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Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide
2008; Elsevier BV; Volume: 284; Issue: 9 Linguagem: Inglês
10.1074/jbc.m805526200
ISSN1083-351X
AutoresRenata Ogusucu, Daniel Rettori, Luís Eduardo Soares Netto, Ohára Augusto,
Tópico(s)Electron Spin Resonance Studies
ResumoPeroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1Δtsa2Δ) are more resistant to hydrogen peroxide than wild-type (WT) cells and consume it faster under fermentative conditions. Also, tsa1Δtsa2Δ cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1Δtsa2Δ cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu,Zn-superoxide dismutase (Sod1), whose expression and activity increased ∼5- and 2-fold, respectively, in tsa1Δtsa2Δ compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1Δtsa2Δ cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of 14C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1Δtsa2Δ cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains. Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1Δtsa2Δ) are more resistant to hydrogen peroxide than wild-type (WT) cells and consume it faster under fermentative conditions. Also, tsa1Δtsa2Δ cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1Δtsa2Δ cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu,Zn-superoxide dismutase (Sod1), whose expression and activity increased ∼5- and 2-fold, respectively, in tsa1Δtsa2Δ compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1Δtsa2Δ cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of 14C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1Δtsa2Δ cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains. Living organisms are constantly exposed to oxygen- and nitrogen-derived reactive species that are produced by normal metabolic activity and in response to external stimuli. To protect themselves against the toxicity of these species, aerobic organisms have evolved a range of defense mechanisms (1Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 4th Ed. Oxford University Press, Oxford, UK2007: 79-186Google Scholar). Among these, a family of cysteine-based peroxidases, currently named peroxiredoxins, has attracted considerable attention because of its ubiquity and versatility. These enzymes have been shown to detoxify hydrogen peroxide, organic peroxides and peroxynitrite through oxidation of their reactive cysteine residues, which are recycled back by reducing equivalents provided by thioredoxin and other thiol-electron donors (reviewed in Refs. 2Hofmann B. Hecht H.-J. Flohé L. Biol. Chem. 2002; 383: 347-364Crossref PubMed Scopus (763) Google Scholar, 3Wood Z.A. Schroder E. Robin-Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar, 4Rhee S.G. Chae H.Z. Kim K. Free Radic. Biol. Med. 2005; 38: 1543-1552Crossref PubMed Scopus (1122) Google Scholar, 5Trujillo M. Ferrer-Sueta G. Thomson L. Flohé L. Radi R. Subcell Biochem. 2007; 44: 83-113Crossref PubMed Scopus (109) Google Scholar). In addition to detoxifying peroxides, specific peroxiredoxins have been shown to act as molecular chaperones (6Jang H.H. Lee K.O. Chi Y.H. Jung B.G. Park S.K. Park J.H. Lee J.R. Lee S.S. Moon J.C. Yun J.W. Choi Y.O. Kim W.Y. Kang J.S. Cheong G.-W. Yun D.-J. Rhee S.G. Cho M.J. Lee S.Y. Cell. 2004; 117: 625-635Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 7Moon J.C. Hah Y.S. Kim W.Y. Jung B.G. Jang H.H. Lee J.R. Kim S.Y. Lee Y.M. Jeon M.G. Kim C.W. Cho M.J. Lee S.Y. J. Biol. Chem. 2005; 280: 28775-28784Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) and to play roles in regulating hydrogen peroxide-mediated cell signaling events (3Wood Z.A. Schroder E. Robin-Harris J. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2085) Google Scholar, 8Woo H.A. Chae H.Z. Hwang S.C. Yang K.-S. Kang S.W. Kim K. Rhee S.G. Science. 2003; 300: 653-656Crossref PubMed Scopus (463) Google Scholar, 9Wood Z.A. Poole L.B. Karplus P.A. Science. 