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Mitochondrial GPx1 Decreases Induced but Not Basal Oxidative Damage to mtDNA in T47D Cells
Biochemical and Biophysical Research Communications 272, 416 – 422 (2000) doi:10.1006/bbrc.2000.2800, available online at http://www.idealibrary.com on
Mitochondrial GPx1 Decreases Induced but Not Basal Oxidative Damage to mtDNA in T47D Cells J. Legault,* C. Carrier,* P. Petrov,* P. Renard,† J. Remacle,† and M.-E. Mirault* ,1 *Unit of Health and Environment, CHUL Research Center and Laval University, Ste-Foy, Que´bec, Canada G1V 4G2; and †Laboratoire de Biochimie et Biologie Cellulaire, Faculte´s Universitaires Notre-Dame de la Paix, 5000 Namur, Belgium
Received May 1, 2000
The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., ⴙ14% 8-oxo-dG versus ⴙ54% in Hygro-3 after 1-h exposure to 25 M menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model. © 2000 Academic Press Key Words: mitochondria; glutathione peroxidase; immunodetection; electron microscopy; antioxidant; oxidative stress; mtDNA damage; 8-oxo-dG; postlabeling assay; DNA strand breaks.
Reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including normal aging and several pathological conditions (1– 4). The mitochondrial respiratory chain is one of the major sources of endogenous ROS, which encompass • O 2⫺, H 2O 2 and hydroxyl radicals (5–7). Mitochondrial ROS generation, which accounts for ⬃1–2% of oxygen 1 To whom correspondence should be addressed at Unite´ de sante´ et environnement, Centre Hospitalier de l’Universite´ Laval, 2705 boulevard Laurier, Sainte-Foy, Que´bec, Canada G1V 4G2. Fax: (418) 654-2159. E-mail: [email protected].
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
consumption under normal physiological conditions, may be stimulated by inhibition of mitochondrial electron transport and impairment of oxidative phosphorylation (8). Accumulation of mtDNA mutations/ deletions resulting from mitochondrial diseases and aging was associated with mitochondrial ROS production (8 –12). Although the mechanism(s) inducing mtDNA deletions is (are) not known, recent data suggest that oxidative damage to mtDNA may contribute to increase their frequency (13), notably via formation of highly mutagenic 8-oxo-7,8-dihydro-2⬘-deoxyguanosine (8-oxo-dG) (14). 8-Oxo-dG is the major base lesion resulting from hydroxyl radical or singlet oxygen DNA attack (15–18). The fact that hydroxyl radicals and singlet oxygen can be formed, respectively, by H 2O 2 metabolism via iron-catalyzed Fenton-type reactions (18, 19), and as by-products of lipid peroxidation (20, 21), points to the potential importance of the peroxidescavenging capacity in mitochondria. The H 2O 2 produced in mitochondria by manganese superoxide dismutase-mediated dismutation of •O 2⫺ can be reduced by two distinct antioxidant pathways: a glutathione (GSH) system composed of GSH peroxidase-1 (GPx1) and GSH reductase (22, 23), and thioredoxincycle enzymes including mitochondrial peroxiredoxins and thioredoxin reductases recently characterized in several species (24 –26). Since GSH appears to be the major cellular antioxidant required to maintain the mitochondrial functions and protect against toxicity of basal endogenous ROS formation (27, 28), we have investigated the cellular distribution of GPx1 in mitochondria and the effect of boosting mitochondrial GPx1 activity on 8-oxo-dG formation in mtDNA, using well characterized GPx1-deficient or -proficient T47D cell transfectants (29 –31). Here we provide evidence that the mitochondrial glutathione/GPx1 system plays a major role in preserving mtDNA from oxidative damage induced by prooxidant conditions, but not from basal oxidative damage, which appears independent of GPx1 status in the T47D model.
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MATERIALS AND METHODS Cell culture and treatments. Human breast carcinoma T47D cells and transfectants were grown in RPMI 1640 medium as previously described (29). The transfectant derivatives T47D-Hygro-3 (Hygro-3) and T47D-GPx-2 (GPx-2) have been described elsewhere (29). Human WI38-VA13 (VA13) cells derived from SV40-transformed normal lung fibroblasts were obtained from the ATCC (Rockville, MD) and grown in MEM containing 10% fetal bovine serum (Gibco BRL, Grand Island, NY) supplemented as the RPMI 1640 medium. Menadione treatments: Hygro-3 and GPx-2 cells were incubated 1 h at 37°C in growth medium containing menadione (Sigma, St. Louis, MO) at the concentration indicated. Then, the cells were washed twice with cold PBS before cell lysis and mtDNA isolation. Cell fractionation. Hygro-3, GPx-2, and VA13 cells were lysed with a Dounce homogenizer and fractionated by differential centrifugation as previously described (32, 33) to yield four fractions: nuclear, mitochondria-lysosomes, peroxysomes and cytoplasm. Mitochondria were further separated from lysosomes by density centrifugation. All manipulations were carried out at 4°C. GPx activity assay. GPx activity was measured as described by Gu¨nzler and Flohe´ (34) and Mbemba et al. (33), using tertbutylhydroperoxide as substrate. Triton X-100, was added to the mitochondrial fractions to a final concentration of 0.5% to lyse these organelles. Protein contents were determined by the Lowry method. All fractions were stored at ⫺80°C. Ultrastructural immunocytochemistry. The cells were collected and centrifuged at 800g for 10 min. The cell pellets were fixed in 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate at 4°C for 25 min. After dehydration with 100% ethanol, the pellets were coated with LOWICRYL-K4M (Chemische Werke Lowi, Germany). Polymerization was carried out as described by Valentino et al. (35). Ultra-fine slices were laid on nickel grids covered with Formvar–Carbon. Immunocytochemistry was carried out by incubation of ultra-fine slices for 2 h at RT with affinity-purified GPx1 antibody