Hemin Metabolism & Toxicity in Astrocytes: Hemorrhagic Stroke Insights

GLIA 59:1540–1550 (2011)
The Metabolism and Toxicity of Hemin in Astrocytes
THERESA N. DANG,1* GLENDA M. BISHOP,1 RALF DRINGEN,1,2 AND STEPHEN R. ROBINSON1
1
Blood-Brain Interactions Group, School of Psychology and Psychiatry, Monash University, Clayton VIC Australia
2
Center for Biomolecular Interactions Bremen, Faculty 2 (Biology/Chemistry), University of Bremen, Bremen, Germany
KEY WORDS
hemorrhagic stroke; rat; brain; heme oxygenase; hydrogen
peroxide; iron
ABSTRACT
Hemin is cytotoxic, and contributes to the brain damage that
accompanies hemorrhagic stroke. In order to better understand the basis of hemin toxicity in astrocytes, the present
study quantified hemin metabolism and compared it to the
pattern of cell death. Heme oxygenase-1 (HO-1) expression
was first evident after 2 h incubation with hemin, with maximal expression being observed by 24 h. Despite the induction
of HO-1, it was found that the proportion of hemin metabolized by astrocytes remained fairly constant throughout the
24 h period, with 70–80% of intracellular hemin remaining
intact. A period of cell loss began after 2 h exposure to hemin,
which gradually increased in severity to reach a maximum
by 24 h. This cell loss could not be attenuated by the iron
chelator, 1,10-phenanthroline, or by several antioxidant
compounds (Trolox, N-acetyl-L-cysteine and N-tert-butyl-aphenylnitrone), indicating that the mechanism of hemin
toxicity does not involve iron. While these results make it
unlikely that hemin toxicity is due to interactions with
endogenous H2O2, hemin toxicity was increased in the presence of supraphysiological levels of H2O2 and this increase
was ameliorated by PHEN, indicating that the iron released
from hemin can be toxic under some pathological conditions.
However, when H2O2 is present at physiological levels, the
toxicity of hemin appears to be caused by other mechanisms
that may involve bilirubin and carbon monoxide in this
model system. V 2011 Wiley-Liss, Inc.
C
INTRODUCTION
Heme provides the prosthetic group of several important proteins (e.g. hemoglobin and cytochrome c) and
forms the catalytic center of some key enzymes (e.g.,
catalase) (Tsiftsoglou et al., 2006). The usefulness of
heme stems from the iron atom that is bound to the center of the molecule; the iron is redox-active and can
readily accept or donate electrons. This reactivity can
also cause injury. After a hemorrhagic stroke for
instance, hemin (oxidized heme) is released from lysed
red blood cells into the extracellular space of the brain.
Hemin is cytotoxic and contributes to the brain damage
that accompanies a hemorrhagic stroke (Regan et al.,
2001, 2002). Astrocytes have the capacity to take up
hemin via the heme carrier protein 1 (HCP1) (Dang
et al., 2010; Lara et al., 2009), and consequently these
brain cells are particularly vulnerable to elevated levels
of extracellular hemin.
C 2011 Wiley-Liss, Inc.
V
The toxicity of hemin is commonly thought to be due to
oxidative stress that arises when iron is released into the
cytosol after the degradation of hemin by heme oxygenases
(HO). This view is supported by evidence that iron chelators
can limit oxidative stress and cell death after neurons and
astrocytes are exposed to hemin (Goldstein et al., 2003;
Laird et al., 2008; Regan et al., 2004). Further support comes
from the demonstration that neurons can be protected from
hemin toxicity by blocking their HO activity (Koeppen et al.,
2004; Wang and Dore, 2007). However, this view has been
challenged by data showing that the inhibition of HO-1 does
not protect astrocytes from hemin, whereas increasing their
expression of HO-1 does (Benvenisti-Zarom and Regan,
2007; Chen and Regan, 2005; Teng et al., 2004). This
difference between astrocytes and neurons indicates that
the basis of hemin toxicity is complex and may involve
several modes of action that differ between cell types.
Studies of hemin toxicity have generally focused on the
metabolic degradation of hemin, yet there are several
ways in which the intact hemin molecule can be toxic to
cells (for overview see Robinson et al., 2009), including
radical generation via interaction between hemin and
hydrogen peroxide (H2O2) or superoxide production as a
byproduct of the glutathione-dependent reduction of
hemin to heme. In order to better understand the basis of
hemin toxicity in astrocytes, the present study has compared the time course of hemin metabolism in astrocytes
with that of their subsequent cell death. In addition, we
have examined the capacity of an iron chelator and various antioxidant compounds to attenuate hemin toxicity,
and have investigated whether exogenous H2O2 potentiates the toxicity of hemin in astrocytes. This combined
approach has helped to shed light on the mechanisms
underlying hemin cytotoxicity.
