Báo cáo khoa học: H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces cerevisiae An experimental and theoretical approach

doi:10.1046/j.1432-1033.2003.03529.x

Eur. J. Biochem. 270, 1578–1589 (2003) (cid:2) FEBS 2003

H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomycescerevisiae An experimental and theoretical approach

Hugo Osorio1,2, Elisabete Carvalho1, Mercedes del Valle1, Marı´a A. Gu¨ nther Sillero1, Pedro Moradas-Ferreira2 and Antonio Sillero1 1Departamento de Bioquı´mica, Instituto de Investigaciones Biome´dicas Alberto Sols UAM/CSIC, Facultad de Medicina, Madrid, Spain; 2Instituto de Biologı´a Molecular e Celular, Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Portugal

When Saccharomyces cerevisiae cells, grown in galactose, glucose or mannose, were treated with 1.5 mM hydrogen peroxide (H2O2) for 30 min, an important decrease in the ATP, and a less extensive decrease in the GTP, CTP, UTP and ADP-ribose levels was estimated. Concomitantly a net increase in the inosine levels was observed. Treatment with 83 mM menadione promoted the appearance of a compound similar to adenosine but no appreciable changes in the nucleotide content of yeast cells, grown either in glucose or galactose.

laboratory pointed to a potential inhibition of glycolysis as the main reason for that effect. This theoretical consideration was reinforced both by the lack of an appreciable effect of 1.5 mM (or even higher concentrations) H2O2 on yeast grown in the presence of ethanol or glycerol, and by the observed inhibition of the synthesis of ethanol promoted by H2O2. Normal values for the adenylic charge, ATP and inosine levels were reached at 5, 30 and 120 min, respectively, after removal of H2O2 from the culture medium. The strong decrease in the ATP level upon H2O2 treatment is an import- ant factor to be considered for understanding the response of yeast, and probably other cell types, to oxidative stress.

Keywords: Saccharomyces cerevisiae; hydrogen peroxide; menadione; glycolysis; oxidative stress.

Changes in the specific activities of the enzymes involved in the pathway from ATP to inosine, in yeast extracts from (un)treated cells, could not explain the effect of H2O2 on the levels of ATP and inosine. Application of a mathematical model of differential equations previously developed in this

Oxygen is both the support to maintain the aerobic metabolism of organisms and a source of damaging reactive free radicals [6]. Molecular oxygen (O2) contains two unpaired electrons, both with the same spin, and its reactivity as a free radical is rather limited. Upon accepting one electron, molecular oxygen generates a very reactive –), with one unpaired electron. superoxide radical (ÆO2 Further additions of electrons and combination with protons generate a variety of oxygen derivatives of biolo- gical interest [6,7].

Our laboratory has been engaged for several years in the study of the metabolism and function of dinucleoside polyphosphates [1,2] and purine nucleotides [3]. Initially, the aim of the work presented here was to investigate potential changes in the level of diadenosine tetraphosphate (Ap4A) in Saccharomyces cerevisiae subjected to oxidative stress, based on previous work by others describing the increase of Ap4A in yeast and in other microorganisms, when subjected to heat shock or oxidative stress [4,5]. However, whereas we did not observe significant changes in the level of Ap4A, important variations in the concentration of other nucleo- tides were noticed; as shown below, this finding prompted us to investigate in more detail the influence of oxidative stress in yeast nucleotide metabolism.

The reduced NADH and FADH2 are reoxidized by molecular oxygen with formation of H2O [8,9]. Although this process is very efficient, the electron flow throughout the respiratory chain may produce reactive oxygen species (ROS) as byproducts, such as superoxide anion radical, hydroxyl radical and hydrogen peroxide. Some of these reactive species can also be formed during the oxidation of arachidonic acid, and in different reactions catalyzed by nitric oxide synthase, xanthine oxidase, glucose oxidase, monoamine oxidase, and P450 enzymes [6,10].

radical

can be

Although H2O2 itself is not a free radical, it can be decomposed through the Fenton reaction to generate (Fe2+ + H2O2 (cid:1)! Fe3+ + ÆOH + hydroxyl OH–). Moreover, H2O2, superoxide and hydroxyl radical (ÆOH) interconverted via the Haber–Weiss reaction.

Correspondence to A. Sillero, Departamento de Bioquı´ mica, Facultad de Medicicina, Universidad Auto´ noma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain. Tel.: + 34 91 3975413; Fax: + 34 91 5854401; E-mail: antonio.sillero@uam.es Abbreviations: ROS, reactive oxygen species; Ino, inosine. Enzymes: adenosine deaminase (EC 3.5.4.4); adenosine kinase (EC 2.7.1.20); AMP deaminase (EC 3.5.4.6); AMP 5¢ nucleotidase (EC 3.1.3.5); IMP 5¢ nucleotidase (EC 3.1.3.5); nucleoside phosphorylase (EC 2.4.2.1); adenylate kinase (EC 2.7.4.3). (Received 19 December 2002, revised 13 February 2003, accepted 20 February 2003)

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Effect of H2O2 and menadione on S. cerevisiae (Eur. J. Biochem. 270) 1579

Fe3þ þ ÆO(cid:1)

2 $ Fe2þ þ O2

ÆO(cid:1)

performed as indicated below. When required, the number of viable cells after H2O2 or menadione exposure was determined by spreading appropriate dilutions of cells onto YEPD plates containing 1.5% agar, and counting the colonies formed after incubation at 30 (cid:4)C for 2–3 days.

Extraction of nucleosides and nucleotides

Fe2þ þ H2O2 ! Fe3þ þ ÆOH þ OH(cid:1) 2 þ H2O2 !ÆOH þ OH(cid:1) þ O2 Menadione is a cytotoxic quinone acting through a cycling reaction, implying its one-electron reduction to a semiquinone radical and subsequent reaction with molecular oxygen with the formation of the quinone and superoxide [11].

The oxygen reactive species may oxidatively damage nucleic acids (producing double-strand breaks, apurinic and apyrimidic bases), lipids (formation of lipid peroxides), and proteins (oxidation of the amino acids side chains) [7,12–15]. The yeast S. cerevisiae has been used as model system to explore the mechanisms underlying the oxidative stress response, such as exposure to H2O2 or menadione [16–19]. In this study we have assessed the effect of H2O2 and menadione on the metabolism of purine nucleotides. Whereas menadione did not alter significantly the levels of these nucleotides, H2O2 promoted a drastic decrease in the level of adenine nucleotides and a concomitant increase in the level of inosine. A plausible explanation of the effect of H2O2 as inhibitor of glycolysis is presented.

