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

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
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awarded by NeuroSciences Victoria. We thank the Depart-