MATERIALS AND METHODS
Materials
Bovine serum albumin (BSA), hemin, bilirubin, ferritin,
NADH, ferrozine [3,(2-pyridyl)-5,6-bis(phenyl sulfonic
Grant sponsor: NHMRC; Grant number: 334129; Grant sponsor: NHMRC Peter
Doherty Fellowship; Grant number: 284393; Grant sponsors: NeuroSciences
Victoria Senior Research Fellowship, School of Psychology and Psychiatry, Monash
University.
*Correspondence to: Theresa N. Dang, School of Psychology and Psychiatry,
Monash University, Wellington Road, Clayton VIC 3800 Australia.
E-mail: [email protected]
Received 27 November 2010; Accepted 18 May 2011
DOI 10.1002/glia.21198
Published online 16 June 2011 in Wiley Online Library (wileyonlinelibrary.com).
acid)-1,2,4-triazine], neocuproine, sodium pyruvate,
TRIZMA base, TRIZMA HCl, b-mercaptoethanol, bromophenol blue, ammonium persulfate, glycine, 3,30 -diaminobenzidine tetrahydrochloride (DAB), TEMED, N-acetyl-Lcysteine (NAC), N-tert-butyl-a-phenylnitrone (PBN),
1,10-phenanthroline (PHEN), TroloxTM and monoclonal
anti-actin antibody from mouse were from Sigma (Australia). HEPES-free Dulbecco’s modified Eagle medium
(DMEM; Cat. # 12100046), fetal calf serum (FCS), and
penicillin/streptomycin were from Gibco Invitrogen
(Carlsbad, CA). Sodium dodecyl sulfate (SDS) and potassium ferrocyanide were from ICN biomedicals (Aurora,
Ohio) and 30% Acrylamide/Bis solution was from BIORAD (Hercules, CA). Paraformaldehyde was from ProSciTech (Thuringowa, QLD, Australia). Sodium hydroxide
and Triton X-100 were from Ajax Finechem (Seven Hills,
Australia). Anti-heme oxygenase-1 (HO-1) was from
Stressgen (Michigan, USA) and anti-ferritin was from
Dako (Glastrup, Denmark). Anti-rabbit and anti-mouse
secondary antibodies and biotinylated-streptavidin
horseradish peroxidase (SB-HRP) were from Amersham
Biosciences (UK). All other chemicals of the highest grade
were purchased from Merck (Kilsyth, VIC, Australia).
Twenty-four-well culture plates and 96-well microtiter
plates were from Greiner Bio-One (Frickenhausen,
Germany).
Primary Astrocyte Cultures
Primary astrocyte cultures were obtained from the
brains of newborn Wistar rat pups (<24 h old) as previously described (Hamprecht and Loffler, 1985). In 24well culture plates, viable astrocytes were seeded at
300,000 cells/well, while in wells containing 10 mm glass
or plastic coverslips, astrocytes were seeded at 150,000
cells/well. All cells were incubated in culture medium
containing 90% DMEM, 10% FCS, 20 U/mL penicillin G,
and 20 lg/mL streptomycin sulfate and were maintained
in a 37°C humidified Hereaus incubator containing 10%
CO2. The culture medium was replaced every seven
days. Cells were cultured for 15–17 days before experimentation. Immunocytochemical staining using the
astrocyte marker glial fibrillary acidic protein (GFAP)
revealed that more than 90% of cells in the cultures contained GFAP (data not shown).
Experimental Incubations
Under the culture conditions used in our laboratory,
concentrations of 30 lM hemin result in a substantial
amount of hemin accumulation by astrocytes, yet this
concentration does not produce substantial levels of cell
loss until 24 h (Dang et al., 2010). Therefore, hemin
concentrations of 30 lM have been used throughout
this study.
Cells were washed twice with 1 mL of warm (37°C)
DMEM before being incubated with 1 mL of DMEM
containing various concentrations of hemin (from a 1 mM
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hemin stock solution dissolved in 10 mM NaOH) for a
number of time points up to 24 h as detailed in the figure
legends. For all investigations on the effect of H2O2, pyruvate-free DMEM was used as the incubation medium. In
experiments requiring incubations with Trolox, it was
included in the preincubation step only, since we have
observed that Trolox decreases cell viability after 24 h
(data not shown). For experiments examining the effect of
NAC on H2O2 toxicity, NAC was included in the preincubation step only since we have observed that NAC readily
detoxifies H2O2 in solution (data not shown).
