acid. The extinction coefficient used in activity calculation was 3.701 M–1cm–1 for cis-aconitate.21 The reactions were started by addition of cell-free extracts. One unit of GR, G6PDH, MDH, IDH, catalase and aconitase activities are defined as the amount of supernatant protein that utilizes or produces 1 µmol of substrate or product per minute. All activities were measured at 25°C and expressed per milligram of soluble protein in the supernatant. Measurement of protein carbonyls The content of carbonyl groups in proteins was measured by determining the amount of 2,4-dinitro-

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phenylhydrazone formed upon the reaction with DNPH.20 Carbonyl content was calculated from the absorbance maximum of 2,4-dinitrophenylhydrazone measured at 370 nm using an extinction coefficient of 22 mM–1cm–1.20 The results are expressed in nanomoles per milligram of protein. Lipid peroxide assay Lipid peroxide (LOOH) content was assayed with xylenol orange.22 Cell supernatants were mixed with 4 volumes of 96% ice-cold (~0°C) ethanol, centrifuged for 5 min at 13,000 g and the resultant supernatants were used for assay. Aliquots of 75 µl of the supernatants were incubated at room temperature for 30 min in a medium containing 0.25 mM FeSO4, 25 mM H2SO4, and 0.1 mM xylenol orange. Blanks contained all components without supernatant. The final sample volume was 1.5 ml. After incubation, absorbance at 580 nm was measured and 5 nM cumene hydroperoxide (CHP) was added. After 60 min incubation, the absorbance at 580 nm was re-read. The content of LOOH is expressed as nanomoles of CHP equivalents per milligram of soluble protein. Low-molecular weight thiol groups (L-SH) Free thiols are widely measured by the Ellman procedure with DTNB.23 For measuring of lowmolecular weight thiol content, 50 µl of cell-free extract were mixed with 25 µl of 30% trichloroacetic acid, centrifuged for 5 min at 8000 g and the whole supernatant was used for the assay. Aliquots of the latter supernatants (50 µl) were incubated with 20 µM DTNB in 50 mM KPi buffer (final volume 1.25 ml) pH 8.0, for 30 min. In control samples, cell-free extract was substituted by the respective volume of trichloroacetic acid solution. Absorption was read at 412 nm and a molar extinction coefficient of 14 x 103 M–1cm–1 was used to calculate the thiol concentration.23 Thiol levels are expressed as micromoles of SH-groups Table 1

per milligram of soluble protein in cell-free supernatant. Protein concentration and statistical analysis Protein concentration was determined by the Coomassie brilliant blue G-250 dye-binding method24 with bovine serum albumin as a standard. Experimental data are expressed as mean ± SEM, and statistical testing used the ANOVA followed Dunnett’s test.

Results Sodium nitroprusside up to a concentration of 10 mM did not affect yeast cell survival (data not shown). Therefore, for experiments, we selected non-toxic SNP concentrations of 1, 5 and 10 mM. Table 1 summarizes the data on the effect of SNP on the indices of oxidative stress – protein carbonyl groups (CP), lipid peroxides (LOOH) and low-molecular weight thiol groups (L-SH) in the YPH250 wild-type strain. None of the concentrations affected these indices except for 1 mM SNP, which increased L-SH levels by 30% compared to the control. Pre-incubation of yeast cells with cycloheximide, an inhibitor of protein synthesis, before SNP treatment did not affect the levels of the oxidative stress markers except for 1 mM SNP, which prevented the increase of L-SH concentration (Table 1). Figure 1 shows the effects of YPH250 yeast cell incubation with 1, 5 and 10 mM SNP for 1 h on the activities of catalase (Fig. 1A), SOD (Fig. 1B) and aconitase (Fig. 1C) in control yeast cells and those pretreated with cycloheximide. All SNP concentrations increased catalase and SOD activities, while aconitase was inactivated. Catalase and SOD activities were increased by 30–35% and 25–35%, respectively. Aconitase activity was reduced in a concentrationdependent manner with only 65% of the control value present after treatment with 10 mM SNP.

