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 . Such changes are indicative of depleted in-
7.4) and then provided with 500 l of incubation buffer (con- tracellular iron levels . 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 . tent of the cell cultures was determined with the method of
To assess the relative contributions of GSH and catalase to Lowry et al.  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 . For BSO + 3AT conditions, cells were stable for at least 1 h . 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;
. 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 . 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.
166 J.R. Liddell et al. / Neuroscience Letters 364 (2004) 164–167
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 . 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
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
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
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
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R.D. was supported by a Senior Research Fellowship oxidative neuronal death by multiple mechanisms, J. Neurochem. 73
awarded by NeuroSciences Victoria. We thank the Depart-