Quantification of Iron and Hemin-Derived Iron
Cellular iron content was determined via a colorimetric ferrozine-based iron estimation assay (Dang et al.,
2010; Riemer et al., 2004). For the detection of iron
within hemin, a complete lysis reagent containing potassium permanganate was required to liberate the iron in
hemin to make it accessible for detection by ferrozine.
When solutions containing hemin were given permanganate-free releasing reagent, less than 3% of the iron
could be detected (data not shown), suggesting that the
iron accumulated by cells was predominantly from
hemin. In each experiment, the concentration of hemin
applied to cells was quantified by determining the concentration of iron present in the sample (each hemin
molecule contains one atom of iron).
Non-hemin-iron (i.e., iron not bound to hemin) was
measured using a modified colorimetric ferrozine-based
iron estimation assay. To detect iron that was not bound
to hemin, potassium permanganate was omitted from
the lysis solution. To determine whether the permanganate-free lysis solution could detect iron contained
within ferritin, the iron content of pure ferritin from
horse spleen was measured using lysis reagent that either contained or lacked permanganate. No difference in
iron content was detected, indicating that permanganate
is not required to liberate iron from ferritin but is
required to liberate iron from hemin (data not shown).
Quantification of Cell Viability and Cellular
Protein Content
The activity of lactate dehydrogenase (LDH) in the
media was measured to determine cell viability. This
method has been described elsewhere (Dringen et al.,
1998). In addition, cellular protein content per well was
determined via the Lowry method as described previously (Lowry et al., 1951), and was used to standardize
the iron and hemin content of incubated cultures, as
well as providing a second measure of cell viability.
Immunocytochemistry for HO-1 and Ferritin
Cells were fixed and immunolabeled for HO-1 and ferritin as previously described (Edwards and Robinson,
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HEMIN METABOLISM AND TOXICITY IN ASTROCYTES
DANG ET AL.
2006). In brief, nonspecific binding sites were blocked,
and cells were incubated with anti-HO-1 (1:1000) or
anti-ferritin (1:500) for 1 h, followed by 1 h incubations
with anti-mouse or anti-rabbit secondary antibodies and
SB-HRP (1:300). Labeling was then visualized by DABNi sulfate intensification. One coverslip per condition
was counterstained with 0.1% (w/v) methylene blue.
Coverslips were then mounted onto glass slides using
Depex. The specificity of immunostaining was verified
by omitting the respective primary antibody. Nonspecific
staining was not observed (data not shown).
Visualization of Cellular Iron
A DAB-enhanced Perls’ stain (Moos and Mollgard,
1993) was used to visualize iron in cells using a protocol
modified for cell culture (Bishop and Robinson, 2001;
Dang et al., 2010). In brief, cells were fixed and then
incubated with 5% (w/v) potassium ferrocyanide
solution, followed by another incubation with 5% (w/v)
potassium ferrocyanide solution containing 1.6 M HCl.
Visualization of iron was achieved via the DAB-Ni sulfate intensification method. One coverslip per condition
was counterstained with 0.1% (w/v) methylene blue.
Coverslips were then mounted onto slides using Depex.
Western Blot Analysis of HO-1 and Ferritin in
Astrocytes
After incubation, cells were washed twice with 1 mL
of ice cold phosphate buffered saline (PBS, 10 mM potassium phosphate buffer pH 7.4 containing 150 mM NaCl)
and the protein content was quantified via the Lowry
method. Samples were prepared as described elsewhere
(Dang et al., 2010). Protein samples (20 lg of astrocyte
lysates) were then loaded onto an SDS-PAGE separation
gel containing 12% acrylamide and run against a broadrange protein ladder at 120 V for 90 min. Proteins were
transferred onto a nitrocellulose membrane at 100 V for
60 min at 4°C. After the transfer, bands were visualized
using the DAB-Ni sulfate intensification method for
Western blot as previously described (Dang et al., 2010)
using antisera directed against HO-1 (1:5,000) or ferritin
(1:500). The membrane was air-dried and digitized using
a HP Scanjet scanner. To ensure equal loading of protein, the membranes were also immunostained for actin
(1:2,000).