The levels of protein carbonyl groups (CP), lipid peroxides (LOOH) and low-molecular weight thiols (L-SH) in S. cerevisiae YPH250 cells treated with SNP at three concentrations for 1 h and treated with SNP after pretreatment with cycloheximide (+CHI) for 30 min Conditions

Control SNP (1 mM) SNP (5 mM) SNP (10 mM) Control + CHI (250 µg/ml) SNP (1 mM) + CHI SNP (5 mM) + CHI SNP (10 mM) + CHI

CP level (nmol/mg protein)

LOOH ([CHP] nmol/mg protein)

L-SH (nmol/mg protein)

4.77 ± 0.28 4.61 ± 0.17 4.58 ± 0.21 4.66 ± 0.24

40.8 ± 7.3 37.9 ± 3.1 33.1 ± 4.7 34.3 ± 5.4

231 ± 21 301 ± 10* 242 ± 23 244 ± 24

ND ND ND ND

26.7 ± 5.6 32.7 ± 2.6 28.0 ± 0.9 27.5 ± 4.1

249 ± 20 234 ± 19 236 ± 24 239 ± 25

*Significantly different from the respective of control (untreated) cells with P < 0.05 (n = 6). ND, not determined.

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Figure 1 Activities of catalase (A), superoxide dismutase (B) and aconitase (C) in wild-type (YPH250) cells after 1-h treatment with 1, 5 and 10 mM SNP (SNP); or pretreated for 30 min with 250 µg/ml cycloheximide SNP exposure (SNP+CHI). Data are shown as mean ± SEM (n = 6). *Significantly different from the control (untreated) cells with P < 0.05

Cycloheximide, an inhibitor of protein synthesis, prevented the activation of catalase (Fig. 1A) but, surprisingly, did not block SOD activation (Fig. 1B) and, in fact, it did not affect aconitase inactivation (Fig. 1C). Extending the time of incubation with 1 mM and 5 mM SNP to 2 h further increased the catalase activation by 1.6- and 1.8-fold, respectively, while at 10 mM the increase in activity was not significant (data not shown).

However, cell treatment with SNP for 2 h did not show any increase in SOD activity (not shown). It should be added that yeast exposure to 5 mM and 10 mM SNP for 2 h increased the concentrations of protein carbonyls for 42% and 50%, respectively (data not shown). In order to check if the SNP effects were related to its decomposition, we diluted SNP with water and kept the solution in the dark at room temperature for 24 h and 48 h. Then, we treated yeast cells with the decomposed SNP solutions. Incubation with both decomposed solutions did not change SOD, catalase and aconitase activities or protein carbonyl concentrations (data not shown). This demonstrates that the SNP effects are connected with decomposition products generated during yeast cell treatment with SNP and that the stable products of its decomposition are not responsible for the observed effects. As mentioned above, •NO may interact with oxygen metabolites. For example, its interaction with O2•– results in the formation of the powerful oxidant peroxynitrite.8 The latter causes oxidation of many cellular components either directly or after its decomposition, for example, to hydroxyl radical. We incubated yeast cells with SNP under anaerobic conditions in order to exclude the involvement of products of interaction between products of SNP decomposition and ROS. Neither SOD nor catalase activities were increased following yeast treatment under anaerobic conditions (Fig. 2A,2B); also, protein carbonyl concentrations were not changed. However, the activity of aconitase was reduced in this case and effects of SNP treatment were even more pronounced than under aerobic conditions (Fig. 2C). For example, 1, 5 and 10 mM caused 39%, 34% and 48% reduction, respectively, which is substantially lower than under aerobic conditions (Fig. 1C). These results clearly show that ROS are involved in the effects found following SNP treatment under anaerobic conditions. Next we tested the activities of GR, G6PDH, IDH and MDH following yeast treatment with SNP. We selected the first two enzymes because they are responsible for the maintenance of glutathione (GSH) levels (an important compound in cellular defense against ROS25,26) and they are possible targets for inactivation by reactive nitrogen and/or oxygen species.27,28 In addition, these two enzymes are upregulated by oxidative stress in yeast.29–31 The activities of GR and G6PDH were 34.5 ± 2.5 mU/mg protein and 224 ± 13 mU/mg protein, respectively. Unexpectedly, neither GR nor G6PDH activities were changed under any of the conditions used herein (data not shown). The activities of IDH and MDH were 117 ± 8 mU/mg protein and 34.5 ± 3.1 mU/mg protein, Redox Report

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respectively. Being a mitochondrial enzyme, IDH plays an important role in energy metabolism and is also a subject to oxidative inactivation.32,33 Again, neither IDH nor MDH activities were affected by SNP at any concentration used in this study (data not shown). The incubation of yeast cells with cycloheximide prior to treatment with SNP did not affect the activities of GR, G6PDH, IDH, and MDH (data not shown).