Materials and methods

Materials

Hydrogen peroxide (30%) solution, menadione sodium bisulfite, auxiliary enzymes, cofactors and substrates were purchased from Sigma or Roche Molecular Biochemicals. Yeast nitrogen base was from Difco (catalogue no. 233520). Hypersil ODS column (4.6 · 100 mm) was from Hewlett- Packard.

The sampling method was essentially as described in [21]. 100-mL portions of the cell culture grown to a density of around 1.5 · 107 cellsÆmL)1 (1.2 mg wet weightÆmL)1), were rapidly collected by filtration on a nitrocellulose membrane filter (Millipore, pore size 1.2 lm, 47 mm diameter) and washed once with 5 mL of a mixture of methanol/water (1 : 1, v/v) at )40 (cid:4)C. The yeast pellicle was immediately gathered with the help of a spatula and immersed in liquid nitrogen. The samples were kept at )70 (cid:4)C until extraction. To prepare the acidic extracts 1.2 M HClO4 was added to the frozen yeast (0.4 mL per 100 mg wet weight) and the suspension was frozen and thawed three times to extract metabolites [22]. Cell debris was removed by centrifugation and the pellet re-extracted once with 0.2 M HClO4 (0.1 mL per 100 mg wet weight). The supernatants were combined, neutralized with KOH/K2CO3 and ana- lyzed by HPLC as described previously [23]. The amount of the nucleosides/nucleotides was determined from the areas of the corresponding peaks, using the absorption coeffi- cients obtained from standard curves; their intracellular concentration was calculated assuming that 1 g of yeast (wet weight) contains 0.6 mL of intracellular volume [24]. NADH did not interfere with NAD+ measurements, because it was destroyed by the acid extraction procedure. Inosine (Ino) was identified by its retention time and its nature confirmed by treating the sample, before analysis by HPLC, with commercial E. coli purine nucleoside phos- phorylase. In our assay conditions the detection limit was 5 nmoles per gram of yeast cell dry weight.

Strain and growth conditions

Energy charge

Energy charge is defined in terms of actual concentrations as ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]) [25].

Preparation of cell extracts

The strain used in this work was the wild-type W303 1A from S. cerevisiae, genotype: MATa leu2-3, 112 his3-11, 15 trp1-1, can1-100, ade2-1, ura3-1 [20]. Cells were grown aerobically at 30 (cid:4)C in a gyratory shaker (at 180 r.p.m), in a minimal medium containing (per litre): yeast nitrogen base without amino acids and ammonium sulfate 1.7 g; ammo- nium sulfate 5 g; galactose, glucose or mannose 20 g; leucine 0.08 g; tryptophan, adenine, histidine and uracil 0.04 g each. For growth on nonfermentable carbon sources the minimal medium contained 3% (v/v) glycerol or 2% (v/v) ethanol. Cell growth was followed by optical absorb- ance readings at 600 nm (D600 ¼ 1 corresponds to a concentration of 1.5 · 107 cellsÆmL)1).

To determine the wet weight, portions of cell cultures grown to different cell densities were rapidly filtered and the filter plus the cells weighed out. One gram of wet yeast has been found to contain an average of 24 mg of protein.

H2O2 and menadione treatment: control of cell viability

Exponentially growing yeast cells, with a density of about 1.5 · 107 cellsÆmL)1, were treated with H2O2 or menadione as indicated in each experiment. The extraction of nucleo- tides and the determination of enzyme activities were

All the procedures were carried out at 0–4 (cid:4)C. Yeast (200 mL) grown to a cell density of around 1.5 · 107 cellsÆ mL)1 was harvested by centrifugation, and washed twice with 10 mL of extraction buffer (20 mM sodium phosphate pH 7.0, 0.1 M KCl; 0.1 mM dithiothreitol). The cells (1 g wet weight) were disrupted in the presence of 2 mL of buffer plus 4 g of glass beads (500 lm diameter) by vortexing at top speed on a tabletop mixer for 6 periods of 1 min separated by 1-min periods of cooling on ice. The homo- genate was centrifuged for 5 min at 750 g and the super- natant centrifuged further at 550 000 g for 30 min. The final supernatant was dialyzed for 2 h against 200 volumes of 20 mM sodium phosphate buffer, pH 7.0; 50 mM KCl; 0.1 mM dithiothreitol, followed by a second dialysis of 12 h against the same buffer. All enzyme determinations were performed with freshly prepared supernatants. Protein content was determined by the method of Bradford [26].

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Enzymatic assays

Except when indicated, the reaction mixtures (0.15 mL) contained: 50 mM imidazole/HCl buffer pH 7.0, 0.1 M KCl; 0.1 mM dithiothreitol and 4 mM MgCl2; the appropriate nucleoside and/or nucleotide, and inorganic phosphate or ribose-1-phosphate, when required. The reaction, initiated by the addition of yeast cytosol (around 0.07 mg protein) was incubated at 30 (cid:4)C and analyzed by HPLC as follows. Aliquots of 20 lL were withdrawn from the reaction mixture at different times of incubation, transferred into 180 lL of water and kept in a boiling water bath for 1.5 min. After chilling, the mixture was filtered and 50 lL injected into a Hypersil ODS column. Elution was per- formed as described previously [3]. The nature and the concentration of the products formed in the course of the reaction were established by comparison with standards. Quantification was made from data obtained under linear conditions of substrate consumption. One unit is defined as 1 lmol of substrate transformed per min. The following enzyme activities were estimated in the presence of the indicated substrates or cofactors: adenosine deaminase (EC 3.5.4.4) (0.5 mM adenosine); adenosine kinase (EC 2.7.1.20) (0.2 mM adenosine and 1 mM ATP); AMP deaminase (EC 3.5.4.6) (5 mM AMP and 1 mM ATP); AMP 5¢ nucleotidase (EC 3.1.3.5) (1 mM AMP); IMP 5¢ nucleotidase (EC 3.1.3.5) (1 mM IMP, 4 mM MgCl2 and 2 mM ATP); nucleoside phosphorylase (EC 2.4.2.1) (0.5 mM inosine and 2 mM inorganic phosphate) or (1 mM hypoxanthine and 2 mM ribose-1-phosphate). Adenylate kinase (EC 2.7.4.3) was determined spectrophotometrically in the presence of 2 mM ADP.

Glucose and ethanol were determined in the medium, after the yeast cells had been removed by centrifugation, by the hexokinase/glucose-6-P dehydrogenase [27] and the alcohol dehydrogenase/acetaldehyde dehydrogenase coupled assays [28], respectively.

