EFFECTS OF BICARBONATE AND ALPHA-KETOGLUTARATE ON SENSITIVITY OF SACCHAROMYCES CEREVISIAE YEAST TO HYDROGEN PEROXIDE AND IRON IONS

The effects of sodium bicarbonate on the sensitivity of Saccharomyces cerevisiae yeast to hydrogen peroxide and ferrous sulfate were studied. Viability of yeast cells treated with 10–25 mM H 2 O 2 and 0.1–0.2 mM FeSO 4 was significantly decreased when 25 or 50 mM NaHCO 3 was added to the medium. In the absence of bicarbonate, the levels of oxidative stress markers, namely protein carbonyls, total and oxidized glutathione in cells exposed to 0.2 mM FeSO 4 did not differ from such levels in control cells (without FeSO 4 ). Yeast cells incubated with 0.2 mM FeSO 4 and 50 mM NaHCO 3 had similar levels of oxidized glutathione and carbonyl groups in proteins but lower level of total glutathione compared to cells treated with FeSO 4 in the absence of NaHCO 3 . Yeast cells exposed to a mixture of “2 mM H 2 O 2 + 2 mM FeSO 4 ” in 50 mM sodium bicarbonate buffer survived better than cells treated with these oxidants in 50 mM potassium phosphate buffer. The addition of 10 mM alpha-ketoglutarate led to the increased yeast survival in both buffers under the treatment with “Fe 2+ /Н 2 О 2 ”. The protective effect of alpha-ketoglutarate can be due to its H 2 O 2 -scavenging activity. The results suggest that bicarbonate ions can enhance or alleviate the toxic effects of redox-active compounds on S. cerevisiae . Pro/antioxidant effects of bicarbonate ions are likely to depend on the kinetics of interaction between HCO 3 ˉ and produced ROS. Abbreviations: AKG (alpha-ketoglutarate); CP (carbonyl proteins); Cu, Zn-SOD (Cu, Zn-superoxide dismutase); KPi (potassium phosphate buffer); OD (optical density); ROS (reactive oxygen species).


INTRODUCTION
Production of the reactive oxygen species (ROS) and carbon dioxide (CO 2 ) is a part of normal aerobic cellular metabolism [19,26]. ROS such as superoxide anion radical (O 2 •− ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical ( • ОН) are potentially dangerous due to their high reactivity and capability to interact with virtually all cellular components. Toxicity of ROS is largely dependent on the presence of ions of transition metals, such as iron and copper. Transition metals can participate in the formation of highly reactive hydroxyl radical in the Fenton reaction [24]: Fe 2+ + H 2 O 2 → Fe 3+ + • OH + OH − . Excessive ROS production and/or decrease in antioxidant defense leads to the development of oxidative stress, which is implicated in aging and many human diseases [19]. Carbon dioxide and its hydrated forms (НСО 3ˉ і СО 3 2ˉ) are components of carbonate buffer system which plays an important role in pH regulation in biological liquids [23]. Bicarbonate buffer that is composed of 1.3 mM CO 2 in equilibrium with 25 mM HCO 3ˉ in serum and 14 mM НСО 3ˉ intracellularly, has well-demonstrated redox effects [20,23]. A number of studies demonstrated that HCO 3ˉ or CO 2 /HCO 3ˉ can stimulate the oxidation, peroxidation, and nitration of various molecules [1,2,5,6,10,14,28]. Carbon dioxide and (bi)carbonate ions enhance metal-catalyzed decomposition of Н 2 О 2 [5,14] and peroxidase activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) [9,15,28]. At the same time, (bi)carbonate-mediated peroxidase activity of Cu,Zn-SOD leads to the formation of carbonate radical (СО 3 •− ), which has strong oxidizing properties [1,3,20,26,28]. СО 3 •− formation was shown to be responsible for the increased oxidation of proteins and lipids in carbonate buffer under exposure to transition metals [2]. It should be noted that articles cited above and many similar articles used in vitro systems. There is a little information about similar processes in vivo. We have previously shown that bicarbonate buffer sensitized Saccharomyces cerevisiae yeast to menadione, a redox-active compound which is able to generate superoxide anion radical [17]. The inactivation of aconitase and the decrease in glutathione level in yeast cells treated with menadione in bicarbonate buffer were observed.