Figure 2 Activity of catalase (A), superoxide dismutase (B) and aconitase (C) in wild-type (YPH250) yeast cells treated with SNP in concentrations of 1, 5 and 10 mM for 1 h under anaerobic conditions. *Significantly different from the control (untreated) cells with P < 0.05

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In order to check which specific catalase isoenzyme was affected by SNP treatment, we used two isogenic strains to YPH250 – YTT7 (∆CTT1) possessing the peroxisomal catalase A, and YIT2 (∆CTA1) which produces the cytosolic catalase T. Figure 3A shows that SNP treatment did not change the activity of catalase T and SOD activity was also not affected (Fig. 3B). However, aconitase activity was reduced in YIT2 cells treated with 5 mM and 10 mM SNP (Fig. 3C). The SNP

Figure 3 Activities of catalase (A), superoxide dismutase (B) and aconitase (C) in cells possessing cytosolic catalase T (YIT2 strain) after 1-h treatment with SNP at concentrations of 1, 5 and 10 mM. Data are shown as mean ± SEM (n = 6). *Significantly different from the control (untreated) cells with P < 0.05

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Table 2 The levels of protein carbonyl groups (CP) and lowmolecular weight thiols (L-SH) in S. cerevisiae YTT7 and YIT2 cells treated with SNP at various concentrations for 1 h Strain and conditions

CP level (nmol/mg of protein)

L-SH (nmol/mg of protein)

Control SNP (1 mM) SNP (5 mM) SNP (10 mM)

6.14 ± 0.48 5.92 ± 0.68 6.55 ± 0.37 6.57 ± 0.74

166 ± 25 157 ± 26 147 ± 20 148 ± 15

Control SNP (1 mM) SNP (5 mM) SNP (10 mM)

6.84 ± 0.32 5.98 ± 0.18* 4.94 ± 0.15* 6.50 ± 0.36

158 ± 12 153 ± 14 178 ± 23 174 ± 10

YIT2

YTT7

*Significantly different from the respective control with P < 0.05 (n = 6).

treatment did not affect the levels of CP and L-SH in the ∆CTA1 strain (Table 2). Figure 4 shows that in the YTT7 strain, defective in cytosolic catalase T, SNP treatment increased the activity of peroxisomal catalase A. Surprisingly, in this strain, SOD activity was not increased by incubation with SNP (Fig. 4B), while aconitase activity again was reduced by SNP treatment (Fig. 4C). Inhibition of translation by pretreatment of yeast cells with cycloheximide did not affect SOD and aconitase behavior, while it cancelled catalase T activation. In YTT7 cells, 1 mM and 5 mM SNP concentrations reduced the CP level, but none of the concentrations used affected L-SH levels (Table 2).

Discussion In this study, we used a biochemical approach to characterize the effects of the •NO donor SNP on S. cerevisiae. Information on how yeast cells survive nitrosative stress is rather scarce. Jakubowski and coworkers16 have described the possible protective role of certain antioxidant enzymes following budding yeast treatment with peroxynitrite and the nitric oxide donor S-nitrosoglutathione. In their study, the SODdeficient strain showed higher sensitivity to nitrosative stress than the wild-type strain as well as strains lacking catalases and with decreased levels of glutathione. In several other studies, the effects of Snitrosoglutathione were examined in S. cerevisiae.34,35 S-nitrosoglutathione did not affect yeast growth up to a concentration of 7 mM. However, when Snitrosoglutathione effects were examined in the fission yeast Shizosaccharomyces pombe, it was found that even relatively low concentrations substantially reduced growth.27 Other RNS donors such as SNP,

Figure 4 Activities of catalase (A), superoxide dismutase (B) and aconitase (C) in cells possessing peroxisomal catalase A (YTT7) after 1-h treatment with SNP at concentrations of 1, 5 and 10 mM (SNP); or pretreated for 30 min with 250 µg/ml cycloheximide before SNP exposure (SNP+CHI). Data are shown as mean ± SEM (n = 6). *Significantly different from the control (untreated) cells with P < 0.05

sodium nitrite (NaNO2), DETA NONOate, and peroxynitrite also affected Shizo. pombe growth. In our experiments, SNP up to a concentration of 10 mM did not change S. cerevisiae YPH250 growth, which may indicate that it possesses powerful protective mechanisms.