Results

Effect of H2O2 on the nucleotide content of yeast cells, grown in the presence of galactose, glucose or mannose

Exponentially growing yeast cells, with galactose as carbon source, were challenged with 1 mM H2O2 for 0, 7, 11, 20 and 30 min incubation (Fig. 1A), and the nucleotide content analyzed by HPLC as described in Material and methods. After 11 min incubation in the presence of 1 mM H2O2, the total amount of adenine nucleotides (AMP, ADP and ATP) decreased by around 50%, with concom- itant appearance of inosine (Fig. 1A). Incubation times longer than 30 min in the presence of H2O2 did not greatly change the ratio SATP + ADP + AMP/Ino. Similar changes in ATP and inosine concentrations were observed when yeast cultures were treated for 30 min with different concentrations (0, 0.3, 0.6, 1.0 and 1.5 mM) of H2O2 (Fig. 1B).

to AMP, ADP, ATP and Ino, the following compounds were also quantified: CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UDP, UTP, UDP-sugars, NAD+, NADP+, ADP-ribose and hypoxanthine. IMP was not detected. Representative HPLC nucleotide profiles obtained from yeast cultures grown in galactose and in the absence (A) or presence (B) of 1.5 mM H2O2 are shown in Fig. 2. Treatment with 1.5 mM H2O2 for 1 h gave rise to a 10-fold increase in the amount of inosine, a 17-fold decrease in the ATP level, and a five- to sixfold decrease in the levels of ADP, CTP, GTP, UTP and ADP-ribose (Table 1). Changes in the concentration of the other nucleotides analyzed were less relevant. The nucleoside mono-, di- and triphosphate pools of adenosine, cytidine, guanosine and uridine in the untreated vs. the H2O2- treated cells decreased around seven-, two-, two- and 1.5-fold, respectively. Although the interpretation of these in the results is currently not possible,

it seems that,

The results presented in Fig. 1 were confirmed by growing several batches of yeast cells in galactose as carbon source, in the absence (6 batches) or presence (7 batches) of 1.5 mM H2O2 for 1 h (Table 1). In addition

Fig. 1. Effect of H2O2 on (AMP + ADP + ATP) and inosine pools of S. cerevisiae grown in the presence of galactose as carbon source. Yeast cells were challenged with 1 mM H2O2 for the indicated times of incubation (A) or with different concentrations of H2O2 for 30 min (B). AMP, ADP, ATP and inosine contents were determined as des- cribed in Materials and methods.

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Table 1. Nucleoside and nucleotide content of S. cerevisiae (strain W303) grown in galactose and treated for 1 h in either 1.5 mM H2O2 or 83 mM menadione. Exponentially growing yeast cells were challenged with either H2O2 or menadione. Analysis of the nucleotide content was performed as described in Materials and methods. The data represent mean values ± SE of 6, 7 and 3 experiments for the control, H2O2-treated and menadione- treated cells, respectively. The concentrations of the indicated compounds are expressed in mM.

Menadione MD/Control Parameters Control H2O2 H2O2/Control

a The concentration (mM) of this compound has been calculated assuming the extinction coefficient of adenosine.

Starting wet weight (mg) Total protein (mg) Adenylic charge AMP ADP ATP S (ATP + ADP + AMP) CMP CDP CTP S (CTP + CDP + CMP) GMP GDP GTP S (GTP + GDP + GMP) UMP UDP UTP S (UTP + UDP + UMP) ADP-Rib NAD+ NADP+ UDP-sugars Hypoxanthine Inosine Unknown a 147 ± 70 3.7 ± 1.5 0.78 ± 0.03 0.21 ± 0.04 0.52 ± 0.18 1.51 ± 0.32 2.24 ± 0.49 0.18 ± 0.03 0.07 ± 0.01 0.21 ± 0.03 0.46 ± 0.08 0.06 ± 0.01 0.18 ± 0.03 0.30 ± 0.02 0.54 ± 0.06 0.15 ± 0.06 0.22 ± 0.04 0.33 ± 0.08 0.70 ± 0.16 0.24 ± 0.11 0.97 ± 0.39 0.06 ± 0.01 1.30 ± 0.16 0.11 ± 0.05 0.20 ± 0.04 – 185 ± 34 3.0 ± 1.2 0.41 ± 0.15 0.14 ± 0.09 0.10 ± 0.02 0.09 ± 0.05 0.33 ± 0.12 0.14 ± 0.09 0.04 ± 0.02 0.04 ± 0.02 0.22 ± 0.03 0.08 ± 0.02 0.11 ± 0.07 0.07 ± 0.03 0.26 ± 0.08 0.31 ± 0.08 0.14 ± 0.08 0.05 ± 0.01 0.50 ± 0.09 0.04 ± 0.03 0.95 ± 0.34 0.05 ± 0.02 1.18 ± 0.16 0.16 ± 0.06 2.07 ± 0.69 – 173 ± 9.0 2.5 ± 0.5 0.86 ± 0.01 0.08 ± 0.01 0.26 ± 0.01 1.15 ± 0.08 1.49 ± 0.08 0.18 ± 0.08 0.03 ± 0.01 0.13 ± 0.01 0.34 ± 0.07 0.03 ± 0.00 0.10 ± 0.03 0.19 ± 0.02 0.32 ± 0.05 0.27 ± 0.03 0.12 ± 0.04 0.07 ± 0.02 0.46 ± 0.05 0.05 ± 0.03 0.88 ± 0.04 0.05 ± 0.02 1.58 ± 0.41 0.11 ± 0.05 0.94 ± 0.57 0.33 ± 0.03 – – 0.53 0.67 0.19 0.06 0.15 0.78 0.57 0.19 0.48 1.33 0.61 0.23 0.48 2.07 0.64 0.15 0.71 0.17 0.98 0.83 0.91 1.45 10.3 – – – 1.10 0.38 0.50 0.76 0.67 1.00 0.43 0.62 0.74 0.50 0.55 0.63 0.59 1.80 0.54 0.21 0.66 0.21 0.91 0.83 1.21 1.00 4.70 –

Fig. 2. HPLC nucleotide profile obtained from yeast cells grown in galactose or glucose, as carbon source and in the absence or presence of H2O2. Yeast cells grown in the presence of galactose or glucose were challenged, when indicated, with 1.5 mM H2O2 for 1 h. Thereafter nucleotides were extracted and analyzed by HPLC as described in Materials and methods. The chromatographic peaks, identified by its UV spectra and time of elution, correspond to (1) Hyp, (2) Ino, (3) NAD+, (4) (unknown compound whose spectrum has a maximum at 280 nm), (5) UDP-sugars, (6) AMP, (7) ADP-rib, (8) NADP+, (9) ADP, (10) GTP, (11) UTP and (12) ATP.