Spectrophotometric Analysis of Cellular Hemin
Content
Cellular hemin content was determined spectrophotometrically, using a modification of the method described
by Regan et al. (2001). After incubation, cells were
washed twice with ice cold PBS before being lysed with
50 mM NaOH for 2 h. Samples were then transferred to
a microtiter plate and their absorbance was measured at
GLIA
340 nm. The amount of hemin present was calculated by
comparison to a standard dilution curve of hemin in 50
mM NaOH (after first subtracting background absorbance from untreated cell lysates). All samples were protected from light. Cellular hemin content is expressed as
nmol/mg protein.
Data Presentation and Statistical Analysis
All experiments were performed on a minimum of 3
independent cultures in triplicate wells, except for the
Perls’ stain, immunocytochemistry, and Western blot
analysis, which were performed on 2 independent cultures. Values are presented as mean 6 SD. ANOVA
using post hoc Tukey and Dunnett tests, and two-tailed
independent samples t-tests were performed with significance defined as P < 0.05.
RESULTS
Metabolism of Hemin by Astrocytes
The capacity of astrocytes to metabolize hemin was
investigated by examining the induction of HO-1 expression in cultures that had been incubated with 30 lM hemin for up to 24 h (see Fig. 1). Untreated astrocytes
expressed no detectable HO-1, yet a mild induction of
HO-1 expression was already evident 2 h after incubation with hemin (see Fig. 1). Faint staining was seen
within the perinuclear region and within some astrocytic
processes, as well as in the perinuclear cytoplasm of
some smaller cells, which may be microglia. Counterstaining with methylene blue revealed that some, but
not all, of the astrocytes contained HO-1 labeling (data
not shown). After 6 h, the staining had increased in intensity, and primary processes were more clearly evident. Methylene blue counterstaining revealed that
most astrocytes expressed HO-1 (data not shown), but
the presumptive microglia were no longer evident. After
24 h, methylene blue counterstaining (not shown)
revealed that all astrocytes were intensely labeled for
HO-1 throughout their somata and primary processes.
Labeling was also evident in the secondary processes
and nuclei of some astrocytes (see Fig. 1). When astrocytes were incubated without hemin (i.e. culture media
only), HO-1 immunostaining was not observed at any of
the time points examined (data not shown).
Western blot analysis confirmed that HO-1 expression
by astrocytes increased following incubation with hemin
(Fig. 2A) in a temporal pattern that paralleled the
increase observed with immunocytochemistry. A faint
band, corresponding to the expected molecular weight of
HO-1, was evident in the lysates of control cells and in
lysates of cells that had been incubated with hemin for
2 h. Thereafter, the intensity of the band increased systematically until after 24 h incubation with hemin. In
lysates of control cells that had been incubated without
hemin, the band intensity did not change from basal levels throughout the 24 h period (data not shown).
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Fig. 1. HO-1, iron, and ferritin staining in astrocytes after incubation with 28–30 lM hemin for 0–
24 h. The presence of HO-1 and ferritin was visualized by immunocytochemistry and is represented as
black labeling. Iron released from hemin was visualized with a modified Perls’ stain and is represented
as black labeling. Scale bar 5 50 lm and applies to all panels.
Since HO-1 is induced by astrocytes in response to
incubation with hemin, the amount of hemin metabolized by astrocytes was quantified by comparing total
iron and hemin content in the cultures. A colorimetric
iron assay revealed that incubation with 29 lM hemin
resulted in a progressive increase in total cellular iron
content over time. Since this iron is either contained
within hemin or released from degraded hemin, it indicates the total amount of hemin accumulated by cells.
After 24 h, cells had accumulated 77.5 6 1.8 nmol iron/
mg protein (Fig. 3A). No increase in iron content was
observed in control cells incubated without hemin (data
not shown).
The amount of intracellular hemin increased over
time, reaching a maximum of 61.0 6 3.2 nmol/mg protein after 24 h (Fig. 3A). However, throughout the 24 h
period the cellular hemin content was always slightly
lower (80%) than the total iron content (Fig. 3A), suggesting that most of the hemin remained intact. To
quantify the amount of non-hemin-iron present in the
cultures, the colorimetric iron assay was performed
without the use of potassium permanganate. When the
Fig. 2. Western blot analysis of HO-1 (A) and ferritin (B) protein.
Cultures were incubated with 30 lM hemin for up to 24 h. HO-1 antisera detected a single distinct band at 32 kDa (the expected size of HO1). Ferritin antisera detected a single distinct band at 22 kDa in cells,
which corresponds with the band obtained using horse spleen ferritin
(Ft). Actin counterstaining revealed that similar amounts of protein
were loaded for all samples.