raised against recombinant human GPx1, or non-immune antibody or GPx1-adsorbed GPx1 antibody for controls, as previously described (29, 36). The slices were then incubated for 1 h at RT with anti-rabbit IgG conjugated with 10-nm colloidal gold. Ultrastructure and immunostaining was visualized in an electron microscope CX1200 JEOL at 60 kV. mtDNA isolation and analysis. About 80% confluent cells in 2 petri dishes (100 mm) were collected in 10 mM Tris–HCl, pH 7.4, 120 mM NaCl, 50 mM EDTA and 10 M deferoxamine mesylate (Sigma, St. Louis, MO) with a rubber policeman and transferred into one ice-cold 1.5 ml microtube. The cells were centrifuged at 1000g for 1 min at 4°C and resuspended in 360 l of cold mtDNA isolation buffer. To each sample, 40 l of cold Triton X-100 10% were added before gentle shaking for 1 min on ice and 2 min centrifugation at 3000g at 4°C. The supernatant was recentrifuged in the same conditions to remove all nuclei. The mitochondria were lysed by addition of 100 l of 5⫻ lysis buffer containing 5% SDS, 50 M deferoxamine mesylate and proteinase K (2.5 mg/ml, Gibco-BRL) for 2 h at 37°C. Mitochondrial DNA was then extracted with 1 vol phenol/chloroform followed by chloroform extraction, and quantitatively precipitated by addition of 1/10 volume of 5 M NaCl and 2.5 vol cold absolute ethanol, and stored 30 min at ⫺70°C. The DNA was collected by centrifugation at 13,000g for 15 min at 4°C and resuspended in 60 l TE (10 mM Tris, 1 mM EDTA, pH 7.4) after washing with 70% ethanol. RNA was removed by incubation with RNase I (2.5 U/l) in 20 l 100 mM Tris–HCl, pH 7.5, 200 mM EDTA for 60 min at 37°C. The mtDNA was phenol extracted once, ethanol precipitated as described above and resuspended in 25 l TE. The DNA concentration was determined by fluorometry using Hoechst 33258 and a TKO-100 DNA fluorometer according to the manufacturer’s instructions (Hoefer Scientific Instruments, Mississauga, Ontario, Canada). The DNA
was stored at ⫺80°C. mtDNA samples (100 ng) were analyzed by field inversion gel electrophoresis (FIGE) according to the instructions of Hoefer Scientific Instruments, using 20-cm-long 0.6% agarose gels in TBE buffer, electric field of 6.9 V/cm, 28 h run time, 1 to 50-s pulse time and F/R ratio 3.0:1 (switchback pulse controller). The DNA was visualized by ethidium bromide staining. Quantification of 8-oxo-dG by 32P-postlabeling. 32P-postlabeling was carried out as described by Devanaboyina and Gupta (37). Briefly, 200 ng mtDNA was digested in 20 l 10 mM sodium succinate, pH 6.0, 5 mM CaCl 2, 2 ng/l micrococcal nuclease and 2 ng/l spleen phosphodiesterase at 37°C for 5 h. To 3 l aliquot of mtDNA digest product (30 ng) were added 7 l of hot kinase mix containing 30 mM Tris–HCl, pH 9.5, 10 mM MgCl 2, 10 mM DTT, 1 mM spermidine, 200 pmol nonradioactive ATP, 16.5 pmol [␥- 32P]ATP and 2 units T4 polynucleotide kinase for 45 min at RT. To the labeled digest were added 15 l containing 83 mM sodium acetate, pH 5, 167 M ZnCl 2 and 167 ng/l nuclease P1 for 1 h at RT. A 20 l aliquot of mtDNA digest (24 ng) was applied to the origin of a PEI-cellulose (20 ⫻ 20). The 32P-8-oxo-dG spots were quantitated with a Packard Instant Imager. The number of 8-oxo-dG residues was calculated from the specific activity of the radioactive nucleotide in the kinase incubation mixture and the radioactivity (cpm) of the 32P-8-oxo-dG spot. The specific activity of the radioactive nucleotide (cpm/mmol) was determined by scintillation counting (10 l of a 1:100 dilution of commercial [␥- 32P]dATP (3000 Ci/mmol). Reference 8-oxo-dG was generously provided by Dr. R. Wagner of Sherbrooke University. The artifactual formation of 8-oxo-dG during the 32P-postlabeling protocol was assessed by increasing incubation times and length of phenol/chloroform treatments. Lysis and digestion of mitochondria was for 2, 4 or 6 h at 37°C; phenol/chloroform extractions for 10, 20, or 30 min at RT; RNase I treatments for 1, 2, or 3 h at 37°C, and micrococcal nuclease and spleen phosphodiesterase digestions for 5, 10, or 15 h at 37°C. All other steps were carried out as described above.
RESULTS AND DISCUSSION Intracellular Distribution of GPx Activity The intracellular distribution of GPx activity was determined by cell fractionation of T47D-GPx-2 (GPx-2) transfectant cells, which express a high level of GPx1 in contrast to parental cells or control transfectant T47D-Hygro-3 (Hygro-3) (29). WI38-VA13 (VA13) fibroblast cells were used as untransfected cells expressing GPx1 at normal level. Total GPx activities measured in Hygro-3, GPx-2 and VA13 were respectively of 0.7 ⫾ 0.2, 75 ⫾ 13 and 36 ⫾ 6 mU/mg protein (Fig. 1). About 78% of GPx activity was found in the cytoplasm of GPx-2 cells (data not shown). A similar distribution was observed in different cell types, e.g., rat liver (38). Average GPx activity in the mitochondria-lysosomes fractions, measured in the presence of Triton X-100 detergent to lyse these organelles, was 16% and 14% (no significant difference) of total GPx activity in GPx-2 and VA13 cells, respectively (Fig. 1). Measurements of GPx activity in corresponding mitochondrial fractions from Hygro-3, at the limit of detection, were not reliable enough to assess accurately the relative distribution of GPx1 in these cells (at least 6% in mitochondria). Importantly, however, GPx activity in this fraction was at least 100-fold lower than in GPx-2. When assayed without Triton
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FIG. 1. GPx activity in mitochondria of T47D-Hygro-3, -GPx-2, and WI38-VA13 cells. The cells were homogenized and fractionated by differential centrifugation to produce a mitochondrial fraction. GPx activity was assayed with tert-butylhydroperoxide in both total homogenates and mitochondrial fractions treated with 0.5% Triton X-100 to release GPx activity from mitochondria. The data are means ⫾ SD of 4 different experiments.