2003; 300: 650-653Crossref PubMed Scopus (1122) Google Scholar). Many organisms have multiple peroxiredoxins. Six peroxiredoxins have been identified in human cells, and five in the yeast Saccharomyces cerevisiae. Peroxiredoxin isoforms are distributed to different locations within the cell, and two of the yeast peroxiredoxins, Tsa1 and Tsa2, are cytosolic (10Park S.G. Cha M.K. Jeong W. Kim I.-H. J. Biol. Chem. 2000; 275: 5723-5732Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Tsa1 was the first peroxiredoxin identified in eukaryotes. It is expressed constitutively and corresponds to 0.7% of total soluble protein content in this species. In contrast, the levels of Tsa2 are very low under normal conditions, but are highly induced upon treatment of the yeast with peroxides (11Munhoz D.C. Netto L.E.S. J. Biol. Chem. 2004; 279: 35219-35227Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Monje-Casas F. Michan C. Pueyo C. Biochem. J. 2004; 383: 139-147Crossref PubMed Scopus (30) Google Scholar). Tsa1 and Tsa2 share 86% identity in their amino acid sequence, and both react with hydrogen peroxide with second order rate constants similar to those of hemoproteins, such as catalase (k ∼ 107 m–1 s–1) (13Ogusucu R. Rettori D. Munhoz D.C. Netto L.E.S. Augusto O. Free Radic. Biol. Med. 2007; 42: 326-334Crossref PubMed Scopus (155) Google Scholar). Deletion of the TSA1 gene increases the expression of Tsa2 and other peroxiredoxin isoforms (10Park S.G. Cha M.K. Jeong W. Kim I.-H. J. Biol. Chem. 2000; 275: 5723-5732Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14Wong C.-M. Zhou Y. Ng R.W.M. Kung H.-F. Jin D.-Y. J. Biol. Chem. 2002; 277: 5385-5394Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 15Demasi A.P. Pereira G.A. Netto L.E.S. FEBS J. 2006; 273: 805-816Crossref PubMed Scopus (57) Google Scholar), suggesting that these peroxidases have both redundant and non-redundant physiological functions. The deletion of one or all of the peroxiredoxin genes in S. cerevisiae is not lethal, probably because of the resulting increase in the levels of other antioxidant enzymes, such as catalase, cytochrome c peroxidase, and Sod1, that has been demonstrated by transcriptional and proteomic analysis (10Park S.G. Cha M.K. Jeong W. Kim I.-H. J. Biol. Chem. 2000; 275: 5723-5732Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 15Demasi A.P. Pereira G.A. Netto L.E.S. FEBS J. 2006; 273: 805-816Crossref PubMed Scopus (57) Google Scholar, 16Wong C.-M. Siu K.L. Jin D.Y. J. Biol. Chem. 2004; 279: 23207-23213Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). A problem arising from this compensatory response, however, is the mutator phenotype of the peroxiredoxin-null strains that display increased spontaneous mutation rates and accumulated gross chromosomal rearrangements (16Wong C.-M. Siu K.L. Jin D.Y. J. Biol. Chem. 2004; 279: 23207-23213Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 17Huang M. Rio A. Nicolas A. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11529-11534Crossref PubMed Scopus (226) Google Scholar, 18Ragu S. Faye G. Iraqui I. Masurel-Heneman A. Kolodner R.D. Huang M.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9747-9752Crossref PubMed Scopus (65) Google Scholar, 19Iraqui I. Faye G. Ragu S. Masurel-Heneman A. Kolodner R.D. Huang M.E. Cancer Res. 2008; 68: 1055-1063Crossref PubMed Scopus (23) Google Scholar). These consequences are particularly conspicuous in TSA1-null cells, suggesting a major contribution of Tsa1 to genome stability. Such protection has been attributed to the ability of Tsa1 to reduce the levels of reactive oxygen species that would otherwise oxidize DNA leading to mutations, chromosomal rearrangements and cell death. These genetic and lethality studies, however, did not provide mechanistic insights on how deletion of peroxiredoxin genes leads to DNA damage nor to its molecular nature (16Wong C.