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HEMIN METABOLISM AND TOXICITY IN ASTROCYTES
DANG ET AL.
Fig. 3. Hemin metabolism by astrocytes. A: Total iron and hemin
were quantified in cells following incubation with 28 lM hemin for up
to 24 h. Total iron was quantified via a colorimetric assay for iron and
represents the total amount of hemin accumulated by cells. Hemin was
quantified via spectrophotometric analysis of the cell lysates and represents the amount of hemin that has not been metabolized by cells. Basal protein (t 5 0 h) was 129.4 6 12.8 lg/well. B: Non-hemin-iron was
examined in cells. Cultures were incubated with 31 lM hemin and the
amount of total iron and non-hemin-iron was quantified in cells. The
proportion of non-hemin-iron was calculated as a percentage of the
total iron content. No significant difference was found in the percentage
of non-hemin-iron at the various time points examined (P > 0.05). Basal protein (t 5 0 h) was 109.2 6 9.5 lg/well.
amount of non-hemin-iron was expressed as a percentage of total iron, the proportion of non-hemin-iron comprised 20% of the total iron content throughout the
24 h period with no significant differences between time
points (P > 0.05; Fig. 3B), indicating that at each time
point, approximately 80% of the total cellular iron was
contained within hemin. Non-hemin-iron was also
detected in the media using this method, and following
the incubation period, the non-hemin-iron content in the
media was found to be 2% of the total iron content
(data not shown). This low value suggests that very little non-hemin-iron had been released from the cells into
the culture media.
GLIA
When hemin is metabolized, the iron released from
hemin can be detected in cells via the Perls’ stain. The
Perls’ method cannot detect iron contained within hemin
(Meguro et al., 2007), therefore any iron staining
observed with this method is non-hemin-iron. Untreated
astrocytes exhibited no iron staining; however, distinct
granular staining of iron was detected after 2 h of hemin
incubation (see Fig. 1). Counterstaining with methylene
blue revealed that the iron was localized in the perinuclear region and some processes of some cells (data not
shown). The intensity of staining was increased after 6
h (see Fig. 1), with more diffuse iron labeling detected in
the somata and processes of most cells (as revealed by
methylene blue counterstaining; data not shown). At
this time point, iron labeling was also present in smaller
cells resembling microglia, although it was no longer
evident at 24 h. After 24 h incubation, the intensity of
iron staining had increased further (see Fig. 1) and was
clearly present within the somata and processes of
nearly all cells within the culture (as revealed by methylene blue counterstaining; data not shown). Intense
Perls’ staining was evident around the perinuclear
region, with defined granular labeling present in the
processes of astrocytes (see Fig. 1). Iron labeling was not
evident in cells that had been incubated without hemin
at any of the time points examined (data not shown).
Since free iron can induce ferritin expression in astrocytes (Hoepken et al., 2004), the induction of ferritin
expression in response to the iron released by the metabolism of hemin was examined. Faint labeling of ferritin was detected in untreated cultures, with at best a
slight increase in labeling evident after 2 h incubation
with hemin (see Fig. 1). Counterstaining with methylene
blue revealed that the labeling was located in the
somata and perinuclear region of a few cells (data not
shown). After 6 h incubation with hemin, the ferritin
immunolabeling had increased in intensity but was still
weak (see Fig. 1), and labeling could be detected in a
larger proportion of cells (as revealed by methylene blue
counterstaining; data not shown). However after 6 h,
ferritin labeling was also detected in smaller cells
resembling microglia, which was no longer evident after
24 h. This pattern of ferritin labeling is similar to that
observed with iron labeling. After 24 h incubation with
hemin, the ferritin immunostaining had greatly
increased in intensity and was clearly located within the
somata and the primary and secondary processes of
astrocytes (see Fig. 1). Methylene blue counterstaining
revealed that labeling was present in nearly all cells
within the culture (data not shown). In control cells
incubated without hemin, the intensity of labeling did
not change from basal levels at any of the time points
examined (data not shown). Western blot analysis of
lysed astrocytes that had been incubated with hemin
also showed increased ferritin content, however unlike
the immunocytochemical labeling, increased ferritin
expression was first detected at a later time point. A
band corresponding to the molecular weight of ferritin
subunits was evident after 14 h of hemin incubation,
which greatly increased in intensity after 24 h (Fig. 2B).
