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Ting Endogenous Glutathione And Catalase Protect Cultured Rat Astrocytes From The Iron Mediated Toxicity Of Hydrogen Peroxide
Ting Endogenous Glutathione And Catalase Protect Cultured Rat Astrocytes From The Iron Mediated Toxicity Of Hydrogen Peroxide
Ting Endogenous Glutathione And Catalase Protect Cultured Rat Astrocytes From The Iron Mediated Toxicity Of Hydrogen Peroxide
Ting Endogenous Glutathione And Catalase Protect Cultured Rat Astrocytes From The Iron Mediated Toxicity Of Hydrogen Peroxide
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Ting Endogenous Glutathione And Catalase Protect Cultured Rat Astrocytes From The Iron Mediated Toxicity Of Hydrogen Peroxide

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Endogenous glutathione and catalase protect cultured rat astrocytes …

Endogenous glutathione and catalase protect cultured rat astrocytes
from the iron-mediated toxicity of hydrogen peroxide

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  1. Neuroscience Letters 364 (2004) 164–167 Endogenous glutathione and catalase protect cultured rat astrocytes from the iron-mediated toxicity of hydrogen peroxide Jeff R. Liddell a , Stephen R. Robinson a , Ralf Dringen a,b,∗ a Department of Psychology, Monash University, Clayton, Vic. 3800, Australia b Interfakultäres Institut für Biochemie der Universität Tuebingen, D-72076 Tuebingen, Germany Received 21 March 2004; received in revised form 15 April 2004; accepted 15 April 2004 Abstract Primary astrocyte cultures from rat brain were exposed to hydrogen peroxide (H2 O2 ) to investigate peroxide toxicity and clearance by astrocytes. After bolus application of H2 O2 (100 M), the peroxide was eliminated from the incubation medium following first-order kinetics with a half-time of approximately 4 min. The rate of peroxide detoxification was significantly slowed by pre-incubating the cells with the glutathione synthesis inhibitor buthionine sulfoximine (BSO), or the catalase inhibitor 3-amino-1,2,4-triazole (3AT), and was retarded further when both treatments were combined. H2 O2 application killed a small proportion of cells, as indicated by the levels of the cytosolic enzyme lactate dehydrogenase in the media 1 and 24 h later. In contrast, cell viability was strongly compromised when the cells were pre-incubated with 3AT and/or BSO before peroxide application. The iron chelator deferoxamine completely prevented this cell loss. These results demonstrate that chelatable iron is involved in the toxicity of H2 O2 and that both the glutathione system and catalase protect astrocytes from this toxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Astrocytes; Catalase; Glutathione; Hydrogen peroxide; Iron; Oxidative stress Hydrogen peroxide is generated during aerobic meta- when acutely applied at concentrations of 100 M [5]. bolism by the action of superoxide dismutases and various These studies have also found that chemical inactivation oxidases [4]. H2 O2 and superoxide, in the presence of iron of either GPx or catalase only slightly retards the rate of ions, can lead to the formation of highly reactive and cyto- H2 O2 detoxification, and does not appreciably increase toxic hydroxyl radicals [3,13]. Such processes contribute to the extent of cell loss during the period of H2 O2 exposure the cell loss associated with reperfusion injury [11], and to [5]. the neurodegeneration associated with chronic conditions The preceding observations question whether cells are such as Alzheimer’s and Parkinson’s diseases [12,15]. To only vulnerable to H2 O2 when both antioxidant systems reduce the likelihood of radical formation from peroxides, have been inactivated, or whether cell loss occurs but brain cells use two antioxidant systems that can rapidly is delayed beyond the first few hours. It is also unclear inactivate H2 O2 . In a reaction catalyzed by glutathione per- from these observations whether endogenous iron is an oxidases (GPx), the tripeptide glutathione (GSH) serves as important factor in peroxide-mediated toxicity, as has an electron donor to reduce H2 O2 to water. The product been suggested by some other studies [1,16,17]. Answers of the oxidation of GSH is glutathione disulfide. Besides to these questions could assist the design of therapeutic the glutathione system, the diffusion-controlled catalase approaches that are aimed at reducing the extent of neu- also inactivates H2 O2 , especially if this peroxide is present rodegeneration following acute episodes, such as stroke in high concentrations [2]. Experiments using cultured and ischemia. The present study therefore investigates: cells have established that endogenous levels of GSH and (i) the relative contributions of GSH and catalase to the catalase are sufficient to rapidly inactivate H2 O2 , even detoxification of H2 O2 by cultured astrocytes; (ii) the con- tributions of both systems to the protection of astrocytes ∗ Corresponding author. Present address: Interfakultäres Institut für from H2 O2 -mediated damage in the 24 h period following Biochemie der Universität Tübingen, Hoppe-Seyler-Str. 4, D-72076 Tübin- peroxide exposure; and (iii) whether the iron chelator de- gen, Germany. Tel.: +49-7071-2973334; fax: +49-7071-295360. feroxamine (DFX) can play a protective role against H2 O2 E-mail address: ralf.dringen@uni-tuebingen.de (R. Dringen). toxicity. 0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.04.042