X-100 detergent, the mitochondrial-lysosomal GPx activities were about 3.5-fold lower than in the presence of detergent (not shown) (39). This last result suggests that most GPx activity resided inside the mitochondria or lysosomes. The GPx activity fully overlapped the cytochrome C oxidase activity pattern of mitochondria but not the N-acetyl-glucoaminidase activity pattern of lysosomes following further fractionation by sucrose gradient density centrifugation (39). No glucose6-phosphate dehydrogenase activity, a cytoplasmic marker, was detected in the mitochondria-lysosomes fraction, therefore excluding a contamination by cytoplasmic GPx activity (data not shown). With respect to GPx1 import in mitochondria, it is of interest that a remarkably similar partition between mitochondria and cytoplasm was observed in GPx-2 and untransfected VA13 cells, indicating that vector-derived recombinant GPx1 was translocated efficiently into mitochondria. In contrast to phospholipid-hydroperoxide GPx, known to use alternative transcription and translation start sites to determine cytoplasm or mitochondrial localization in different tissues (40), no such pattern of differential expression was reported for GPx1, nor did we find any evidence for a mitochondrial presequence in GPx1. Thus, GPx1 appears to be imported by an unknown mechanism, which does not involve the classic presequence cleavage pathway described for protein import in mitochondria (41, 42). Ultrastructural Localization of GPx1 To confirm the preferential localization of GPx1 in mitochondria, and compare the relative contents of mitochondrial GPx1 in Hygro-3 and GPx-2 cells, the
ultrastructural distribution of GPx1 was investigated by immuno-electron microscopy. Immuno-staining was performed with an immunoaffinity-purified GPx1 antibody (29, 36) and colloidal gold-conjugated secondary antibody, as described under Materials and Methods. Figure 2A shows relatively intense gold staining in GPx-2 mitochondria (b, c), in contrast to barely detectable staining in Hygro-3 cells (a). These observations are in excellent agreement with the GPx activity distribution determined by cell fractionation (see above). Figure 2B shows that, after subtraction of the background obtained with adsorbed antibody, the colloidalgold grain density per surface was about 8-fold higher in mitochondria than in the cytoplasm of GPx-2 cells. Conversion of this 2-dimensional factor 8 (per surface) to a 3-dimensional factor (per volume, i.e., 8 3/2) indicate a 20- to 25-fold higher concentration of GPx1-like antigen in the mitochondria. Importantly, comparison of the mitochondrial grain density in the two cell lines also yields an ⬃8-fold higher density in GPx-2 (Fig. 2B), thus indicating that the apparent mitochondrial concentration of GPx1 in these cells was 20 to 25-fold higher than in Hygro-3 mitochondria. Compared to the corresponding higher difference in GPx activity (more than 100-fold), the apparent difference in GPx1 protein level obtained by immuno-detection is most likely underestimated. Although the immuno-staining observed in Hygro-3 mitochondria was very low, any possible cross-reaction of the antibody to non-GPx1 proteins including, for example, PHGPx would contribute to increase non-specific immuno-staining signal in Hygro-3, despite the very good specificity of the affinity-purified antibody (29, 36). Effect of GPx1 Expression on Basal Levels of 8-Oxo-dG in mtDNA The frequency of 8-oxo-dG lesions was assessed as an index of oxidative damage to mtDNA. To this end, we used the 32P-postlabeling assay described by Devanaboyina and Gupta (37). The major advantage of this method is that it requires very little DNA for detection of 8-oxo-dG. However, this assay involves a series of incubations at 37°C, which may introduce 8-oxo-dG lesions artifactually. Indeed, the suggestion that the quantification of 8-oxo-dG is prone to artifactual oxidation during DNA isolation and analysis, was recently discussed in the context of the current controversy on the relative sensitivity of mtDNA to oxidative damage (43). Therefore, the artifactual formation of 8-oxo-dG during the overall postlabeling assay was assessed by measuring the time-dependent increase in 8-oxo-dG content resulting from doubling and tripling the enzymatic incubation periods at 37°C as well as phenol/chloroform extraction time used in the original protocol. Figure 3 shows that the level of 8-oxo-dG determined in mtDNA prepared from normally grow-
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FIG. 2. Ultrastructural localization of GPx1-like antigen in Hygro-3 and GPx-2 cells. The cells were fixed and coated as described under Materials and Methods. Immunocytochemistry was carried out with an immuno-affinity-purified GPx1 primary antibody and secondary anti-rabbit IgG conjugated with 10-nm colloidal gold, visualized by electron microscopy (A). (a) Hygro-3 cells (scale bar, 0.5 m); (b) GPx-2 cells (scale bar, 0.5 m); (c) GPx-2 cells (scale bar, 0.2 m). The arrows point to mitochondrial immunostaining. (B) Histogram of colloidal gold grain counts over mitochondria and cytoplasm area, after subtraction of background determined with GPx1-adsorbed primary antibody. Means ⫾ SD, P ⬍ 0.05 for the differences between mitochondria and corresponding cytoplasm, and between Hygro-3 and GPx-2 mitochondria; P ⬍ 0.05 for the differences between signal and background of Hygro-3 mitochondria and GPx-2 cytoplasm; P ⬍ 0.001 for the differences between signal and background for GPx-2 mitochondria (Student’s t tests).