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Fig. 4. Hemin toxicity and the effect of PHEN and various antioxidants on cell viability. A and B: Cell viability was assessed following incubation with or without 28 lM hemin for up to 24 h. LDH released into the
media at each time point was determined and compared against the
initial LDH content of cells (A), while the amount of protein present in
the culture wells after the incubation period was quantified as an additional measure of cell viability (B). *Significant difference between cells
incubated with 28 lM hemin and 0 lM hemin as revealed by planned
comparisons t-tests (P < 0.05). Basal protein (t 5 0 h) was 128.6 6
12.5 lg/well. C and D: Cultures were preincubated without (control) or
with 0.1 mM PHEN, 0.5 mM Trolox, 1 mM NAC, or 1 mM PBN for 1 h
before being incubated with 28 lM hemin in the absence of Trolox or with
PHEN, NAC or PBN for an additional 24 h. Cell viability was assessed by
measuring LDH release (C) or quantifying the protein content of culture
wells (D) after 24-h incubation. ANOVA with post hoc Dunnett one-sided
tests using the hemin only condition as the control revealed no significant
difference in the LDH release or protein content in any condition
(P > 0.05). Basal protein (t 5 0 h) was 129.4 6 12.7 lg/well.
The Effect of PHEN and Antioxidants on
Hemin Toxicity
approximately 36.5%, which equates to that observed
with the LDH assay.
To examine whether the iron chelator PHEN or
antioxidants could attenuate the toxicity of hemin,
cells were preincubated for 1 h with PHEN or various
antioxidants (Trolox, NAC or PBN) before being incubated with 28 lM hemin for a further 24 h. It was
found that PHEN did not attenuate the LDH release
in response to hemin incubation (P > 0.05; Fig. 4C).
Trolox and PBN also failed to significantly attenuate
LDH release (P > 0.05; Fig. 4C). NAC decreased LDH
release by approximately 25%, but the extent of this
attenuation did not reach significance (P > 0.05;
Fig. 4C). No differences were detected in the protein
content of culture wells across the various conditions
(P > 0.05; Fig. 4D), indicating that PHEN and the
antioxidants did not affect cell viability following hemin incubation. These results suggest that the toxicity
of hemin is not mediated by standard Fenton chemis-
Hemin kills astrocytes, causing them to release LDH
into the media. After 6 h of incubation with 28 lM hemin, the percentage of cellular LDH released into the
culture media (Fig. 4A) was significantly greater than
that of control wells that had been incubated without
hemin (23.1 6 6.8% vs. 9.3 6 1.5%; P < 0.05). This difference increased further after 24 h incubation (37.6 6
5.6% vs. 7.8 6 0.9%; P < 0.05). Cell viability was also
assessed by quantifying the amount of protein present
in the culture wells. Incubation with hemin for 6 or 24 h
resulted in a decrease in protein content compared to
untreated culture wells (Fig. 4B). After 24 h, culture
wells incubated with hemin had significantly less protein content than culture wells incubated without hemin
(61.1 6 23.0 lg/well and 98.2 6 4.8 lg/well, respectively;
P < 0.05; Fig. 4B), indicating a loss of cell viability of
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HEMIN METABOLISM AND TOXICITY IN ASTROCYTES
DANG ET AL.
Fig. 5. Cell viability in H2O2 treated cells after preloading with
hemin or FAC. Cultures were incubated for 4 h with incubation medium
alone or with 29 lM hemin or 30 lM FAC before application of the indicated final concentrations of H2O2 and incubated for 2 (A), 6 (B) and 24 h
(C and D). The LDH activity released into the media (A–C) and the cellular protein content per well (D) was determined. Basal protein (t 5 0 h)
was 122.9 6 7.9 lg/well. One-way ANOVA were conducted using either
Tukey or Dunnett post hoc tests as appropriate. *Significant difference
compared to control and FAC-loaded cells (P < 0.05). Significant
difference compared to the 0 lM H2O2 condition within each control,
FAC-loaded or hemin-loaded cell condition; P < 0.05). àSignificant difference compared with control and hemin-loaded cells (P < 0.05).
try, indicating that it must be mediated by other
mechanisms.
increase in toxicity at 2 h is due to the direct effects of
H2O2 rather than to an interaction with hemin.
After 6 h, hemin toxicity was evident, as cells preloaded with hemin had a significantly greater LDH
release than control cells, even in the absence of H2O2
(P < 0.05; Fig. 5B). At this time point, incubation with
50 or 100 lM H2O2 significantly increased cell death in
cells that had been preloaded with hemin (P < 0.05; Fig.