  2. J.R. Liddell et al. / Neuroscience Letters 364 (2004) 164–167 165 H2 O2 was applied in a concentration of 100 M to as- ability of astrocyte cultures, DFX was applied in a final trocyte primary cultures in 24-well plates that had been concentration of 2 mM either 2 h before the application prepared from the brains of newborn Wistar rats, as de- of H2 O2 (pre-peroxide) and/or during the 24 h incubation scribed previously [8,9]. To measure the rate of peroxide following the H2 O2 exposure (post-peroxide). Exposure of clearance, cells were washed with 2 ml of incubation buffer cultured astrocytes to 1 mM DFX is sufficient to up-regulate (20 mM HEPES, 145 mM NaCl, 1.8 mM CaCl2 , 5.4 mM transferrin receptor expression and down-regulate ferritin KCl, 1 mM MgCl2 , 0.8 mM Na2 HPO4 , 5 mM glucose, pH expression [10]. Such changes are indicative of depleted in- 7.4) and then provided with 500 l of incubation buffer (con- tracellular iron levels [3]. Since 2 mM DFX was used in the taining 100 M H2 O2 ) at 37 ◦ C. During the incubation the present experiments, it is likely that most of the chelatable extracellular concentration of H2 O2 was repeatedly mea- iron was effectively bound by the DFX. The protein con- sured by the colorimetric method described previously [6]. tent of the cell cultures was determined with the method of To assess the relative contributions of GSH and catalase to Lowry et al. [14] using bovine serum albumin as a standard. H2 O2 toxicity, astrocyte cultures were pre-incubated either When untreated astrocyte cultures (control) were exposed for 24 h with 1 ml of Dulbecco’s modified Eagles’s medium to 100 M H2 O2 , the peroxide was depleted from the incuba- (DMEM) containing the GSH synthesis inhibitor buthionine tion buffer (Fig. 1A) following first-order kinetics (Fig. 1B), sulfoximine (BSO; 1 mM), or for 2 h in 1 ml DMEM with with a half-time of about 4 min (Table 1). This result is con- the catalase inhibitor 3-amino-1,2,4-triazole (3AT; 10 mM). sistent with previous reports concerning the rate of H2 O2 Under these conditions BSO reduces the GSH content of detoxification by cultured astrocytes [5,6,9]. In the absence astrocytes to 14% of controls and 3AT completely inhibits of cells, but otherwise under identical conditions, H2 O2 is catalase activity [5]. For BSO + 3AT conditions, cells were stable for at least 1 h [7]. LDH measurements indicated incubated for 22 h with BSO and for an additional 2 h with that control astrocytes which had been treated with 100 M BSO plus 3AT. The subsequent incubation with H2 O2 was H2 O2 did not undergo substantial cell damage in the subse- carried out in the absence of inhibitors. quent 24 h incubation period (Fig. 2B, control). Cell viability was analyzed by determining the extracel- Astrocytes that had been pre-incubated with BSO or 3AT lular activity of lactate dehydrogenase (LDH) in the cultures disposed of the H2 O2 more slowly than controls (Fig. 1; [6]. The LDH activity in the incubation buffer after 60 min Table 1). Semi-logarithmic plots of the data obtained dur- incubation with H2 O2 (or in DMEM 24 h after removal of ing the initial 8 min of H2 O2 detoxification showed that un- the peroxide) was compared to the LDH activity in lysates der these conditions the clearance of the peroxide follows of untreated astrocyte cultures. These lysates were obtained first-order kinetics (Fig. 1B). The rate of peroxide disposal by exposing cultures to 1% Triton X-100 for 30 min [6]. was normalized to the protein content of the respective wells Pre-incubation of astrocytes with BSO, DFX or 3AT did by calculating the specific H2 O2 detoxification rate constant not alter the total LDH activity of astrocyte cultures (data (D = 1/(half-time in minutes × initial protein content in not shown). To investigate the effect of DFX on the vi- milligrams)). D is proportional to the capacity of cells to Fig. 1. Disposal of exogenous H2 O2 by cultured astrocytes. The cells were pre-incubated for 24 h in DMEM (control), for 24 h in DMEM containing 1 mM BSO, for 2 h in DMEM containing 3AT (10 mM) or for 24 h with BSO (1 mM) as well as for 2 h with 10 mM 3AT (BSO + 3AT). Subsequently, astrocytes were incubated with 500 l of incubation buffer containing 100 M H2 O2 . (A) Time course of the concentration of H2 O2 in the incubation buffer of the cultures. (B) Semi-logarithmic representation of the data obtained. This figure shows a representative experiment performed on a 20 days old culture. The mean protein content was 146 ± 9 g per well.