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FIG. 3. Basal 8-oxo-dG levels in mtDNA and artifactual formation during incubations inherent to the postlabeling procedure. Mitochondrial DNA was isolated from normally growing Hygro-3 and GPx-2 cells. 8-Oxo-dG contents were determined by 32P-postlabeling assay (see Materials and Methods) and related to the total period of enzymatic incubation and phenol/chloroform extraction required in the original postlabeling protocol (490 min), which was either doubled (980 min) or tripled (1470 min) for test. The graph displays the linear regression of 8-oxo-dG formation in function of total incubation time (r 2 ⫽ 0.99).
tive in terms of mtDNA strand breaks since a 200 M menadione treatment was required to induce a significant relaxation of supercoiled mtDNA. Thus, the relative oxidoresistance of mtDNA in GPx-2 cells was not only evident at the level of oxidative base damage, but could also be demonstrated at the level of DNA strand breaks. The data would be consistent with at least two distinct sources of ROS mediating oxidative damage to mtDNA: (i) H 2O 2 (GPx1-sensitive)-derived ROS, e.g., hydroxyl radicals derived from Fenton-type reactions produced during stimulation of cyanide-insensitive respiration (e.g., menadione treatment), and (ii) nonhydroperoxide-derived ROS, possibly hydroxyl radicals derived from (GPx1-insensitive) reactions between •O 2⫺ and NO • that would be responsible for most basal oxidative damage detected in both GPx-deficient and
ing (unstressed) Hygro-3 and GPx-2 cells, increased in direct relation to overall incubation time (r 2 ⫽ 0.99), and very similarly for both cell lines. Interpolation of both curves to 0-incubation time indicated no significant difference in basal frequency of 8-oxo-dG lesions, i.e., regardless of GPx1 level. Oxidative Stress-Induced Damage to mtDNA Is Reduced in GPx1-Proficient Cells Oxidative stress was induced in Hygro-3 and GPx-2 cells by incubating the cells in the presence of 25 or 50 M menadione for 1 h. Induced formation of 8-oxo-dG in mtDNA was then determined as described above, after subtraction of the basal levels measured in unexposed control cells. Figure 4A shows that the induction of 8-oxo-dG lesions in mtDNA was menadione dosedependent, and, importantly, was much reduced in the GPx1-proficient cells (GPx-2), i.e., by respectively ⫺74 ⫾ 8% and ⫺53 ⫾ 6% after 1-h exposure to 25 and 50 M menadione. To confirm these results, DNA strand breakage was also assessed in mtDNA from these cells exposed 1 h to menadione, but using higher menadione concentrations (100 and 200 M) to permit detection of strand breaks revealed by relaxation of supercoiled mtDNA. Figure 4B shows similar levels of supercoiled and relaxed mtDNA in unexposed control Hygro-3 and GPx-2 cells. Menadione exposure induced a dose-dependent relaxation of supercoiled mtDNA in the GPx1-deficient Hygro-3 cells, with a clear effect already seen at 100 M menadione. In contrast, GPx-2 cells are less sensi-
FIG. 4. Decreased formation of 8-oxo-dG lesions and mtDNA strand breaks in GPx-2 cells exposed to oxidative stress. (A) Hygro-3 and GPx-2 cells were incubated or not (controls) with 25 or 50 M menadione for 1 h at 37°C. mtDNA was isolated and 8-oxo-dG content determined by 32P-postlabeling assay. The 8-oxo-dG backgrounds of control cells were subtracted from the 8-oxo-dG determinations of menadione-treated cells. Means ⫾ SD of three determinations, P ⬍ 0.05, for the differences between Hygro-3 and GPx-2 (Student’s t test). (B) Hygro-3 and GPx-2 cells were incubated or not (controls) with 100 or 200 M menadione at 37°C for 1 h. Mitochondrial DNA was isolated, analyzed by FIGE in 0.6% agarose gel and visualized by ethidium bromide staining.
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GPx-proficient cells. The observation that the parental T47D cells and Hygro-3 derivative grow normally in the quasi absence of GPx1 activity suggests that other mitochondrial peroxidases may compensate for the lack of GPx1 activity. These peroxidases may include a mitochondria-targeted phospholipid hydroperoxide glutathione peroxidase (44) and a mitochondrial thioredoxin peroxidase such as Prx III (24). On the other hand, we found no significant alteration in catalase, CuZnSOD (SOD1) and MnSOD (SOD2) expression or activity in GPx-2, compared to Hygro-3 or T47D (29) (Bilodeau and Mirault, unpublished). The results presented here suggest that the mitochondrial glutathione/GPx1 system may be of particular importance in conditions of oxidative stress resulting from excess • O 2⫺/H 2O 2 production. Future studies will be required to evaluate the respective role of each mitochondrial peroxidase system in different cell types, in both unstressed cells and cells challenged by various oxidant stresses.
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ACKNOWLEDGMENTS The work was supported by the National Cancer Institute of Canada with funds provided by the Canadian Cancer Society. J.L. was recipient of studentships from the Natural Sciences and Engineering Research Council of Canada and from the FCAR. This research was also supported by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. P.R. is a postdoctoral researcher of the Fonds National de la Recherche Scientifique, Belgium.