-M. Siu K.L. Jin D.Y. J. Biol. Chem. 2004; 279: 23207-23213Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 17Huang M. Rio A. Nicolas A. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11529-11534Crossref PubMed Scopus (226) Google Scholar, 18Ragu S. Faye G. Iraqui I. Masurel-Heneman A. Kolodner R.D. Huang M.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9747-9752Crossref PubMed Scopus (65) Google Scholar, 19Iraqui I. Faye G. Ragu S. Masurel-Heneman A. Kolodner R.D. Huang M.E. Cancer Res. 2008; 68: 1055-1063Crossref PubMed Scopus (23) Google Scholar). Here, contrary to the common notion, we show that deletion of both TSA1 and TSA2 genes in S. cerevisiae increases resistance to toxic concentrations of hydrogen peroxide. In parallel, hydrogen peroxide consumption and ethanol- and DNA-derived radical formation are increased mainly through higher expression of the enzyme Sod1. Materials—All chemicals were purchased from Sigma-Aldrich, Merck, or Fisher and were analytical grade or better. Yeast nitrogen base (20Sherman F. Methods Enzymol. 1991; 194: 3-19Crossref PubMed Scopus (2526) Google Scholar) was from Difco and media supplements (amino acids, adenine and uracil) were from Sigma-Aldrich or Synth. Desferrioxamine was purchased from Novartis. Bathocuproine disulfonic acid and [2-13C]ethanol were from Sigma-Aldrich. Hydrogen peroxide concentration was determined spectrophotometrically at 240 nm (∊240 = 43.6 m–1 cm–1) (21Claiborne A. Greenwald R.A. Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL1985: 283-284Google Scholar). All solutions were prepared with water purified in a Millipore Milli-Q system. All buffers were treated with Chelex-100 to remove trace amounts of metal ion contaminants prior to use. S. Cerevisiae Strains and Growth Conditions—The wild-type strain employed was BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), whereas the mutant strains were: tsa1Δ (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YML028W::KAN MX4), tsa2Δ (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YDR453C::KAN MX4), and tsa1Δ tsa2Δ (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; tsa1::KAN MX4; tsa2::LEU2). The first three strains were obtained from EUROSCARF, whereas the double mutant (generated according to Ref. 14Wong C.-M. Zhou Y. Ng R.W.M. Kung H.-F. Jin D.-Y. J. Biol. Chem. 2002; 277: 5385-5394Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) was kindly donated by Dr. Jin (University of Hong Kong, China University). Yeasts overexpressing Sod1 were obtained by standard transformation (22Rabizadeh S. Gralla E.B. Borchelt D.R. Gwinn R. Valentine J.S. Sisodia S. Wong P. Lee M. Hahn H. Bredesen D.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3024-3028Crossref PubMed Scopus (327) Google Scholar) of the BY47411 strain with the YEp600 plasmid containing one copy of the human sod1 gene with its own promoter. This plasmid was extracted from EGy118 yeasts provided by Dr. Edith B. Gralla. The strains were grown in complete synthetic media (yeast nitrogen base) supplemented with 2% glucose, amino acids, adenine, and uracil under shaking at 300 rpm, 30 °C (11Munhoz D.C. Netto L.E.S. J. Biol. Chem. 2004; 279: 35219-35227Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). For all experiments, strains were grown overnight, diluted to A600 nm = 0.2, and collected as soon as they reached mid-log phase (A600 nm = 0.8). Growth curves under fermentative conditions were obtained for the four strains by diluting overnight cultures to A600 nm = 0.2 and by monitoring the optical density at 2-h intervals. Viability Assay—The viability of the cells was monitored by the number of colony forming units. Cell cultures at mid-log phase of growth were washed with 0.1 m phosphate buffer containing 0.1 mm DTPA, 3The abbreviations used are: DTPA, diethylenetriamine-N,N,N,N-pentaacetic; FOX, ferrous oxidation xylenol orange assay; DMPO, 5,5-dimethylpyrroline-N-oxide; POBN, α-(4-pyridil-1-oxide)-N-tert-butylnitrone; Tsa1 