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the adenine nucleotide content

Effect of menadione on the nucleotide content of yeast cells

is presence of H2O2, diverted towards inosine, and that the adenylic charge value decreases, from a standard value of around 0.8 to a value of around 0.4.

results

Similar

to those obtained with galactose, concerning variations in the levels of ATP and Ino (Fig. 1) were obtained when yeast cells grown in glucose were treated with different concentrations (0, 0.5, 1.0 and 1.5 mM) of H2O2 (results not shown). As was the case for the experiments were performed using five galactose, different batches of yeast cells, in the absence or presence of 1.5 mM H2O2 for 1 h (Table 2). In the presence of H2O2 there was a decrease of about threefold in the content of adenosine, cytidine and uridine, and twofold for guanosine nucleotides. The nucleoside triphosphates were the most affected by the H2O2 treatment. The decrease in ATP (5.6-fold) was almost coincident with the increase in Ino (5.7-fold). By contrast, the concentration of NAD+ remained almost constant after H2O2-treatment of yeast cells growing either in galactose or glucose. A representative chromatographic profile of a batch of yeast cells growing in glucose, in the absence or presence of 1.5 mM H2O2 is also depicted in Fig. 2C,D.

Here we tried to compare the effect of H2O2 on yeast cells with that of menadione, a different oxidative agent. As for H2O2, we started by assaying the effect of different concentrations of menadione (10, 30, 83, 90 and 110 mM) on cell viability (results not shown) and noticed that 83 mM menadione produced a viability similar to that evoked by 1.5 mM H2O2 (around 40% after 60 min treatment.) Based on these experiments, three batches of yeast cells growing exponentially in a medium containing galactose (Table 1) or glucose (Table 2), were treated for 1 h with 83 mM mena- dione. In general, the variations in the concentration of the nucleoside triphosphates induced by menadione are lower than those promoted by H2O2 treatment. In all the chromatograms corresponding to menadione-treated yeast cells, a new peak with a retention time of around 4.0 min was observed (Tables 1 and 2, and results not shown). Although its UV spectrum coincides with that of adenosine, both compounds are different because (a) they elute in a slightly different chromatographic position (not shown) and (b) they behave differently as substrates of adenosine deaminase: the new chromatographic peak is insensitive to the enzyme, in the same experimental conditions that adenosine is transformed to inosine (results not shown).

When mannose was used as a carbon source, similar results to those described for glucose were obtained (results not shown).

Table 2. Nucleoside and nucleotide content of S. cerevisiae (strain W303) grown in glucose and treated for 1 h with either 1.5 mM H2O2 or 83 mM menadione. Exponentially growing yeast cells were challenged with either H2O2 or menadione. Analysis of the nucleotide content was performed as described in Materials and methods. The data represent mean values ± SE of 5, 5 and 3 experiments for the control, H2O2-treated and menadione- treated cells, respectively. The concentrations of the indicated compounds are expressed in mM.

Menadione MD/ Control Parameters Control H2O2 H2O2/ Control

a The concentration (mM) of this compound has been calculated assuming the extinction coefficient of adenosine.

Starting wet weight (mg) Total protein (mg) Adenylic charge AMP ADP ATP S (ATP + ADP + AMP) CMP CDP CTP S (CTP + CDP + CMP) GMP GDP GTP S (GTP + GDP + GMP) UMP UDP UTP S (UTP + UDP + UMP) ADP-Rib NAD+ NADP+ UDP-sugars Hypoxanthine Inosine Unknowna 123 ± 42 2.7 ± 0.8 0.89 ± 0.02 0.05 ± 0.01 0.18 ± 0.02 1.07 ± 0.08 1.30 ± 0.09 0.04 ± 0.01 0.04 ± 0.02 0.22 ± 0.03 0.30 ± 0.04 0.02 ± 0.01 0.05 ± 0.01 0.16 ± 0.02 0.23 ± 0.14 0.06 ± 0.03 0.06 ± 0.01 0.53 ± 0.07 0.65 ± 0.07 0.11 ± 0.03 0.62 ± 0.12 0.04 ± 0.02 0.49 ± 0.09 0.10 ± 0.01 0.20 ± 0.06 – 124 ± 6 3.2 ± 1.4 0.53 ± 0.10 0.16 ± 0.04 0.12 ± 0.03 0.19 ± 0.10 0.47 ± 0.12 0.03 ± 0.02 0.02 ± 0.01 0.05 ± 0.03 0.10 ± 0.03 0.03 ± 0.02 0.05 ± 0.01 0.06 ± 0.03 0.14 ± 0.04 0.07 ± 0.06 0.06 ± 0.01 0.10 ± 0.04 0.23 ± 0.05 0.02 ± 0.00 0.97 ± 0.12 0.02 ± 0.01 0.59 ± 0.19 0.15 ± 0.05 1.16 ± 0.28 – 147 ± 17 4.7 ± 1.6 0.85 ± 0.05 0.08 ± 0.04 0.18 ± 0.05 0.90 ± 0.14 1.16 ± 0.06 – 0.05 ± 0.01 0.28 ± 0.01 – – 0.05 ± 0.01 0.18 ± 0.02 – – 0.06 ± 0.01 0.41 ± 0.08 – 0.05 ± 0.01 0.64 ± 0.12 0.04 ± 0.00 0.40 ± 0.12 0.04 ± 0.01 0.22 ± 0.06 0.44 ± 0.09 – – 0.60 3.20 0.67 0.18 0.36 0.75 0.50 0.23 0.33 1.50 1.00 0.37 0.61 1.17 1.00 0.19 0.35 0.18 1.59 0.50 1.20 1.50 5.80 – – – 0.96 1.60 1.00 0.84 0.89 – 1.25 1.27 – – 1.00 1.12 – – 1.00 0.77 – 0.45 1.05 1.00 0.82 0.40 1.10 –

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This new unknown chromatographic peak, not present in the preparation of menadione used, may correspond to a derivative of adenosine.

Effect of H2O2 on yeast cells growing in the presence of glycerol or ethanol

To obtain further insight into the oxidative effect of H2O2 (see below), yeast cells were grown in the presence of 3% glycerol as carbon source, and challenged with 1, 2, 3 and 4 mM H2O2. A concentration of H2O2 as high as 4 mM did not change the HPLC nucleotide profile obtained with untreated cells (results not shown). In a different experi- ment, yeast cells were grown in the presence of 2% ethanol as a carbon source, and challenged with 1.5 mM H2O2 for 1 h; again, no significant changes in the nucleotide content were observed in relation to the control cells (results not shown). It seems that, with respect to nucleotide metabo- lism, yeast cells grown in ethanol or glycerol as carbon sources are more resistant to H2O2 than those grown in the presence of galactose or glucose.