Taking into account that bicarbonate ions can intensify free radical processes, it seems possible that the exogenous antioxidant compounds can alleviate these processes. Recently, the antioxidant properties for alpha-ketoglutarate (AKG) as an important intermediate in the Krebs cycle were demonstrated. In particular, the ability to scavenge hydrogen peroxide was shown for AKG [4].
This study was aimed at studying the effects of sodium bicarbonate on sensitivity of yeast S. cerevisiae to hydrogen peroxide, iron ions and their mixture. The ability of AKG to prevent yeast cell death in bicarbonate buffer under combined treatment with H 2 O 2 and Fe 2+ was also studied.
Cell extracts were prepared by vortexing yeast cells with glass beads (0.5 mm), as described [17]. The content of carbonyl groups in the proteins (CP) was measured by determining the amount of 2,4-dinitrophenylhydrazone formed upon the reaction with 2,4-dinitrophenylhydrazine. Carbonyl content was calculated from the absorbance maximum of 2,4-dinitrophenylhydrazone at 370 nm with molar extinction coefficient of 22 mM -1 ·cm -1 [13]. The level of total glutathione was measured as described in the paper [18]. Yeast cells were suspended in 1.3 % dinitrosalicilic acid and disrupted by vortexing with glass beads (0.5 mm) for three cycles (1 min of disruption and 3 min of cooling on ice). For determination of oxidized glutathione, the aliquots of supernatants were incubated with 5 % 2-vinylpyridine for 1 h at room temperature. Protein concentration was determined by Bradford [7] basing on binding of Coomassie brilliant blue G-250 dye with protein.
Experimental data are expressed as mean of 4-6 independent experiments ± the standard error of the mean (SEM), and statistical analysis used Dunnett's test and Student's t-test [8].

RESULTS AND DISCUSSION
The survival of yeast cells upon treatment with hydrogen peroxide or ferrous sulfate in the presence of sodium bicarbonate at different concentrations was studied (Fig. 1). Hydrogen peroxide decreased yeast survival in both control and bicarbonate-supplemented suspensions (Fig. 1, A). The survival was decreased with increasing of Н 2 О 2 concentration. In particular, cell viability was 79 and 35 % in the control suspensions treated with 10 and 25 mМ Н 2 О 2 , respectively. The addition of 10 mМ NaHCO 3 did not influence yeast resistance to Н 2 О 2 , whereas 25 mМ NaHCO 3 enhanced sensitivity of yeast cells to 10 mМ and 15 mМ Н 2 О 2 . Yeast cells were the most sensitive to Н 2 О 2 in the presence of 50 mМ NaHCO 3 with 73 and 17 % of survival after treatment with 10 and 25 mМ Н 2 О 2 , respectively.
The incubation of yeast cells with 0.1-0.2 мМ FeSO 4 did not affect cell survival in the control (without bicarbonate) and in the medium, containing 10 mМ NaHCO 3 ( Fig. 1, B). However, the treatment with ferrous sulfate in the presence of 25 or 50 mМ NaHCO 3 decreased yeast viability with more sensitizing effect of 50 mМ NaHCO 3 . Thus, the survival decreased by 19 and 56 % after treatment with 0.2 mM FeSO 4 in the presence of 25 and 50 mМ NaHCO 3 , respectively. The obtained results suggest that bicarbonate ions can enhance sensitivity of S. cerevisiae cells to hydrogen peroxide and iron ions.