and -2, cytosolic thioredoxin peroxidases 1 and 2; WT, wild type. pH 7.4, and resuspended in growth media to a density of 5 × 107 cells/ml. Half of the cell suspension was treated with 1 mm hydrogen peroxide under shaking at 350 rpm for 30 min at 30 °C; the other half was treated with the same volume of buffer and incubated as above. The cell suspensions were then serially diluted, and 100-μl aliquots of 1/104 and 1/105 dilution were plated onto complete synthetic media supplemented with 2% glucose and 2% agar. The plates were incubated at 30 °C for 2 days, and the colony forming unit was number counted. The results shown are expressed as the percentage of colony forming units of hydrogen peroxide-treated cultures in relation to the corresponding controls. Hydrogen Peroxide Consumption—Hydrogen peroxide consumption was determined by the ferrous oxidation xylenol orange assay (FOX) (23Wolff S.P. Methods Enzymol. 1994; 233: 182-189Crossref Scopus (1039) Google Scholar). Cells at mid-log phase were treated with 1 mm hydrogen peroxide as above. At the specified times, 50-μl aliquots were diluted with 500 μl of growth media and mixed with 950 μl of FOX solution (100 μm xylenol orange, 250 μm ammonium ferrous sulfate, 100 mm sorbitol, and 25 mm sulfuric acid). After 30-min incubation at room temperature, the absorbance of the samples was read at 560 nm. Calibration was performed with a standard solution of hydrogen peroxide. Detection of Ethanol-derived Radicals—Production of the 1-hydroxyethyl radical formed from the oxidation of ethanol, a glucose metabolite, was detected by EPR spin-trapping experiments with POBN (24Nakao L.S. Augusto O. (1998) Chem. Res. Toxicol. 1998; 11: 888-894Crossref PubMed Scopus (21) Google Scholar, 25Roe J.A. Wiedau-Pazos M. Moy V.N. Goto J.J. Gralla E.B. Valentine J.S. Free Radic. Biol. Med. 2002; 32: 169-174Crossref PubMed Scopus (29) Google Scholar). Cells (5 × 107 cells/ml) in growth media containing glucose were preincubated with 90 mm POBN for 5 min and 1 mm hydrogen peroxide was added under shaking at 30 °C. After 30 min, aliquots were transferred to flat cells, and the EPR spectra were scanned. In some experiments, cells were incubated with POBN as above, and 171 mm [2-13C]ethanol was added together with hydrogen peroxide. In other experiments, cells in growth media containing glucose were preincubated with 2 mm desferrioxamine, 2 mm bathocuproine disulfonic acid, or with 10 mm diethyldithiocarbamate for 30 min or 1 h before addition of the components specified above. Also, cells in media alone were heated at 90 °C for 20 min and brought to 30 °C before addition of glucose, POBN, and hydrogen peroxide. In the case of purified enzymes, 10 μm catalase, horseradish peroxidase, cytochrome c, or bovine Sod1 was incubated with 90 mm POBN, 1 mm hydrogen peroxide, 0.1 mm DTPA, and 171 mm ethanol in 100 mm phosphate buffer, pH 7.4, at 30 °C. After 30-min incubation, the samples were transferred to flat cells, and the EPR spectra were scanned. In the case of Sod1, kinetic experiments were also performed. EPR spectra were recorded at room temperature (25 ± 2 °C) on a Bruker EMX spectrometer equipped with an ER4122 SHQ 9807 high sensitivity cavity. Radical adduct quantification was performed by double integration of the EPR spectra and comparison with a standard solution of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy. Computer simulation analyses of some spectra were performed by using a program written by Duling (26Duling D.R. J. Magn. Res. 1994; 104: 105-110Crossref Scopus (885) Google Scholar). Chelatable Iron Ion Measurements—Chelatable iron levels in the strains were determined by low temperature EPR after chelation with desferrioxamine (27Srinivasan C. Liba A. Imlay J.A. Valentine J.S. Gralla E.B. J. Biol. Chem. 2000; 275: 29187-29192Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 28Linares E. Nakao L.S. Augusto O. Kadiiska