Search for a plausible mechanism

A mechanism to explain the different effects of H2O2 on the nucleotide content of yeast grown in the presence of hexoses (galactose, glucose or mannose), glycerol or ethanol was sought.

To explore the reasons for the decrease in ATP and the increase in Ino promoted by H2O2 in yeast cells growing in the presence of galactose, glucose or mannose, we followed an approach partially based on a previous study from our laboratory [3]. In that work, the metabolic pathways of AMP, GMP, IMP and XMP catalyzed by rat brain cytosol were explored using two complementary (experimental and theoretical) approaches.

Experimental approach – determination of enzyme acti- vities related to adenine metabolism. Enzyme activities related to adenine metabolism were determined in the cytosol of yeast cells, grown in glucose and in the absence or presence of 1.5 mM H2O2.

The pathways considered here to approach the meta- bolism of adenine nucleotides in yeast cells subjected (or not) to oxidative stress, together with the differential equation describing these pathways are represented in Figs 3 and 4, respectively. The enzymes considered in the pathway from ATP to Ino were E1 (AMP 5¢-nucleoti- dase), E2 (IMP-GMP specific 5¢–nucleotidase), E3 (AMP deaminase), E4 (adenosine deaminase), E5 (purine nucleo- side phosphorylase), E6 (adenylate kinase), E7 (adenosine kinase), E8 (a hypothetical enzyme catalyzing two general and reversible reactions), E8d (synthesis of ATP through the glycolytic pathway) and V8r (degradation of ATP through general anabolic processes).

Fig. 3. Adenine and hypoxanthine nucleotide metabolism in yeast cyto- sol. The pathways considered are those shown in the Figure. The enzymes involved are E1, 5¢-nucleotidase acting on AMP and IMP; E2, IMP-GMP specific 5¢-nucleotidase; E3, AMP deaminase; E4, adeno- sine deaminase; E5, purine nucleoside phosphorylase; E6, adenylate kinase; E7, adenosine kinase; E8, hypothetical enzyme recycling ATP.

kinetic constants taken from the literature are compiled in Table 3. As a representative example, mixtures containing 1.8 mM ATP, 0.8 mM ADP and 0.34 mM AMP were incubated with cytosol from yeast cells grown in glucose in

Enzyme activities were determined as described in Materials and methods. To avoid enzyme inactivation, only fresh (not frozen) cytosol was used. Reaction mixtures were set up containing the yeast cytosol, and the concen- tration of substrate(s) and buffering conditions that we considered pertinent (based on the literature) to render linear formation of products. The results obtained and the

Fig. 4. Differential equations describing the fluxes operating in the pathways from ATP to hypoxanthine, as described in Fig. 3. Vn, maximum velocity of the reaction catalyzed by En, on the substrate indicated. The velocity equations considered for E1, E3, E4, E5, E6 and E7 were as in Torrecilla et al. [3]; those for E2 and E8 are indicated in the text and Table 3.

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Table 3. Vmax, Km and Ki values of enzymes involved in the adenine and hypoxanthine metabolism in yeast cytosol. Vmax values represent the average of a minimum of three determinations obtained from different batches of yeast cells grown in glucose, with H2O2 results determined in the yeast cytosol, and grown for 30 min in the presence of 1.5 mM H2O2. Ki values of the products were assumed equal to the Km values of the substrate in the cases of enzymes E2, E5, E6, E7 and E8. En represents the enzymes as specified in Fig. 3.

Vmax (mUÆmg)1)

Enzyme Substrate Control H2O2 Km (lM) Ki (lM)

AMP IMP 10.4 ± 1.4 12.7 ± 0.9 11.6 ± 2.2 11.8 ± 2.2 200 [29] 540 [29] E1 5¢-AMP nucleotidase

IMP 5.7 ± 0.8a 3.1 ± 0.7 a

E2 5¢-nucleotidase IMP-GMP specific 640 (Ado) [29] 8600 (Ino) [29] 27.2 ± 5.4 (ATP) 37.5 ± 8.9 (ADP) 400 (Ino) 300 (Ino) 2000 (Ino) 400 [30] 300 [30] a 2000 b 1800 a,b

AMP 29.2 ± 2.4 a 24.4 ± 7.5 a 5000 (Pi) [30] 4700 (IMP) [31]

Ado 0.8 ± 0.2 0.6 ± 0.2 2670 [31] 500 [31] a 40.7 [32] 28 (Ino) [32,33]

10.9 ± 1.7 11.6 ± 1.0 E3 AMP deaminase E4 Adenosine deaminase E5 Nucleoside phosphorylase 29.4 ± 1.4 33.4 ± 0.6

E6 Adenylate kinase

Ino Pi Hyp Rib-1P AMP ADP ATP Ado 4000 c 2414 ± 621 4000 c 203 ± 97 4297 c 2593 ± 557 4297 c 216 ± 61 E7 Adenosine kinase 166 [34] 1600 22 [35] 320 [35] 34 [36] 23 [36] 63 [36] 2.8 [35] 220 (ATP) [35]

E8

a Values obtained in the presence of ATP. b Km values determined in yeast cytosol, and used in the theoretical simulation depicted in Fig. 5. c Values calculated using MATHEMATICA-3.0 program.

for the AMP deaminase. Accordingly [3] the velocity equation used for 5¢-IMP nucleotidase was settled as:

m2 ¼

V2IMP½IMP(cid:7)n ½IMP(cid:7)n þ ½S0:5(cid:7)n

the absence (Fig. 5A) or presence of 1.5 mM H2O2 (not shown). The rate of adenine nucleotide degradation and the appearance of intermediate products were essentially the same in both cases and, above all, no appreciable differences in the rates of disappearance of ATP or appearance of Ino were observed.

where n ¼ 1.7–1.2[ATP]/(Ka2ATP + [ATP]) and S0.5 ¼ Km2IMP – (F2K [ATP]/(Ka2ATP + [ATP])).

From both the enzyme properties reported by Itoh [30], and experiments from this laboratory (not shown), the following values were used:

Ka2ATP ¼ 1250; F2K ¼ 200 (Fig. 5) or 100 (Fig. 6) (see [3], and Table 3, for further explanations on the significance of these parameters).