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36. Tre´panier, G., Furling, D., Puymirat, J., and Mirault, M.-E. (1996) Immunocytochemical localization of seleno-glutathione peroxidase in the adult mouse brain. Neuroscience 75, 231–243. 37. Devanaboyina, U., and Gupta, R. C. (1996) Sensitive detection of 8-hydroxy-2⬘deoxyguanosine in DNA by 32P-postlabeling assay and the basal levels in rat tissues. Carcinogenesis 17, 917–924. 38. Zakowski, J. J., and Tappel, A. L. (1978) Purification and properties of rat liver mitochondrial glutathione peroxidase. Biochim. Biophys. Acta 526, 65–76. 39. Carrier, C. (1995) in Department of Medicine, pp. 1–111. Laval University, Que´bec City. 40. Pushpa-Rekha, T. R., Burdsall, A. L., Oleksa, L. M., Chisolm, G. M., and Driscoll, D. M. (1995) Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites. J. Biol. Chem. 270, 26993–26999. 41. Hachiya, N., Komiya, T., Alam, R., Iwahashi, J., Sakaguchi, M., Omura, T., and Mihara, K. (1994) MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria. EMBO J. 13, 5146 –5154. 42. Neupert, W. (1997) Protein import into mitochondria. Annu. Rev. Biochem. 66, 863–917. 43. Beckman, K. B., and Ames, B. N. (1999) Endogenous oxidative damage of mtDNA. Mutat. Res. 424, 51–58. 44. Arai, M., Imai, H., Koumura, T., Yoshida, M., Emoto, K., Umeda, M., Chiba, N., and Nakagawa, Y. (1999) Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J. Biol. Chem. 274, 4924 – 4933.
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Mitochondrial GPx1 Decreases Induced but Not Basal Oxidative Damage to mtDNA in T47D Cells J. Legault,* C. Carrier,* P. Petrov,* P. Renard,† J. Remacle,† and M.-E. Mirault* ,1 *Unit of Health and Environment, CHUL Research Center and Laval University, Ste-Foy, Que´bec, Canada G1V 4G2; and †Laboratoire de Biochimie et Biologie Cellulaire, Faculte´s Universitaires Notre-Dame de la Paix, 5000 Namur, Belgium
Received May 1, 2000
The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., ⴙ14% 8-oxo-dG versus ⴙ54% in Hygro-3 after 1-h exposure to 25 M menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model. © 2000 Academic Press Key Words: mitochondria; glutathione peroxidase; immunodetection; electron microscopy; antioxidant; oxidative stress; mtDNA damage; 8-oxo-dG; postlabeling assay; DNA strand breaks.
Reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including normal aging and several pathological conditions (1– 4). The mitochondrial respiratory chain is one of the major sources of endogenous ROS, which encompass • O 2⫺, H 2O 2 and hydroxyl radicals (5–7). Mitochondrial ROS generation, which accounts for ⬃1–2% of oxygen 1 To whom correspondence should be addressed at Unite´ de sante´ et environnement, Centre Hospitalier de l’Universite´ Laval, 2705 boulevard Laurier, Sainte-Foy, Que´bec, Canada G1V 4G2. Fax: (418) 654-2159. E-mail: [email protected].
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
consumption under normal physiological conditions, may be stimulated by inhibition of mitochondrial electron transport and impairment of oxidative phosphorylation (8). Accumulation of mtDNA mutations/ deletions resulting from mitochondrial diseases and aging was associated with mitochondrial ROS production (8 –12). Although the mechanism(s) inducing mtDNA deletions is (are) not known, recent data suggest that oxidative damage to mtDNA may contribute to increase their frequency (13), notably via formation of highly mutagenic 8-oxo-7,8-dihydro-2⬘-deoxyguanosine (8-oxo-dG) (14). 8-Oxo-dG is the major base lesion resulting from hydroxyl radical or singlet oxygen DNA attack (15–18). The fact that hydroxyl radicals and singlet oxygen can be formed, respectively, by H 2O 2 metabolism via iron-catalyzed Fenton-type reactions (18, 19), and as by-products of lipid peroxidation (20, 21), points to the potential importance of the peroxidescavenging capacity in mitochondria. The H 2O 2 produced in mitochondria by manganese superoxide dismutase-mediated dismutation of •O 2⫺ can be reduced by two distinct antioxidant pathways: a glutathione (GSH) system composed of GSH peroxidase-1 (GPx1) and GSH reductase (22, 23), and thioredoxincycle enzymes including mitochondrial peroxiredoxins and thioredoxin reductases recently characterized in several species (24 –26). Since GSH appears to be the major cellular antioxidant required to maintain the mitochondrial functions and protect against toxicity of basal endogenous ROS formation (27, 28), we have investigated the cellular distribution of GPx1 in mitochondria and the effect of boosting mitochondrial GPx1 activity on 8-oxo-dG formation in mtDNA, using well characterized GPx1-deficient or -proficient T47D cell transfectants (29 –31). Here we provide evidence that the mitochondrial glutathione/GPx1 system plays a major role in preserving mtDNA from oxidative damage induced by prooxidant conditions, but not from basal oxidative damage, which appears independent of GPx1 status in the T47D model.