5B). The increase in LDH released by cells preloaded
with hemin was significantly greater than control cells
for the 50 and 100 lM H2O2 condition, indicating that
cells preloaded with hemin were more vulnerable to
H2O2 toxicity (P < 0.05; Fig. 5B). After 24 h, the higher
susceptibility of cells preloaded with hemin to H2O2 toxicity is clearly evident, with these cells having a significantly greater LDH release than control cells, when
incubated with 50 or 100 lM H2O2 (P < 0.05; Fig. 5C).
At this time point, incubation of cells preloaded with
100 lM H2O2 resulted in a significant decrease in pro-
Hemin Toxicity Via Interactions with H2O2
It has been suggested that the interaction between
hemin and H2O2 may contribute to hemin toxicity
(Robinson et al., 2009); therefore, this hypothesis was
examined by preloading cells with hemin for 4 h before
subsequently incubating cells with various concentrations of H2O2 for up to 24 h. Incubation for 2 h with 50
or 100 lM H2O2 significantly increased the LDH release
from cells preloaded with hemin when compared with
0 lM H2O2 (P < 0.05; Fig. 5A). It should be noted that
there was no significant difference in the LDH release
following incubation with 100 lM H2O2 between control
cells and cells that were preloaded with hemin (P >
0.05; Fig. 5A), which suggests that the apparent
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1546
1547
Fig. 6. The effect of antioxidant compounds on H2O2 toxicity in cells
preincubated with hemin or FAC. Cultures were preincubated for 4 h
with 34 lM hemin (A and B) or 30 lM FAC (C and D), without or with
0.1 mM PHEN, 1 mM NAC, 0.5 mM Trolox or 1 mM PBN, before 0 lM or
100 lM H2O2 was applied and further incubated for up to 24 h without
(in NAC and Trolox preincubated cells) or with PHEN or PBN. LDH
released into the media (A and C) and cellular protein content (B and D)
were used as indicators of cell death and were assessed at 2, 6, and 24 h.
Basal protein (t 5 0 h) was 105.4 6 18.0 lg/well. One way ANOVA with
Dunnett one-sided post hoc tests (< hemin/FAC 1 H2O2 for LDH data; >
hemin/FAC 1 H2O2 for protein data) were conducted for each individual
time point. *Significant difference when compared to the hemin/FAC 1
H2O2 condition (P < 0.05). Independent samples t-tests were also conducted to compare the significantly different conditions (as determined
by the ANOVA) against the hemin/FAC only condition. Significant
difference when compared to the hemin/FAC only condition (P < 0.05).
tein content when compared with those incubated with
lower concentrations of H2O2, as well as when compared
with control and FAC-treated cells (70.7 6 6.5 lg/well,
106.1 6 16.9 lg/well and 106.6 6 19.4 lg/well, respectively; P < 0.05; Fig. 5D), confirming the decrease in cell
viability observed with the LDH assay.
To investigate whether free iron could be mediating
the increase in hemin toxicity in the presence of H2O2,
cells were preloaded with equivalent concentrations of
iron from FAC. Previous results from our laboratory
have shown that astrocytes accumulate similar amounts
of iron when using similar concentrations of hemin and
FAC. After 4 h, cells incubated with 34 lM hemin accumulated 37 6 4 nmol iron/mg protein (Dang et al.,
2010), while cells incubated with 30 lM FAC accumulated 44 6 7 nmol iron/mg protein. These amounts were
not significantly different from each other, as revealed
by an independent samples t-test (P > 0.05). Also, preincubating cells for 4 h with hemin, FAC or incubation
medium alone resulted in less than 5% LDH release
with no significant differences being detected between
control conditions (data not shown). After 2, 6 or 24 h,
the LDH release from cells preloaded with FAC was significantly greater when incubated with 100 lM H2O2
than without H2O2 (P < 0.05; Fig. 5A–C). However, after
2 and 6 h, the LDH release from cells preloaded with
FAC was not significantly different from control cells for
any of the H2O2 conditions, indicating that cells preloaded with FAC were no more susceptible than control
cells to concentrations of <100 lM H2O2 (P > 0.05; Fig.
5A,B). A significant difference was only detected after
24 h incubation with 100 lM H2O2 between cells preloaded with FAC and control cells (22.7 6 3.6% vs. 14.6
6 1.7%; P < 0.05; Fig. 5C), however the LDH release
from cells preloaded with FAC in this condition was still
significantly lower than that from cells preloaded with
hemin (P < 0.05; Fig. 5C). Since the FAC-loaded cells
contain more free iron, this result suggests that the
higher susceptibility to H2O2 of cells preloaded with
hemin is unlikely to be caused by the iron released
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HEMIN METABOLISM AND TOXICITY IN ASTROCYTES
DANG ET AL.
when hemin is metabolized. It should be noted that
there was no difference in the protein content detected
in culture wells preloaded with FAC and control cells
that were subsequently incubated with 100 lM H2O2
(Fig. 5D), suggesting that despite of the increased LDH
release observed under these conditions, the cells did
not detach from the culture wells.