The equation described as V8d and V8r, and the corres- ponding substrates and products were not considered at this stage (i.e. V8d ¼ V8r ¼ 0, see below). Taking into account the above values, application of the MATHEMATICA-3.0 program [37] to the case of a reaction mixture containing ATP, ADP and AMP (at the same concentrations as those present in the experimental approach, Fig. 5A) produced

Theoretical approach – mathematical simulation of some metabolic pathways related to adenine nucleotide meta- bolism. This was a theoretical approach. The simulation was started by stating the metabolic pathways from ATP to Ino (Fig. 3), writing the opportune differential equations (Fig. 4) and solving them with the help of the MATHEMAT- ICA-3.0 program [37]. The equation velocities considered for the enzymes involved were essentially as described in [3], with the following main modifications. The kinetic proper- ties of E2 (5¢-nucleotidase for IMP) from yeast [30] are different to those described for the enzyme from rat brain [38]. The sigmoidal kinetic toward IMP reported for the yeast enzyme changed to near-hyperbolic in the presence of ATP [30], i.e. a behavior similar to that previously described

ATP X ADP X-P 200 c 200 c 4000 c 4000 c 33 100 33 100 166 1600 22 320 34 23 63 2.8 (Ado) 220 (ATP) 200 (AMP) [35] 1200 (ADP) [35] 33 100 33 100

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Effect of H2O2 and menadione on S. cerevisiae (Eur. J. Biochem. 270) 1585

speculated that changes in the relative rates of both processes could affect the actual concentration of ATP and hence the rate of synthesis of inosine. It is here assumed that the complex processes of syntheses and degradation of ATP in vivo (involving many enzymes) is carried out by a hypothetical unique enzyme (E8) catalyzing both the synthesis of ATP in the direct reaction (E8d) and the phosphorylation/transformation of substrates with partici- pation of ATP in the reverse direction (E8r):

ADP þ X-P $ ATP þ X

where X and X-P represent a pool of unphosphorylated and phosphorylated unspecified substrates, respectively. This hypothetical enzyme has been used to test, with the help of the mathematical model described in [3], whether different rates of synthesis of ATP would modify the intracellular pool of inosine.

The reaction catalyzed by E8 is here supposed to be similar to that catalyzed by adenylate kinase, i.e. random- bireactant [41], and the corresponding velocity equation is:

m8

(cid:1)

[ADP][S(cid:1)P]V8d Km8ADPKm8S(cid:1)P

¼

1 þ

þ

þ

þ

þ

þ

[ADP] Ki8ADP

[S] Ki8S

[S][ATP] Km8SKi8ATP

[ATP][S]V8r Km8ATPKi8S [ATP] Ki8ATP

[S(cid:1)P] Ki8s(cid:1)p

[ADP][S(cid:1)P] Km8ADPKi8S(cid:1)P

The following kinetic constants were established to solve the equation:

Km8ADP ¼ Ki8ADP ¼ Km8ATP ¼ Ki8ATP ¼ 0:033 mm Km8S(cid:1)P ¼ Ki8S(cid:1)P ¼ Km8S ¼ Ki8S ¼ 0:1 mm ½X(cid:7) ¼ ½X-P(cid:7) ¼ 0:1 mm:

As this hypothetical activity represents the activity of many enzymes, we have chosen representative mean values for the kinetic constants of the enzyme E8, in the order of mM, while the concentrations of ATP and ADP were considered as variables.

similar rates of disappearance of substrates and appearance of products (Fig. 5B).

Inhibition of glycolysis as a plausible theoretical explan- ation for increased inosine. Yeast cells treated with H2O2 and grown in galactose, glucose or mannose showed an increase in inosine level, for which the inhibition of glycolysis was proposed as a possible theoretical explanation.

With these characteristics, the maximum velocities in the direct (V8d, synthesis of ATP) and in the reverse (V8r, synthesis of ADP) directions were mathematically adjusted, using the MATHEMATICA-3.0 program (to 4000 and 200, respectively) to keep the level of ATP during the application of the mathematical procedure nearly constant (Fig. 6B). Metabolic situations conveying diminution in the rate of formation of ATP from ADP (i.e. inhibition of glycolysis) were simulated by decreasing V8d from 4000 (Fig. 6B) step by step to 500, and leaving constant V8r at 200 (Fig. 6C–F). The graph in Fig. 6A represents an extreme situation in which V8d ¼ V8r ¼ 0. Together, the graphs depicted in Fig. 6B–F show that the inhibition in the rate of synthesis of ATP from ADP is accompanied by an increase in the rate of synthesis of Ino, without any need to modify the kinetic parameters or activities of the enzymes involved in the pathway from ATP to Ino.

The results from Fig. 1 and Table 1, suggested that (a) the main if not unique source of inosine is the intracellular pool of adenine nucleotides, (b) the decrease in ATP and the increase in inosine, promoted by H2O2, cannot be explained solely by a change in the level of the enzymes more directly involved in the nucleotide pathway from ATP to inosine (Fig. 3), so that (c) other factors should account for those changes.

Being aware of the simplifications involved in these calculations (where so many more enzymes participate in vivo), these results would indicate that H2O2 diminishes the rate of synthesis of ATP, probably through inhibition of glycolysis. It is worth noting that H2O2 has no appreciable effect on the level of ATP on yeast cells grown in ethanol or glycerol, that are metabolized through an oxidative pathway.

When yeast cells are grown in glucose, galactose or mannose, ATP is generated mainly through the glycolytic pathway, and used in diverse anabolic pathways [39,40]. We

Fig. 5. Metabolism of ATP, ADP, AMP in the presence of cytosol from yeast growing in glucose, in the absence or presence of H2O2 – theoretical simulation. The reaction mixtures contained: 50 mM imidazole/HCl buffer, pH 7.0; 0.1 M KCl; 0.1 mM dithiothreitol; 4 mM MgCl2; 1.8 mM ATP; 0.8 mM ADP; 0.34 mM AMP and cytosol from yeast cells grown in the absence (A) or presence (result not shown) of 1.5 mM H2O2, for 60 min. Aliquots were taken at the indicated times and analyzed by HPLC. In (B) application of the theoretical model was performed with the MATHEMATICA-3.0 program, as described in the text and in [3]. The V-values, from control cells, and the kinetic parameters described in Table 3 were used.

1586 H. Osorio et al. (Eur. J. Biochem. 270)

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Effect of H2O2 on glycolysis

From the above, it seemed obvious to verify in our experimental conditions the effect of H2O2 on either the rate of glucose consumption or on the rate of synthesis of ethanol. At the usual concentrations of both glucose (2%) and yeast cells (around 1.2 D600 units per mL), at which the effect of H2O2 was previously tested, the consumption of glucose (in control and treated cells) was so low that its disappearance from the culture medium could not be detected. However, at higher yeast cells (91 D600 units per mL) and H2O2 (15 mM) concentrations, a decrease in the consumption of glucose was clearly observed a few minutes after the onset of the H2O2 treatment (results not shown).