  3. 166 J.R. Liddell et al. / Neuroscience Letters 364 (2004) 164–167 Table 1 Half-times of H2 O2 and specific H2 O2 detoxification rate constant D of astrocyte cultures Treatment No DFX DFX n Half-time (min) D (minutes × milligrams of protein)−1 Half-time (min) D (minutes × milligrams of protein)−1 Control 4.0 ± 0.4 1.78 ± 0.19 4.1 ± 0.6 1.74 ± 0.23 18 BSO 5.1 ± 0.8 1.35 ± 0.21∗ 5.6 ± 0.6 1.19 ± 0.10∗ 9 3AT 6.1 ± 1.0 1.19 ± 0.23∗ 5.7 ± 0.8 1.26 ± 0.18∗ 9 BSO + 3AT 11.1 ± 2.5 0.64 ± 0.18∗ 9.8 ± 2.6 0.72 ± 0.17∗ 18 The cells were pre-incubated for 24 h in DMEM (control) or for 24 h in DMEM containing 1 mM BSO and/or for 2 h in the presence of 10 mM 3AT and/or 2 mM DFX. Subsequently, astrocytes were incubated with 100 M H2 O2 for 1 h. The decline in extracellular concentration of the peroxide was monitored and half-times of the peroxide were calculated from the semi-logarithmic representations of the data. The specific detoxification rate constant D is defined as 1/(half-time in minutes × initial protein content in milligrams). The data represent mean values ± S.D. of (n) wells derived from three to six experiments. The significance of the differences of the D values compared to those of the respective controls (control) was calculated by ANOVA followed by Bonferroni’s post hoc test (∗ P < 0.001). Corresponding data obtained for cells treated with and without DFX were not significantly different, as calculated by Student’s t-test. detoxify a peroxide [7]. The higher the D value, the better damage than control astrocytes (Fig. 2). 1 h after application the capacity of the cultured cells to dispose of a peroxide. of the peroxide about 20% of total LDH was measured ex- The specific detoxification rate constant D of astrocytes that tracellularly for cells that had been treated with inhibitors, were treated with BSO or 3AT was significantly reduced compared to 13% for controls (Fig. 2A). Cell loss was con- compared to that in control cultures, indicating that the rate siderably greater 24 h later, with extracellular LDH values of peroxide disposal was reduced by pre-incubating the cells reaching up to 53% of the total (Fig. 2B). These findings with either BSO or 3AT (Table 1). Combined treatment with demonstrate that while astrocytes treated with BSO and/or both inhibitors slowed the rate of peroxide detoxification 3AT can effectively detoxify H2 O2 , they nonetheless sustain to a much greater extent than was achieved with either in- oxidative injury, which results in their death several hours hibitor alone. This result provides clear evidence that both later. It is noteworthy that BSO-treated cells were signifi- GSH and catalase participate in the inactivation of H2 O2 by cantly more sensitive to peroxide treatment (Fig. 2B) than astrocytes. cells which had been pre-incubated with 3AT (P < 0.001), Astrocytes that had a reduced rate of H2 O2 disposal due indicating that under the conditions used the GSH system is to pre-incubation with either BSO, 3AT, or BSO + 3AT more effective at preventing peroxide-mediated damage than (Table 1) underwent significantly higher amounts of cell catalase. A reason for this difference could be that catalase can only inactivate H2 O2 , whereas GSH can also inactivate radicals [4]. Iron has been implicated in the potentiation of peroxide- induced cell damage in brain cells [1,16,17]. In order to examine the contribution of cellular iron to the toxicity observed after H2 O2 treatment, astrocyte cultures were in- cubated with DFX before and after H2 O2 treatment. DFX treatment did not affect the rate of clearance of H2 O2 under the various conditions investigated, as indicated by identical D values and half-times for the peroxide (Table 1). These observations demonstrate that chelatable iron does not con- tribute to the disappearance of H2 O2 from the incubation Fig. 2. LDH release from astrocyte cultures after peroxide stress. The