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MATERIALS AND METHODS Cell culture and treatments. Human breast carcinoma T47D cells and transfectants were grown in RPMI 1640 medium as previously described (29). The transfectant derivatives T47D-Hygro-3 (Hygro-3) and T47D-GPx-2 (GPx-2) have been described elsewhere (29). Human WI38-VA13 (VA13) cells derived from SV40-transformed normal lung fibroblasts were obtained from the ATCC (Rockville, MD) and grown in MEM containing 10% fetal bovine serum (Gibco BRL, Grand Island, NY) supplemented as the RPMI 1640 medium. Menadione treatments: Hygro-3 and GPx-2 cells were incubated 1 h at 37°C in growth medium containing menadione (Sigma, St. Louis, MO) at the concentration indicated. Then, the cells were washed twice with cold PBS before cell lysis and mtDNA isolation. Cell fractionation. Hygro-3, GPx-2, and VA13 cells were lysed with a Dounce homogenizer and fractionated by differential centrifugation as previously described (32, 33) to yield four fractions: nuclear, mitochondria-lysosomes, peroxysomes and cytoplasm. Mitochondria were further separated from lysosomes by density centrifugation. All manipulations were carried out at 4°C. GPx activity assay. GPx activity was measured as described by Gu¨nzler and Flohe´ (34) and Mbemba et al. (33), using tertbutylhydroperoxide as substrate. Triton X-100, was added to the mitochondrial fractions to a final concentration of 0.5% to lyse these organelles. Protein contents were determined by the Lowry method. All fractions were stored at ⫺80°C. Ultrastructural immunocytochemistry. The cells were collected and centrifuged at 800g for 10 min. The cell pellets were fixed in 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate at 4°C for 25 min. After dehydration with 100% ethanol, the pellets were coated with LOWICRYL-K4M (Chemische Werke Lowi, Germany). Polymerization was carried out as described by Valentino et al. (35). Ultra-fine slices were laid on nickel grids covered with Formvar–Carbon. Immunocytochemistry was carried out by incubation of ultra-fine slices for 2 h at RT with affinity-purified GPx1 antibody raised against recombinant human GPx1, or non-immune antibody or GPx1-adsorbed GPx1 antibody for controls, as previously described (29, 36). The slices were then incubated for 1 h at RT with anti-rabbit IgG conjugated with 10-nm colloidal gold. Ultrastructure and immunostaining was visualized in an electron microscope CX1200 JEOL at 60 kV. mtDNA isolation and analysis. About 80% confluent cells in 2 petri dishes (100 mm) were collected in 10 mM Tris–HCl, pH 7.4, 120 mM NaCl, 50 mM EDTA and 10 M deferoxamine mesylate (Sigma, St. Louis, MO) with a rubber policeman and transferred into one ice-cold 1.5 ml microtube. The cells were centrifuged at 1000g for 1 min at 4°C and resuspended in 360 l of cold mtDNA isolation buffer. To each sample, 40 l of cold Triton X-100 10% were added before gentle shaking for 1 min on ice and 2 min centrifugation at 3000g at 4°C. The supernatant was recentrifuged in the same conditions to remove all nuclei. The mitochondria were lysed by addition of 100 l of 5⫻ lysis buffer containing 5% SDS, 50 M deferoxamine mesylate and proteinase K (2.5 mg/ml, Gibco-BRL) for 2 h at 37°C. Mitochondrial DNA was then extracted with 1 vol phenol/chloroform followed by chloroform extraction, and quantitatively precipitated by addition of 1/10 volume of 5 M NaCl and 2.5 vol cold absolute ethanol, and stored 30 min at ⫺70°C. The DNA was collected by centrifugation at 13,000g for 15 min at 4°C and resuspended in 60 l TE (10 mM Tris, 1 mM EDTA, pH 7.4) after washing with 70% ethanol. RNA was removed by incubation with RNase I (2.5 U/l) in 20 l 100 mM Tris–HCl, pH 7.5, 200 mM EDTA for 60 min at 37°C. The mtDNA was phenol extracted once, ethanol precipitated as described above and resuspended in 25 l TE. The DNA concentration was determined by fluorometry using Hoechst 33258 and a TKO-100 DNA fluorometer according to the manufacturer’s instructions (Hoefer Scientific Instruments, Mississauga, Ontario, Canada). The DNA
was stored at ⫺80°C. mtDNA samples (100 ng) were analyzed by field inversion gel electrophoresis (FIGE) according to the instructions of Hoefer Scientific Instruments, using 20-cm-long 0.6% agarose gels in TBE buffer, electric field of 6.9 V/cm, 28 h run time, 1 to 50-s pulse time and F/R ratio 3.0:1 (switchback pulse controller). The DNA was visualized by ethidium bromide staining. Quantification of 8-oxo-dG by 32P-postlabeling. 32P-postlabeling was carried out as described by Devanaboyina and Gupta (37). Briefly, 200 ng mtDNA was digested in 20 l 10 mM sodium succinate, pH 6.0, 5 mM CaCl 2, 2 ng/l micrococcal nuclease and 2 ng/l spleen phosphodiesterase at 37°C for 5 h. To 3 l aliquot of mtDNA digest product (30 ng) were added 7 l of hot kinase mix containing 30 mM Tris–HCl, pH 9.5, 10 mM MgCl 2, 10 mM DTT, 1 mM spermidine, 200 pmol nonradioactive ATP, 16.5 pmol [␥- 32P]ATP and 2 units T4 polynucleotide kinase for 45 min at RT. To the labeled digest were added 15 l containing 83 mM sodium acetate, pH 5, 167 M ZnCl 2 and 167 ng/l nuclease P1 for 1 h at RT. A 20 l aliquot of mtDNA digest (24 ng) was applied to the origin of a PEI-cellulose (20 ⫻ 20). The 32P-8-oxo-dG spots were quantitated with a Packard Instant Imager. The number of 8-oxo-dG residues was calculated from the specific activity of the radioactive nucleotide in the kinase incubation mixture and the radioactivity (cpm) of the 32P-8-oxo-dG spot. The specific activity of the radioactive nucleotide (cpm/mmol) was determined by scintillation counting (10 l of a 1:100 dilution of commercial [␥- 32P]dATP (3000 Ci/mmol). Reference 8-oxo-dG was generously provided by Dr. R. Wagner of Sherbrooke University. The artifactual formation of 8-oxo-dG during the 32P-postlabeling protocol was assessed by increasing incubation times and length of phenol/chloroform treatments. Lysis and digestion of mitochondria was for 2, 4 or 6 h at 37°C; phenol/chloroform extractions for 10, 20, or 30 min at RT; RNase I treatments for 1, 2, or 3 h at 37°C, and micrococcal nuclease and spleen phosphodiesterase digestions for 5, 10, or 15 h at 37°C. All other steps were carried out as described above.