M.B. Free Radic. Biol. Med. 2003; 34: 766-773Crossref PubMed Scopus (14) Google Scholar). WT and tsa1Δtsa2Δ cells were collected at mid-log growth phase. Approximately 109 cells of each strain were resuspended in 10 ml of fresh growth medium without glucose, but containing 2 mm desferrioxamine. After 30-min incubation at 30 °C, cells were collected and washed with 10 ml of cold Tris-HCl buffer (20 mm), pH 7.4. The cell pellet was resuspended in 400 μl of the same buffer containing 10% glycerol, transferred to a 1-ml disposable syringe, and frozen in liquid nitrogen. The samples were extruded from the syringe into a finger-tip Dewar flask containing liquid nitrogen and examined by EPR at 77 K in the region of g ∼4.0 (28Linares E. Nakao L.S. Augusto O. Kadiiska M.B. Free Radic. Biol. Med. 2003; 34: 766-773Crossref PubMed Scopus (14) Google Scholar). The concentration of the desferrioxamine-iron complex present in the cell suspensions was obtained by double integration of the EPR signal and comparison with a standard curve constructed with known concentrations of the iron(III)-desferrioxamine complex. This was prepared by mixing different concentrations of ferrous ammonium sulfate with 2 mm desferrioxamine; the complex concentration was determined spectrophotometrically (∊430 = 2865 m–1 cm–1) (29Yegorov D.Yu. Kozlov A.V. Azizova O.A. Vladimirov Y.A. Free Radic. Biol. Med. 1993; 15: 565-574Crossref PubMed Scopus (74) Google Scholar). Total Iron, Copper, and Zinc Ion Measurements—Levels of iron, copper, and zinc ions in each strain were determined by atomic absorption spectrometry. Approximately 109 cells from each strain were washed in metal-free water, resuspended in a solution of nitric acid, hydrogen peroxide, and water (2:1:3, v/v), and digested in a microwave oven. The metal contents of the digested samples were determined in an Analytikjena AG, AAS ZEEnit 60 instrument. Iron, copper, and zinc were detected at 324.8, 248.3, and 213.9 nm, respectively (30Naozuka J. Oliveira P.V. J. Braz. Chem. Soc. 2007; 18: 1547-1553Crossref Scopus (38) Google Scholar). Total Peroxide Measurements—Levels of total peroxides in WT and tsa1Δ tsa2Δ strains were determined by the FOX assay (23Wolff S.P. Methods Enzymol. 1994; 233: 182-189Crossref Scopus (1039) Google Scholar) as adapted to yeasts (31Salmon J.-M. Fornairon-Bonnefond C. Mazauric J.-P. Moutounet M. Food Chem. 2000; 71: 519-528Crossref Scopus (49) Google Scholar). Cells (5 × 107 cells/ml) of each strain were washed and resuspended in growth media and treated or not with 1 mm hydrogen peroxide. After 30-min incubation, the cells were washed twice and resuspended in 100 μl of 50 mm phosphate buffer. An equal volume of glass beads (425–600 μm) was added, and the samples were vortexed (1 min) and ice-cooled. After addition of 900 μl of cool methanol containing 4 mm butylated hydroxytoluene, the samples were submitted to two cycles of vortexing (1 min) and ice cooling. Next, the samples were centrifuged (1000 × g, 10 min). The supernatants were collected (100 μl) and mixed with 900 μl of the FOX reagent (100 μm xylenol orange, 250 μm ammonium ferrous sulfate, 25 mm sulfuric acid, and 4 mm butylated hydroxytoluene in 90% (v/v) methanol). After 30-min incubation at room temperature, the absorbance of the samples was read at 560 nm. According to previous calibrations performed with standard peroxides, a mean apparent extinction coefficient of 4.5 × 104 m–1 cm–1 was employed (31Salmon J.-M. Fornairon-Bonnefond C. Mazauric J.