Our results are consistent with previous reports in vitro which showed the ability of bicarbonate ions participate in redox-processes [3,20,23,26]. In particular, the increase in peroxidase activity of Cu, Zn-SOD was shown in the presence of bicarbonate. The enzyme decomposes Н 2 О 2 with the formation of superoxide anion radical which is a direct substrate of SOD: At this process, the enzyme is converted to intermediate inactive form SOD-Cu 2+ -• ОН which can undergo further oxidative inactivation or can be restored to initial form (SОD-Cu 2+ ) by interaction with (bi)carbonate ions. НСО 3ˉ and/or СО 3 2ˉ undergo one-electron oxidation to carbonate radical СО 3 15,20,26,28]. It was also shown that in vitro СО 3

•−
can be formed in the reaction of carbonate ions with peroxinitrite (ONOO − ) or directly with hydroxyl radical ( • ОН) [3,16,20]. It was shown that the sensitivity of Escherichia coli bacteria and S. сerevisiae yeast to γ-radiation was significantly increased in the bicarbonate buffer. That was due to the formation of carbonate radical in the reaction of НСО 3ˉ with products of water photolysis [12]. Our results suggest that the enhanced cytotoxic action of H 2 O 2 and Fe 2+ in the presence of bicarbonate ions can be associated with the intracellular generation of carboxyl radical, because there is no information regarding direct non-enzymatic reaction between НСО 3ˉ and H 2 O 2 or iron ions [11]. It was assumed that bicarbonate ions can enter yeast cells through mammalian Slc4-like proteins which were also indentified in yeast as bicarbonate transporters [21]. In cells, НСО 3ˉ can enhance H 2 O 2 -scavenging activity of Cu,Zn-SОD, as it was shown in vitro [15]. Thus, СО 3 •− can be produced in this reaction. СО 3 •− is more reactive compound than H 2 O 2 , and this fact can explain a higher sensitivity of yeast cells to hydrogen peroxide in the presence of bicarbonate ions. The enhanced sensitivity of S. сerevisiae to ferrous sulfate treatment in the presence of bicarbonate (Fig. 1, B) can also be explained by СО 3 •− formation. It is known, the toxicity of Fe 2+ is connected with its ability to generate hydroxyl radical in Fenton reaction [24]. In turn, hydroxyl radical can react with 16,20]. Despite СО 3 •− is less reactive compound than • ОН, СО 3 •− has a much longer half-life and can therefore diffuse further and oxidatively modify distant cellular targets [16]. Since the ability of bicarbonate ions to potentiate toxicity of hydrogen peroxide and iron ions could be connected with the intensification of free radical processes, the levels of oxidative stress markers such as protein carbonyl groups and glutathione were measured in yeast cells. Content of carbonyl group in proteins (CP) is a widely used parameter of oxidative damages of proteins [9,17,22]. Glutathione (GSH) is a low molecular mass antioxidant which plays an important role in the maintenance of redox homeostasis in S. cerevisiae [25]. CP levels and levels of oxidized glutathione (GSSG) were similar control cells and in cells treated 0.2 mМ FeSO 4 in the absence or presence of  Table). At the same time, total GSH was decreased in cells treated with 0.2 mМ FeSO 4 in the presence of NaHCO 3 at higher concentrations. Accordingly, the total GSH was 22 % lower in cells treated with 0.2 mМ FeSO 4 and 50 mM NaHCO 3 .
Similar results were obtained when the ability of bicarbonate to modulate sensiti vity of yeast cells to menadione was studied [17]. Bicarbonate enhanced cytotoxicity of menadione that was accompanied by decreased GSH level in cells without changes in CP levels. The absence of changes in CP level could suggest that СО 3 •− generated in bicarbonate buffer might promote other types of protein damages which are different from carbonylation. For example, СО 3 •− was found can form tyrosyl radical and tyrosine cross-links and oxidize SH-groups of cysteine [1,6,26]. СО 3 •− can also damage DNA by reacting with guanine base producing 8-oxoguanine [27]. The decrease in level of GSH which is a cysteine-containing tripeptide seems not to be connected with its oxidation because the level of GSSG was unchanged in cells co-treated with ferrous sulfate and NaHCO 3 (See Тable). Obviously, the synthesis GSH de novo can be decreased under these conditions. The decreased GSH level can lead to disturbing redox balance in cells and reduce antioxidant defense. It could enhance yeast sensitivity to oxidative stress inductors in bicarbonate buffer. In next step of experiments, the survival of yeast cells treated with mixture of "2 mМ Н 2 О 2 + 2 mМ FeSO 4 " was studied. Hydroxyl radicals are directly generated in this mixture. The survival of YPH250 cells treated with "Fe 2+ /Н 2 О 2 " in 50 mМ KPi (рН 7.5) or in 50 mМ sodium bicarbonate buffer (рН 7.5) was calculated (Fig. 2). The number of the viable cells was significantly decreased in both buffers but the cells treated in sodium bicarbonate buffer were more resistant to "Fe 2+ /Н 2 О 2 " with 1.6-fold higher survival compared to the one in KPi. The results suggest that bicarbonate can alleviate toxic action of Fe 2+ /Н 2 О 2 system. Given that НСО 3ˉ can react with • ОН forming СО 3