medium and that DFX does not compromise the capacity cells were pre-incubated for 24 h in DMEM (control) or for 24 h in DMEM containing 1 mM BSO and/or for 2 h in the presence of 10 mM of astrocytes to inactivate H2 O2 . Nonetheless, DFX com- 3AT and/or 2 mM DFX. Subsequently, astrocytes were incubated with pletely prevented the elevated LDH release that normally 500 l of incubation buffer containing 100 M H2 O2 for 1 h. Cells were resulted when astrocytes were exposed to H2 O2 following then incubated for a further 24 h in 1 ml DMEM in the absence or pre-incubation with BSO, 3AT, or BSO + 3AT (Fig. 2). presence of 2 mM DFX. Cell viability was assessed by determining the This prevention of cell damage by DFX was evident both activity of extracellular LDH 1 h (A) or 24 h (B) after the cells had been incubated with hydrogen peroxide. The data represent mean values ± S.D. 1 h (Fig. 2A) and 24 h (Fig. 2B) after H2 O2 treatment. of 8 or 9 wells of the respective treatments obtained from experiments To determine when the protective action of DFX is most performed on three independently prepared cultures. The significance of critical, DFX application was limited to either the pre- or the differences in LDH release between peroxide-treated control cells and the post-peroxide period. It was found that pre-incubation cells pre-incubated with BSO and/or 3AT was calculated by ANOVA with DFX provides protection against the toxicity of H2 O2 followed by Bonferroni’s post hoc test (+ P < 0.001). The significance of the differences in LDH release obtained from astrocyte cultures treated whereas post-incubation with DFX provides no significant with 2 mM DFX to those treated without DFX was calculated by Student’s protection (Table 2). However, the presence of DFX dur- t-tests (∗ P < 0.001). ing both the pre- and post-peroxide incubation periods
  4. J.R. Liddell et al. / Neuroscience Letters 364 (2004) 164–167 167 Table 2 ment of Psychology, Monash University, for financial sup- Effects of DFX treatment on the LDH release from peroxide-treated port of the research conducted in this study. Jan Riemer astrocytes and Hans-Hermann Hoepken provided valuable technical Treatment LDH release (percent of total LDH) n advice. No DFX Pre- Post- Pre- and peroxide peroxide post-peroxide Control 13 ± 4 6±3 20 ± 10 13 ± 5 9 References BSO + 3AT 42 ± 7∗ 19 ± 4∗,+ 36 ± 5∗ 10 ± 4+ 9 The cells were pre-incubated for 24 h in DMEM (control) or for 24 h [1] K. Abe, H. Saito, Characterization of t-butyl hydroperoxide toxicity in DMEM containing BSO (1 mM) plus for 2 h in the presence of 3AT in cultured cortical neurones and astrocytes, Pharmacol. Toxicol. 83 (10 mM). Subsequently, astrocytes were incubated with 100 M H2 O2 for (1998) 40–46. 1 h and for further 24 h in DMEM. DFX (2 mM) was present 2 h before [2] H.E. Aebi, Catalase, in: H.U. Bergmeyer, J. Bergmeyer, M. Grassl peroxide application (pre-peroxide) and/or during the 24 h incubation af- (Eds.), Methods of Enzymatic Analysis, vol. 3, Verlag Chemie, ter removal of the peroxide (post-peroxide). Cell viability was assessed Weinheim, Germany, 1984, pp. 273–286. by monitoring the amount of LDH release after the 24 h incubation that [3] R.R. Crichton, S. Wilmet, R. Legssyer, R.J. Ward, Molecular and followed the peroxide treatment. The data represent mean values ± S.D. cellular mechanisms of iron homeostasis and toxicity in mammalian of (n) wells obtained from experiments performed on three independently cells, J. Inorg. Biochem. 91 (2002) 9–18. prepared cultures. The significance of the differences in LDH release ob- [4] R. Dringen, Metabolism and functions of glutathione in brain, Prog. tained for control or BSO + 3AT-treated cells was calculated by Student’s Neurobiol. 