-P. Moutounet M. Food Chem. 2000; 71: 519-528Crossref Scopus (49) Google Scholar). Expression of Sod1—The level of Sod1 expression in WT and tsa1Δ tsa2Δ strains was determined by Western blot analysis. To this end, cell extracts of the strains were obtained as previously described (11Munhoz D.C. Netto L.E.S. J. Biol. Chem. 2004; 279: 35219-35227Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, cells collected at mid-log growth phase were washed and resuspended in 50 mm Hepes buffer containing 50 mm NaCl, 2 μg/ml leupeptin, and 1 μg/ml of pepstatin. Next, an equal volume of glass beads (425–600 μm) was added. After 2 cycles of vortexing (6 min) and ice-cooling (6 min), the samples were centrifuged (16,000 × g, 5 min). The supernatants were collected and centrifuged again (16,000 × g, 30 min) to remove precipitated material. The supernatants are referred to as cell extracts, and their protein contents were determined by the Bradford method with a Bio-Rad Kit. Cell extracts (40 μg of protein) were submitted to electrophoresis (12% acrylamide gel) and transferred onto a nitrocellulose membrane (Hybond-D Extra, Amersham Biosciences). After washing (3 times, 10 min) with TBS (50 mm Tris-HCl, pH 7.5, 150 mm NaCl), the membrane was blocked with TBST (TBS plus 0.05% Tween 20) containing 5% nonfat milk, at room temperature for 90 min. The primary antibody was anti-human Sod1 (Calbiochem) prepared in TBST containing 0.1% nonfat milk (1:1.500 dilution). Excess antibody was removed by washing three times with TBST. The membrane was then incubated with the secondary antibody (anti-sheep IgG, peroxidase conjugated, Calbiochem) prepared in TBST containing 0.1% nonfat milk (1:7,000 dilution). After 1-h incubation, the membrane was washed three times with TBST and treated for 5 min with the solutions from the kit SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposed to an x-ray film. Relative quantification of the bands was performed by densitometry (ImageQuaNT V5.2, Molecular Dynamics). Superoxide Dismutase Activity—Superoxide dismutase activity in WT and tsa1Δ tsa2Δ cell extracts was determined by native PAGE staining (32Beauchamp C. Fridovich I. Anal. Biochem. 1971; 44: 276-287Crossref PubMed Scopus (9524) Google Scholar) and inhibition of cytochrome c reduction (33McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar, 34Oberley L.W. Spitz D.R. Greenwald R.A. Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL1985: 217-220Google Scholar). After electrophoresis of cell extracts (40 μg of protein) under non-reducing conditions, the gel was incubated in the dark for 20 min with a solution composed of 0.25 mg/ml nitro blue tetrazolium plus 0.1 mg/ml riboflavin. Then, a solution of 10 mg/ml N,N,N′,N′-tetramethylethylenediamine was added, and the gel was kept under shaking and light until bands became visible (32Beauchamp C. Fridovich I. Anal. Biochem. 1971; 44: 276-287Crossref PubMed Scopus (9524) Google Scholar). Relative quantification of the bands was performed by densitometry (ImageQuaNT V5.2). The cytochrome c reduction assay was performed as previously described (33McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar, 34Oberley L.W. Spitz D.R. Greenwald R.A. Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL1985: 217-220Google Scholar). The incubations contained 100 μm xanthine and xanthine oxidase in amounts that cause an absorbance change of 0.025/min, 1 unit of catalase, and cell extracts (0–50 μg protein) in 100 mm phosphate buffer, pH 7.4, 30 °C. Bicarbonate-dependent Peroxidase Activity—This activity was evaluated in whole cells by EPR monitoring of the oxidation of the spin-trap 5,5-dimethylpyrroline-N-oxide (DMPO) to DMPO/·OH radical adduct was monitored (35Zhang H. Joseph J. Felix C. Kalyanaraman B. J. Biol. Chem. 2000; 275: 14038-14045Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 36Medinas D.B. Cerchiaro G. Trindade D.F. Augusto O. IUBMB Life. 2007; 59: 255-262Crossref PubMed Scopus (142) Google Scholar). Cells (5 × 107 cells/ml) of each strain were resuspended in 100 mm phosphate buffer containing 0.1 mm DTPA, pH 7.4, and incubated with 80 mm DMPO at room temperature for 5 min. After addition of 25 mm sodium bicarbonate and 1 mm hydrogen peroxide, the samples were incubated at 30 °C for 15 min and transferred to flat cells, and their EPR spectra were scanned at room temperature as described above. Quantification of radical adduct was performed by double integration of the EPR spectra and comparison with a standard solution of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy. Detection of DNA-derived Radicals—Formation of DNA-derived radicals was monitored by immuno-spin trapping employing DMPO and the antibody against oxidized DMPO adducts (37Ramirez D.C. Gomez-Mejiba S.E. Mason R.P. Nat. Protoc. 