RESULTS AND DISCUSSION Intracellular Distribution of GPx Activity The intracellular distribution of GPx activity was determined by cell fractionation of T47D-GPx-2 (GPx-2) transfectant cells, which express a high level of GPx1 in contrast to parental cells or control transfectant T47D-Hygro-3 (Hygro-3) (29). WI38-VA13 (VA13) fibroblast cells were used as untransfected cells expressing GPx1 at normal level. Total GPx activities measured in Hygro-3, GPx-2 and VA13 were respectively of 0.7 ⫾ 0.2, 75 ⫾ 13 and 36 ⫾ 6 mU/mg protein (Fig. 1). About 78% of GPx activity was found in the cytoplasm of GPx-2 cells (data not shown). A similar distribution was observed in different cell types, e.g., rat liver (38). Average GPx activity in the mitochondria-lysosomes fractions, measured in the presence of Triton X-100 detergent to lyse these organelles, was 16% and 14% (no significant difference) of total GPx activity in GPx-2 and VA13 cells, respectively (Fig. 1). Measurements of GPx activity in corresponding mitochondrial fractions from Hygro-3, at the limit of detection, were not reliable enough to assess accurately the relative distribution of GPx1 in these cells (at least 6% in mitochondria). Importantly, however, GPx activity in this fraction was at least 100-fold lower than in GPx-2. When assayed without Triton
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FIG. 1. GPx activity in mitochondria of T47D-Hygro-3, -GPx-2, and WI38-VA13 cells. The cells were homogenized and fractionated by differential centrifugation to produce a mitochondrial fraction. GPx activity was assayed with tert-butylhydroperoxide in both total homogenates and mitochondrial fractions treated with 0.5% Triton X-100 to release GPx activity from mitochondria. The data are means ⫾ SD of 4 different experiments.
X-100 detergent, the mitochondrial-lysosomal GPx activities were about 3.5-fold lower than in the presence of detergent (not shown) (39). This last result suggests that most GPx activity resided inside the mitochondria or lysosomes. The GPx activity fully overlapped the cytochrome C oxidase activity pattern of mitochondria but not the N-acetyl-glucoaminidase activity pattern of lysosomes following further fractionation by sucrose gradient density centrifugation (39). No glucose6-phosphate dehydrogenase activity, a cytoplasmic marker, was detected in the mitochondria-lysosomes fraction, therefore excluding a contamination by cytoplasmic GPx activity (data not shown). With respect to GPx1 import in mitochondria, it is of interest that a remarkably similar partition between mitochondria and cytoplasm was observed in GPx-2 and untransfected VA13 cells, indicating that vector-derived recombinant GPx1 was translocated efficiently into mitochondria. In contrast to phospholipid-hydroperoxide GPx, known to use alternative transcription and translation start sites to determine cytoplasm or mitochondrial localization in different tissues (40), no such pattern of differential expression was reported for GPx1, nor did we find any evidence for a mitochondrial presequence in GPx1. Thus, GPx1 appears to be imported by an unknown mechanism, which does not involve the classic presequence cleavage pathway described for protein import in mitochondria (41, 42). Ultrastructural Localization of GPx1 To confirm the preferential localization of GPx1 in mitochondria, and compare the relative contents of mitochondrial GPx1 in Hygro-3 and GPx-2 cells, the
ultrastructural distribution of GPx1 was investigated by immuno-electron microscopy. Immuno-staining was performed with an immunoaffinity-purified GPx1 antibody (29, 36) and colloidal gold-conjugated secondary antibody, as described under Materials and Methods. Figure 2A shows relatively intense gold staining in GPx-2 mitochondria (b, c), in contrast to barely detectable staining in Hygro-3 cells (a). These observations are in excellent agreement with the GPx activity distribution determined by cell fractionation (see above). Figure 2B shows that, after subtraction of the background obtained with adsorbed antibody, the colloidalgold grain density per surface was about 8-fold higher in mitochondria than in the cytoplasm of GPx-2 cells. Conversion of this 2-dimensional factor 8 (per surface) to a 3-dimensional factor (per volume, i.e., 8 3/2) indicate a 20- to 25-fold higher concentration of GPx1-like antigen in the mitochondria. Importantly, comparison of the mitochondrial grain density in the two cell lines also yields an ⬃8-fold higher density in GPx-2 (Fig. 2B), thus indicating that the apparent mitochondrial concentration of GPx1 in these cells was 20 to 25-fold higher than in Hygro-3 mitochondria. Compared to the corresponding higher difference in GPx activity (more than 100-fold), the apparent difference in GPx1 protein level obtained by immuno-detection is most likely underestimated. Although the immuno-staining observed in Hygro-3 mitochondria was very low, any possible cross-reaction of the antibody to non-GPx1 proteins including, for example, PHGPx would contribute to increase non-specific immuno-staining signal in Hygro-3, despite the very good specificity of the affinity-purified antibody (29, 36). Effect of GPx1 Expression on Basal Levels of 8-Oxo-dG in mtDNA The frequency of 8-oxo-dG lesions was assessed as an index of oxidative damage to mtDNA. To this end, we used the 32P-postlabeling assay described by Devanaboyina and Gupta (37). The major advantage of this method is that it requires very little DNA for detection of 8-oxo-dG. However, this assay involves a series of incubations at 37°C, which may introduce 8-oxo-dG lesions artifactually. Indeed, the suggestion that the quantification of 8-oxo-dG is prone to artifactual oxidation during DNA isolation and analysis, was recently discussed in the context of the current controversy on the relative sensitivity of mtDNA to oxidative damage (43). Therefore, the artifactual formation of 8-oxo-dG during the overall postlabeling assay was assessed by measuring the time-dependent increase in 8-oxo-dG content resulting from doubling and tripling the enzymatic incubation periods at 37°C as well as phenol/chloroform extraction time used in the original protocol. Figure 3 shows that the level of 8-oxo-dG determined in mtDNA prepared from normally grow-
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FIG. 2. Ultrastructural localization of GPx1-like antigen in Hygro-3 and GPx-2 cells. The cells were fixed and coated as described under Materials and Methods. Immunocytochemistry was carried out with an immuno-affinity-purified GPx1 primary antibody and secondary anti-rabbit IgG conjugated with 10-nm colloidal gold, visualized by electron microscopy (A). (a) Hygro-3 cells (scale bar, 0.5 m); (b) GPx-2 cells (scale bar, 0.5 m); (c) GPx-2 cells (scale bar, 0.2 m). The arrows point to mitochondrial immunostaining. (B) Histogram of colloidal gold grain counts over mitochondria and cytoplasm area, after subtraction of background determined with GPx1-adsorbed primary antibody. Means ⫾ SD, P ⬍ 0.05 for the differences between mitochondria and corresponding cytoplasm, and between Hygro-3 and GPx-2 mitochondria; P ⬍ 0.05 for the differences between signal and background of Hygro-3 mitochondria and GPx-2 cytoplasm; P ⬍ 0.001 for the differences between signal and background for GPx-2 mitochondria (Student’s t tests).