The rate of ethanol production by yeast cells growing in glucose was also determined as a parameter to measure potential inhibition of glycolysis by H2O2. As shown in Fig. 7, treatment of yeast cells with 0, 0.05, 0.1, 0.3, 0.5 and 1.5 mM H2O2 promoted a dose-dependent decrease in the rate of synthesis of ethanol.

Fig. 6. Influence of the hypothetical enzyme (E8) recycling ATP, on the rate of synthesis of inosine. Application of the theoretical model was performed with the MATHEMATICA-3.0 program, as described in the text and in [3]. Simulation was made considering the kinetic values for the enzymes E1–E7 determined in the cytosol of control cells, grown in glucose. In the case of enzyme E2, the Km values described in [30] were used (Table 3). Graphs A–F were computer made using the following additional values, respectively, for V8d and V8r: A (0,0); B (4000, 200); C (3500, 200); D (3000, 200); E (2500, 200); F (2000, 200).

Recovery of yeast cells after the oxidative stress caused by H2O2

the absence of H2O2, the recovery of ATP was almost complete, while the return of inosine to normal values was much slower.

Fig. 7. Effect of H2O2 on the synthesis of ethanol by S. cerevisiae. Yeast cells, grown in glucose as carbon source, were challenged with 0; 0.05; 0.1; 0.3; 0.5 and 1.5 mM H2O2. Ethanol was determined in the medium, at the indicated times, as described in Materials and methods.

Discussion

The results presented above are clear, concerning the effect of H2O2 on the yeast strain W303 of Saccharomyces cerevisiae. In the presence of glucose, galactose or mannose,

A yeast culture grown in glucose was challenged with 1.5 mM H2O2 for 30 min. After this treatment, cells were separated by centrifugation, resuspended in fresh medium without H2O2, and aliquots taken at 0, 30, 60, 90 and 120 min incubation. As expected, the ATP content was very low after the H2O2 treatment (time zero) and the inosine concentration very high (Fig. 8). After 30 min incubation in

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Effect of H2O2 and menadione on S. cerevisiae (Eur. J. Biochem. 270) 1587

untreated control) in yeast cells subjected to H2O2 treatment and grown in glycerol or glucose, respectively [42]. A similar observation concerning carbonylation of key metabolic enzymes by H2O2 has been described recently by Costa et al. [43]. These authors observed an 80% reduction of glyceraldehyde 3-phosphate dehydrogenase upon incuba- tion of yeast cells with 1.5 mM H2O2 [43]. In this regard, preliminary results from our laboratory showed a fivefold increase in the level of fructose 1,6-bisphosphate concentra- tion in H2O2 treated cells (unpublished results). Moreover, it seems to us important to emphasize that the effect of H2O2 on glycolysis is likely to be reversible, as ATP and inosine levels are restored upon washing H2O2 from the cells and resuspending them in fresh medium. The recovery appears quite fast, which probably suggests covalent modification of protein(s) (i.e. glyceraldehyde 3-phosphate dehydrogenase) and precludes any in vivo protein synthesis.

Considering that ATP is the center of a very important metabolic crossroads [44], other possibilities could be contemplated to explain the decrease of ATP promoted by H2O2, such as the inhibition of the transport of hexoses (what could be considered as an inhibition of glycolysis) or an increase in ATPase activity. This latter possibility does not seem to be operative in this case, as the ATPase activities found by us in the cytosol from untreated or H2O2-treated cells were 5.2 ± 2.3 and 5.5 ± 1.9 mUÆmg)1 protein, respectively. Moreover, application of the theoretical method, taking into accounts these values, did not alter significantly the rate of ATP degradation.

The decrease in ATP promoted by H2O2 could be also compared with the decrease of this nucleotide promoted by the mutation in the gene responsible for the synthesis of trehalose 6-phosphate (TPS-1), which is accompanied also by an increase in glucose 6-phosphate. In the case of tps-1 mutants, the decrease in ATP and the increase in glucose 6-phosphate in yeast grown in glucose could be explained by an enhanced activity of hexokinase produced by both the release of its inhibition by trehalose 6-phosphate and/or by the proper effect of the TPS-1 gene product [45–49], two conditions most probably not prevalent in the H2O2-treated yeast cells, where the decrease of ATP is accompanied by a decrease of about twofold in the glucose 6-phosphate level (unpublished results from this laboratory).

H2O2 evokes a decrease and an increase in the intracellular concentration of ATP and inosine, respectively. Searching for the rationale for these phenomena, possible changes in the specific activities of enzymes directly involved in the pathway from ATP to Ino were explored in extracts from normal and oxidatively stressed cells (Table 3). At first glance, the changes in the activities of those enzymes did not account for the changes in the ATP or inosine levels. This impression was quantified with the help of a mathematical model of differential equations describing the changes in substrate and product concentration in a metabolic path- way as a function of the kinetic constants of the enzymes involved in that pathway [3]. Application of this method pointed to the inhibition of the rate of synthesis of ATP by the glycolytic route as a potential reason for the changes in ATP and inosine levels, provoked by H2O2. This assump- tion was experimentally tested by measuring the consump- tion of glucose and the synthesis of ethanol in yeast cells treated with H2O2, which produced a decrease in both the consumption of glucose and synthesis of ethanol. The apparent effect of H2O2 on glycolysis was further confirmed by the lack of effect of H2O2 when yeast cell were grown in glycerol or ethanol, two oxidative substrates. The possibility that the resistance to H2O2 in these last two cases may be due to a stronger expression of antioxidant enzymes has not been explored.

glyceraldehyde

Godon et al. [50] approached the effect of H2O2 on S. cerevisiae in a different way. Yeast grown in minimal medium containing 2% glucose were treated with 0.4 mM H2O2 for 15 min and subsequently pulse-labeled with [35S]methionine from 15 to 30 min. Total proteins were then extracted and subjected to two-dimensional gel elec- trophoresis. They observed that at least 115 proteins were repressed and 52 induced by this treatment. Two isozymes 3-phosphate dehydrogenase were of repressed by this treatment. Godon et al. [50] did not perform the same experiment growing yeast in the presence of glycerol or ethanol as carbon sources.