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In bacteria, it is known that RNS activate certain antioxidant enzymes at the transcriptional level.9,10 Sarver and DeRisi36 exposed S. cerevisiae cells to the • NO-generating compound dipropylenetriamine NONOate and found the induction of both a general stress response as well as a specific response, including the yeast flavohemoglobin (YHB1), plasma membrane sulfite pump (SSU1), and three additional uncharacterized open reading frames. Deletion of zinc-finger transcription factor Fzf1p eliminated the specific response to nitrosative stress at the transcriptional level, whereas overexpression of Fzf1p recapitulated this response in the absence of exogenously supplied •NO. The authors concluded that •NO and/or its derivatives activate Fzf1p leading to transcriptional induction of discrete genes that function to protect the cell against • NO-mediated stress.36 Horan and colleagues37 investigated the consequence of RNS exposure on whole-genome transcriptional response in S. cerevisiae. No involvement of ROS-scavenging enzymes was found. However, genes of the glutathione system were up-regulated among 177 genes showed a persistent response to nitrosative stress. They also found that many of the up-regulated genes are known to be under the control of the transcription factor Hap1p. The regulatory proteins Msn2/4p and Yap1p, key regulators of the response to general stress and oxidative stress, respectively, played a role, in mediating nitrosative stress.37 With this information, we checked the activities of the main antioxidant enzymes, superoxide dismutase and catalase, in S. cerevisiae treated with SNP. Incubation with SNP increased the activities of both enzymes (Fig. 1). Because cycloheximide blocked the activation of catalase, one may suggest that the effect is connected with catalase synthesis de novo. On the other hand, SOD activation was not blocked by cycloheximide and the activity increase might be associated with activation of pre-existing molecules. Similar activation of pre-existing catalase and SOD proteins was discussed by ourselves previously.38 Catalase and SOD are critically important for survival under oxidative stress conditions and are involved in protection of cellular components from oxidative damage.8–10,20,29,39–41 Up-regulation of SODs and catalases in yeast occurs via oxidative modification of the transcription regulators Yap1p42,43 and Skn7p.43,44 Each of these pathways includes oxidative steps. Therefore, one could suggest that yeast treatment with SNP induced oxidative stress. However, it looks as if this was not the case. In fact, none of the well-known markers of oxidative stress, namely protein carbonyls, lipid peroxides and low-molecular weight thiols

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showed the expected changes. Moreover, the increased L-SH levels demonstrated a possible increase in the reducing power of the cell. Further, we checked the activities of GR, G6PDH, IDH and MDH, which are known to be inactivated by ROS. None of the activities were changed, which is also consistent with an absence of oxidative stress in SNP-treated cells. Although GR and G6PDH are known to be up-regulated by oxidative stress in S. cerevisiae,29–31,44 we did not find any activation. However, the inactivation of aconitase, which is mainly connected with its oxidation, may support the idea of the development of oxidative/nitrosative stress induced by SNP. In summary, we have observed both the evidence of possible induction of oxidative stress response and its absence under SNP treatment. To our knowledge this is the first study demonstrating that yeast cell incubation with the •NO donor SNP results in: (i) the inactivation of aconitase; and (ii) activation of antioxidant enzymes at the transcriptional and post-transcriptional levels. The interaction between RNS and ROS may be involved in modulation of yeast response to incubation with SNP in vivo. The treatment of yeast cells with decomposed SNP solutions, which did not result in SOD and catalase activity increase, demonstrates that SNP, but not stable products of its decomposition, induced the changes observed. It is well known that the character of SNP decomposition depends very much on the environment; the use of chemically decomposed reagent gives some clues in understanding its effects on biological systems. In addition, yeast treatment with SNP under anaerobic conditions cancelled the observed changes excepting for aconitase inactivation. Therefore, one may suggest that the interaction between oxygen and/or reactive oxygen species with reactive nitrogen species might be involved in the described SNP effects. The activity of aconitase is a good marker of oxidative stress and, therefore, we also used it in our work. We have found that the activity of aconitase was decreased in all experimental approaches: at different SNP concentrations, with inhibition of translation and in all three strains examined, YPH250 (wild-type), YIT2 (∆CTA1) and YTT7 (∆CCT1). In its catalytic center, this enzyme contains a [4Fe–4S] cluster, which is sensitive to oxidation.1,4,9–13 When oxidized, iron ions are released the enzyme loses its activity. In a model system, Castro and colleagues45 found that aconitase was inactivated by peroxynitrite •OONO–, but not • NO. Tortora and colleagues46 demonstrated that the treatment of recombinant pig heart aconitase with • NO, S-nitrosoglutathione, and peroxynitrite