2007; 2: 512-522Crossref PubMed Scopus (48) Google Scholar). Cells (5 × 107 cells/ml) of each strain were washed, resuspended in fresh media, and treated with 100 mm DMPO and 1 mm hydrogen peroxide at 30 °C. After 30-min incubation, the cells were washed with distilled water (2 times) and resuspended in 800 μl of a buffer to digest cell walls (0.9 m sorbitol, 0.1 m EDTA, 50 mm dithiothreitol, and 3.5 μg/ml zymolyase 20T) (38Philippsen P. Stotz A. Scherf C. Methods Enzymol. 1991; 194: 169-182Crossref PubMed Scopus (268) Google Scholar). After 2-h incubation at 37 °C, samples were centrifuged (1,000 × g, 10 min), and the spheroplasts obtained were resuspended in lysis buffer (1% SDS, 100 mm NaCl, 25 mm DTPA in 10 mm Tris-HCl, pH 8.0). From this point on, DNA extraction and transfer onto nitrocellulose membrane followed the previously described protocol (37Ramirez D.C. Gomez-Mejiba S.E. Mason R.P. Nat. Protoc. 2007; 2: 512-522Crossref PubMed Scopus (48) Google Scholar). DNA extracted from WT cells (100 ng) treated with 50 mm DMPO, 1 mm CuCl2, and 20 μm hydrogen peroxide in phosphate buffer, pH 7.4, for 30 min at 30 °C was employed as a positive control. DNA radicals/DMPO nitrone adducts blotted onto the nitrocellulose membrane were detected with the anti-DMPO nitrone adduct antibody provided by Dr. Ronald P. Mason (37Ramirez D.C. Gomez-Mejiba S.E. Mason R.P. Nat. Protoc. 2007; 2: 512-522Crossref PubMed Scopus (48) Google Scholar). First, the membrane was blocked for 1 h with phosphate-buffered saline buffer (0.725 m NaCl, 2.7 mm KCl, 80 mm Na2HPO4 and 14 mm KH2PO4) containing 3% nonfat milk. After washing for 10 min with phosphate-buffered saline supplemented with 0.05% nonfat milk and 0.1% Tween 20, the membrane was incubated with the anti-DMPO/adduct antibody for 1 h. The antibody was prepared in phosphate-buffered saline supplemented with 0.05% nonfat milk and 0.1% Tween 20 (1:10,000 dilution). Excess antibody was removed by three 10-min washes with the same buffer, and the membrane was incubated 1 h with the secondary antibody (anti-rabbit IgG, peroxidase-conjugated). After one phosphate-buffered saline wash, the membrane was incubated with the solutions from the kit SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) for 5 min and exposed to an x-ray film. Relative quantification of the bands was performed by densitometry (ImageQuaNT V5.2). Detection of DNA Adducts—To probe for the addition of glucose metabolites to yeast DNA, we monitored incorporation of 14C at the DNA by the strains grown in media supplemented with [14C]glucose (Schwarz/Mann; 1 mCi/ml and 230 mCi/mm). Cells (2 × 108) harvested at the exponential growth phase were resuspended in 500 μl of fresh media containing four times less glucose than usual and supplemented with 50 μCi of [14C]glucose (39Sentandreu R. Northcote D.H. Biochem. J. 1969; 115: 231-240Crossref PubMed Scopus (28) Google Scholar). These samples were treated with 1 mm hydrogen peroxide for 30 min at 30 °C, in the absence or presence of POBN (90 mm). DNA from the samples was extracted as above. DNA (100 ng) from each experimental condition was transferred to filter paper that was incubated with scintillation liquid. Radioactivity was measured in a liquid scintillation analyzer (166 TR, Packard). tsa1Δtsa2Δ Cells Are More Resistant to Hydrogen Peroxide but Metabolize It Faster Producing Ethanol-derived Radicals—As previously reported (11Munhoz D.C. Netto L.E.S. J. Bi
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