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FIG. 3. Basal 8-oxo-dG levels in mtDNA and artifactual formation during incubations inherent to the postlabeling procedure. Mitochondrial DNA was isolated from normally growing Hygro-3 and GPx-2 cells. 8-Oxo-dG contents were determined by 32P-postlabeling assay (see Materials and Methods) and related to the total period of enzymatic incubation and phenol/chloroform extraction required in the original postlabeling protocol (490 min), which was either doubled (980 min) or tripled (1470 min) for test. The graph displays the linear regression of 8-oxo-dG formation in function of total incubation time (r 2 ⫽ 0.99).
tive in terms of mtDNA strand breaks since a 200 M menadione treatment was required to induce a significant relaxation of supercoiled mtDNA. Thus, the relative oxidoresistance of mtDNA in GPx-2 cells was not only evident at the level of oxidative base damage, but could also be demonstrated at the level of DNA strand breaks. The data would be consistent with at least two distinct sources of ROS mediating oxidative damage to mtDNA: (i) H 2O 2 (GPx1-sensitive)-derived ROS, e.g., hydroxyl radicals derived from Fenton-type reactions produced during stimulation of cyanide-insensitive respiration (e.g., menadione treatment), and (ii) nonhydroperoxide-derived ROS, possibly hydroxyl radicals derived from (GPx1-insensitive) reactions between •O 2⫺ and NO • that would be responsible for most basal oxidative damage detected in both GPx-deficient and
ing (unstressed) Hygro-3 and GPx-2 cells, increased in direct relation to overall incubation time (r 2 ⫽ 0.99), and very similarly for both cell lines. Interpolation of both curves to 0-incubation time indicated no significant difference in basal frequency of 8-oxo-dG lesions, i.e., regardless of GPx1 level. Oxidative Stress-Induced Damage to mtDNA Is Reduced in GPx1-Proficient Cells Oxidative stress was induced in Hygro-3 and GPx-2 cells by incubating the cells in the presence of 25 or 50 M menadione for 1 h. Induced formation of 8-oxo-dG in mtDNA was then determined as described above, after subtraction of the basal levels measured in unexposed control cells. Figure 4A shows that the induction of 8-oxo-dG lesions in mtDNA was menadione dosedependent, and, importantly, was much reduced in the GPx1-proficient cells (GPx-2), i.e., by respectively ⫺74 ⫾ 8% and ⫺53 ⫾ 6% after 1-h exposure to 25 and 50 M menadione. To confirm these results, DNA strand breakage was also assessed in mtDNA from these cells exposed 1 h to menadione, but using higher menadione concentrations (100 and 200 M) to permit detection of strand breaks revealed by relaxation of supercoiled mtDNA. Figure 4B shows similar levels of supercoiled and relaxed mtDNA in unexposed control Hygro-3 and GPx-2 cells. Menadione exposure induced a dose-dependent relaxation of supercoiled mtDNA in the GPx1-deficient Hygro-3 cells, with a clear effect already seen at 100 M menadione. In contrast, GPx-2 cells are less sensi-
FIG. 4. Decreased formation of 8-oxo-dG lesions and mtDNA strand breaks in GPx-2 cells exposed to oxidative stress. (A) Hygro-3 and GPx-2 cells were incubated or not (controls) with 25 or 50 M menadione for 1 h at 37°C. mtDNA was isolated and 8-oxo-dG content determined by 32P-postlabeling assay. The 8-oxo-dG backgrounds of control cells were subtracted from the 8-oxo-dG determinations of menadione-treated cells. Means ⫾ SD of three determinations, P ⬍ 0.05, for the differences between Hygro-3 and GPx-2 (Student’s t test). (B) Hygro-3 and GPx-2 cells were incubated or not (controls) with 100 or 200 M menadione at 37°C for 1 h. Mitochondrial DNA was isolated, analyzed by FIGE in 0.6% agarose gel and visualized by ethidium bromide staining.
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GPx-proficient cells. The observation that the parental T47D cells and Hygro-3 derivative grow normally in the quasi absence of GPx1 activity suggests that other mitochondrial peroxidases may compensate for the lack of GPx1 activity. These peroxidases may include a mitochondria-targeted phospholipid hydroperoxide glutathione peroxidase (44) and a mitochondrial thioredoxin peroxidase such as Prx III (24). On the other hand, we found no significant alteration in catalase, CuZnSOD (SOD1) and MnSOD (SOD2) expression or activity in GPx-2, compared to Hygro-3 or T47D (29) (Bilodeau and Mirault, unpublished). The results presented here suggest that the mitochondrial glutathione/GPx1 system may be of particular importance in conditions of oxidative stress resulting from excess • O 2⫺/H 2O 2 production. Future studies will be required to evaluate the respective role of each mitochondrial peroxidase system in different cell types, in both unstressed cells and cells challenged by various oxidant stresses.
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ACKNOWLEDGMENTS The work was supported by the National Cancer Institute of Canada with funds provided by the Canadian Cancer Society. J.L. was recipient of studentships from the Natural Sciences and Engineering Research Council of Canada and from the FCAR. This research was also supported by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. P.R. is a postdoctoral researcher of the Fonds National de la Recherche Scientifique, Belgium.
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