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inactivated the enzyme. They found several types of modifications and only a few affected the activity; in addition, aconitase [4Fe–4S] clusters were mainly affected by •NO-donors.46 Therefore, it can be expected that, in our case, SNP treatment could remove iron from the catalytic center of aconitase inactivating the enzyme. This is supported by the report of Joannou and colleagues,2 who found that, in the bacterium Clostridium sporogenes, SNP treatment reduced signals from [4Fe–4S] clusters. To be removed from the [4Fe–4S] cluster, Fe2+ should be oxidized to Fe3+ and, therefore, it is widely accepted that inactivation of proteins containing these clusters is a good marker of oxidative insult. However, our results to some extent do not correspond to those of Radi and colleagues.45 In our hands, in vivo, the system behaved differently under aerobic and anaerobic conditions. If SNP decomposition gives •NO, it can be responsible for aconitase inactivation under anaerobic conditions (Fig. 2C). Under aerobic conditions SNP treatment also reduced aconitase activities, but to a smaller extend that under anaerobic conditions. This demonstrates that the presence of oxygen and/or its metabolites can substantially modify cellular net response to treatment by •NO-donors. It was interesting to determine which of the yeast catalases was specifically activated by SNP. For this reason, we used strains isogenic to YPH250, defective in either cytosolic (YTT7) or peroxisomal (YIT2) catalase. The results led us to conclude that SNP activated the peroxisomal catalase. Its counterpart, the cytosolic form, was not activated by SNP. That was unexpected, because usually either both or the cytosolic catalase T are/is activated under oxidative stress.29,44 It is also worth mentioning that, unlike wildtype yeast, in both catalase-defective strains SNP did not activate SOD. Similar results were obtained earlier when we studied the effects of hydrogen peroxide on SOD and catalase activities in the same yeast strains used in this work.47 Unfortunately, we cannot at this time explain this phenomenon and can only suggest that catalases are in some way involved in the regulation of SOD expression. It can be speculated that catalases change the ratio between different ROS forms in a manner appropriate for expression regulation. The interpretation of the effect of SNP on S. cerevisiae is complicated because of the presence of defense/response mechanisms. Yeast possess flavohemoglobin which metabolizes •NO. This protein functions as a •NO oxygenase under aerobic conditions and as a •NO reductase anaerobically.14

Yeast cells carrying a deletion in YHB1, the structural gene for YHb, become more sensitive to oxidative and other stresses.12 Antimycin A or menadione did not affect the expression of YHB1, while H2O2, diamide, dithiothreitol, and Cu2+ increased the expression.48 In the latter work, it was concluded, that Yhb1p appears to protect cells against the damage caused by Cu2+ and dithiothreitol, while sensitizing them to H2O2. It also was found that oxidative stress increased the expression of YHB1, but it was not clear if the other stresses were effective.48

Conclusions Our results suggest that S. cerevisiae possesses a system of protection against SNP because, even at a concentration of 10 mM SNP, toxicity was not observed. It did not cause intensive oxidative stress as shown by unchanged levels of oxidized proteins, lipids and low-molecular weight thiols. The activity of enzymes typically sensitive to oxidation, due to reactive thiol groups present in their catalytic centers, also confirms that no induction of oxidative stress occurred. However, increased activities of the antioxidant enzymes SOD and catalase may indicate the development of oxidative stress. This proposal is strengthened by the observation of aconitase inactivation. Therefore, one may suggest, that SNP treatment of S. cerevisiae cells results in the type of stress that allows an up-regulation of selected antioxidant enzymes, but without a direct chemical effect on oxidizable cell components. However, the mechanisms involved in up-regulation of the antioxidant enzymes remain to be studied. Yap1 might be one of the possible candidates for the observed upregulation. It is a functional homolog of Pap1 which is reported to be involved in the Shizo. pombe response to nitrosative stress.27 Our preliminary data with a S. cerevisiae strain with disrupted YAP1 gene demonstrate that Yap1p might be associated with upregulation of catalase activity under SNP-induced stress.

Acknowledgements The authors would like to thank Drs H. Semchyshyn, J. Fukuto and Y. Inoue for fruitful comments and suggestions on the manuscript, and O. Kubrak for excellent technical assistance.

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