62 (2000) 649–671. t-test (∗ P < 0.001). The significance of the differences between cells that [5] R. Dringen, B. Hamprecht, Involvement of glutathione peroxidase were treated in the absence of DFX (no DFX) or with DFX for the given and catalase in the disposal of exogenous hydrogen peroxide by incubation periods was calculated by ANOVA followed by Bonferroni’s cultured astroglial cells, Brain Res. 759 (1997) 67–75. post hoc test (+ P < 0.001). [6] R. Dringen, L. Kussmaul, B. Hamprecht, Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by a microtiter plate assay, Brain Res. Protoc. 2 (1998) completely rescued cells from peroxide-mediated damage 223–228. and was significantly (P < 0.01) more protective than [7] R. Dringen, L. Kussmaul, J.M. Gutterer, J. Hirrlinger, B. Hamprecht, pre-incubation with DFX (Table 2). These data demon- The glutathione system of peroxide detoxification is less efficient strate that the presence of chelatable iron is a key factor in neurons than in astroglial cells, J. Neurochem. 72 (1999) 2523– 2530. in the peroxide-mediated damage of astrocytes that have [8] B. Hamprecht, F. Löffler, Primary glial cultures as a model system a compromised capacity to clear H2 O2 . Since chelatable for studying hormone action, Methods Enzymol. 109 (1985) 341– iron catalyzes the formation of hydroxyl radicals in the 345. presence of superoxide and H2 O2 by the Fenton reaction [9] J. Hirrlinger, A. Resch, J.M. Gutterer, R. Dringen, Oligodendroglial or the Haber–Weiss cycle [3,13], the protection provided cells in culture effectively dispose of exogenous hydrogen peroxide: comparison with cultured neurones, astroglial and microglial cells, to H2 O2 -treated astrocytes by DFX is probably due to a J. Neurochem. 82 (2002) 635–644. reduction in the amount of iron that is available to catalyze [10] H.H. Hoepken, T. Korten, S.R. Robinson, R. Dringen, Iron accumu- the formation of cytotoxic hydroxyl radicals. lation, iron-mediated toxicity and altered levels of ferritin and trans- In summary, both GSH and catalase contribute to the ferrin receptor in cultured astrocytes during incubation with ferric clearance of H2 O2 by astrocytes in vitro. Impairment of ammonium citrate, J. Neurochem. 88 (2004) 1194–1202. [11] P.A. Hyslop, Z. Zhang, D.V. Pearson, L.A. Phebus, Measurement of either antioxidant system increases the vulnerability of as- striatal H2 O2 by microdialysis following global forebrain ischemia trocytes to H2 O2 -induced injury, and even small reductions and reperfusion in the rat: correlation with the cytotoxic potential of in the rate of peroxide disposal can lead to substantial cell H2 O2 in vitro, Brain Res. 671 (1995) 181–186. damage over the following 24 h period. It is of particular [12] P. Jenner, Oxidative stress in Parkinson’s disease, Ann. Neurol. interest that peroxide-induced cell loss in astrocyte cultures 53 (Suppl. 3) (2003) S26–S36. [13] M. Kruszewski, Labile iron pool: the main determinant of cellular can be completely prevented with DFX. Viewed in the con- response to oxidative stress, Mutat. Res. 531 (2003) 81–92. text of the brain, the present results suggest that chelatable [14] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein mea- iron, as well as endogenous levels of GSH and catalase in surement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) astrocytes, may represent useful therapeutic targets in the 265–275. treatment of certain neurodegenerative conditions. [15] N.G. Milton, Role of hydrogen peroxide in the aethiology of Alzheimer’s disease: implications for treatment, Drugs Aging 21 (2004) 81–100. [16] S.J. Robb, J.R. Connor, An in vitro model for analysis of oxidative Acknowledgements death in primary mouse astrocytes, Brain Res. 788 (1998) 125–132. [17] W. Ying, S.K. Han, J.W. Miller, R.A. Swanson, Acidosis potentiates R.D. was supported by a Senior Research Fellowship oxidative neuronal death by multiple mechanisms, J. Neurochem. 73 (1999) 1549–1556. awarded by NeuroSciences Victoria. We thank the Depart-

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