The effect of H2O2 on yeast cells had been previously analyzed from several perspectives. Cabiscol et al. [42] observed the formation of carbonyl groups in several amino acid side chains of proteins after treatment of yeast with H2O2 and menadione. Here, mitochondrial proteins (E2 subunits of both pyruvate kinase and a-ketoglutarate dehydrogenase, aconitase and heat shock protein 60) and the cytosolic fatty acid synthetase and glyceraldehyde 3-phosphate dehydrogenase were the enzymes mainly affec- ted by the H2O2 treatment [42]. In line with the results reported in this study, the activity of glyceraldehyde 3-phos- phate dehydrogenase (one of the two enzymes in glycolysis responsible for the synthesis of ATP through substrate level phosphorylation) was 85 and 53% (in relation to an

The response of S. cerevisiae to stress is also dependent on its redox state. However, as shown in [51] the metabolic basis for this behavior is still not clear. Deficiency in glutathione reductase promotes a higher imbalance in the ratio of reduced glutathione to total glutathione than that produced by glucose 6-phosphate dehydrogenase deficiency. However, in contrast to what would be expected, cells

Fig. 8. Recovery of ATP after treatment of yeast cells with H2O2. Yeast cells grown in glucose, were treated with 1.5 mM H2O2 for 30 min, collected by centrifugation, resuspended in fresh medium (without H2O2) and incubated further for 120 min. At the times indicated, the adenylic charge, ATP and inosine were determined as described in Materials and methods.

1588 H. Osorio et al. (Eur. J. Biochem. 270)

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deficient in this enzyme are comparatively more sensitive to H2O2 stress than those deficient in glutathione reductase. Izawa et al. [51] concluded that glucose 6-phosphate dehydrogenase appears to play other important roles in the adaptive response to H2O2 stress besides supplying NADPH for the recycling of glutathione.

5. Rubio-Texeira, M., Varnum, J.M., Bieganowski, P. & Brenner, C. (2002) Control of dinucleoside polyphosphates by the FHIT- homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae. BMC Mol. Biol. 3, 7–17. 6. Davies, K.J.A. (1995) Oxidative stress: the paradox of aerobic life. Biochem. Soc. Symp 61, 1–31. 7. Fridovich, I. (1978) The biology of oxygen radicals. Science 201, 875–880. 8. Nelson, D.L. & Cox, M. (2000) Lehninger Principles of Biochem- istry. Worth Publishers, New York, USA. 9. Metzler, D. (2001) Biochemistry. Hartcourt/Academic Press, New York, USA.

Our work is also in line with previous reports indicating that H2O2 and menadione have different effects on yeast [52–54]. S. cerevisiae cells subjected to treatment with H2O2 (0.2 mM for 60 min) were more resistant to 4 mM menadi- one. However, pretreatment with menadione did not induce resistance to H2O2 and different polypeptides were synthe- sized as response to treatment with menadione or H2O2 [53]. Partially different results were reported later [54] using Schizosaccharomyces pombe. Cells pretreated with a low dose of menadione became resistant to a lethal dose of H2O2, whereas cells pretreated with H2O2 became only partially resistant to a lethal dose of menadione. The pattern of induction of several oxidative defence enzymes promoted by H2O2 or menadione was also slightly different [54].

10. Dugan, L.L. & Choi, D.W. (1998) Hypoxic-ischemic brain injury and oxidative stress. In Basic Neurochemistry. Molecular, Cellular and Medical Aspects (Siegel, G.J., Agranoff, B.W., Albers, R.W., Fisher, S.K. & Uhler, M.D., eds), pp. 711–729. Lippincot-Raven, Philadelphia, USA

11. Thor, H., Smith, M.T., Hartzell, P., Bellomo, G., Jewell, S.A. & Orrenius, S. (1982) The metabolism of menadione (2-methyl-1,4- naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells. J. Biol. Chem. 257, 12419–12425.

12. Wolff, S.P. & Dean, R.T. (1986) Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis. Biochem. J. 234, 399–403.

13. Storz, G., Christman, M.F., Sies, H. & Ames, B.N. (1987) Spon- taneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium. Proc. Natl Acad. Sci. USA 84, 8917–8921. 14. Halliwell, B. (1992) Reactive oxygen species and the central ner- vous system. J. Neurochem. 59, 1609–1623. 15. Halliwell, B. & Gutteridge, J.M.C. (1999) Free Radicals in Biology and Medicine. Oxford University Press, London, UK. 16. Jamieson, D.J. (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14, 1511–1527.

The study of oxidative response of S. cerevisiae, and of other cell types, to stress can be focused under different aspects: the oxidative defence systems of the cell, inducible adaptive responses and their genetic regulation, signal transduction, etc. One of the main conclusions that can be derived from this report is that the steady state of the nucleotide level is an important factor to be considered in relation to the general response of S. cerevisiae to oxidative stress, as illustrated by the different response to H2O2, depending on whether the yeast uses glucose or glycerol as carbon source. It seems to us evident that the intracellular concentration of nucleotides is a key factor to be considered in the understanding of the cellular response of yeast to the oxidative aggression.

transduction in Saccharomyces cerevisiae:

17. Costa, V. & Moradas-Ferreira, P. (2001) Oxidative stress and insights signal into ageing, apoptosis and diseases. Mol. Aspects Med. 22, 217– 246.

Acknowledgements

18. Moradas-Ferreira, P. & Costa, V. (2000) Adaptive response of the yeast Saccharomyces cerevisiae to reactive oxygen species: defences, damage and death. Redox Report 5, 277–285.

19. Moradas-Ferreira, P., Costa, V., Piper, P. & Mager, W. (1996) The molecular defences against reactive oxygen species in yeast. Mol. Microbiol. 19, 651–658. 20. Thomas, B.J. & Rothstein, R. (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56, 619–630.

This investigation was supported by grants from Direccio´ n General de Investigacio´ n Cientı´ fica y Te´ cnica (PM98/0129, BMC2002-00866) and Comunidad de Madrid (08.9/0004/98; 08/0021.1/2001). We thank Anabel de Diego, Vero´ nica Domingo and Georgia Afonso (from the E´ cole Nationale Chimie, Physique, Biologie of Paris) for their capable technical assistance, and Dr Claudio F. Heredia for helpful discussions. H. O. was supported by a Fellowship from Fundac¸ a˜ o para a Cieˆ ncia e a Tecnologı´ a (SFRH/BD/1477/2000).

21. Saez, M.J. & Lagunas, R. (1976) Determination of intermediary metabolites in yeast. Critical examination of the effect of sampling conditions and recommendations for obtaining true levels. Mol. Cell Biochem. 13, 73–78.

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