Thus far the results demonstrate that the interaction
between hemin and H2O2 is a possible mechanism of hemin toxicity. To further investigate how hemin and H2O2
may be mediating toxicity, we examined whether PHEN,
NAC, Trolox, or PBN were able to attenuate this toxicity. Cells were preincubated for 4 h with hemin or FAC
together with PHEN or the antioxidant compounds
before being incubated with 100 lM H2O2. Preincubating cells for 4 h with hemin or FAC together with PHEN
or the antioxidant compounds, did not significantly alter
the cellular accumulation of iron from hemin or FAC
(data not shown); nor did these compounds significantly
affect the LDH released by cells that had been preincubated with either hemin or FAC alone (data not shown).
In cells preloaded with hemin, PHEN greatly attenuated
LDH release following incubation with 100 lM H2O2.
This attenuation was found to be significant after 6 h
(from 46.5 6 11.5% to 16.3 6 2.9%; P < 0.05; Fig. 6A)
and 24 h (from 55.1 6 9.1% to 30.0 6 6.1%; P < 0.05;
Fig. 6A); however, PHEN did not completely ameliorate
H2O2 toxicity, since the LDH release was still significantly greater than that of cells incubated without H2O2
(P < 0.05; Fig. 6A). NAC, Trolox, and PBN did not
attenuate LDH release in response to H2O2 in cells preloaded with hemin at any of the time points examined.
The protective effect of PHEN against hemin and H2O2
toxicity is confirmed with the Lowry protein assay. After
24 h, there was significantly greater protein content in
wells incubated with hemin alone or incubated with hemin, PHEN and 100 lM H2O2 when compared with cells
incubated with hemin and 100 lM H2O2 (100.5 6 6.3
lg/well, 82.7 6 7.7 lg/well and 56.2 6 11.5 lg/well,
respectively; Fig. 6B; P < 0.05). However, the protein
content of cells incubated with NAC, Trolox or PBN in
addition to hemin and H2O2 was not significantly
greater than cells incubated with hemin and H2O2 (Fig.
6B; P > 0.05).
PHEN was also able to protect cells preloaded with
FAC against H2O2 toxicity, with significant decreases in
LDH release being detected after 2 and 6 h (P < 0.05;
Fig. 6C). These decreased amounts of LDH release were
not significantly different from that of cells incubated
without H2O2, indicating that PHEN completely ameliorated H2O2 toxicity after 2 and 6 h in cells preloaded
with FAC (P > 0.05; Fig. 6C). There was also a trend for
Trolox to decrease the amount of LDH released from
cells preloaded with FAC after incubation with H2O2,
with a significant difference being detected at 24 h (from
21.5 6 1.9% to 11.1 6 4.5%; P < 0.05; Fig. 6C). NAC
and PBN did not significantly attenuate LDH release
after H2O2 incubation in cells preloaded with FAC at
any of the time points examined (P > 0.05; Fig. 6C).
When assessing the protein content of these culture
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wells at 24 h, no significant increases in protein were
detected between cells incubated with FAC and H2O2
compared to all other conditions (P > 0.05; Fig. 6D).
Therefore, a protective effect of PHEN and Trolox
against H2O2 toxicity in cells preloaded with FAC could
not be demonstrated with the Lowry protein assay.
DISCUSSION
This study aimed to better understand the mechanisms of hemin toxicity by examining hemin metabolism
and subsequent toxicity in primary astrocyte cultures. It
was demonstrated that astrocytes have the capacity to
metabolize 20% of the accumulated hemin. This study
found that the iron released from hemin is unlikely to
mediate toxicity and that hemin is unlikely to mediate
toxicity through direct interactions with H2O2 except
when peroxide concentrations are supraphysiological.
These findings highlight the potential of other byproducts of hemin metabolism to mediate toxicity in this culture system.
This study is the first to investigate the metabolism of
hemin by astrocytes. It was found that cells take up hemin rapidly, and that free iron can be detected in astrocytes within 30 min of exposure to hemin, indicating
that astrocytes have sufficient endogenous HO activity
to metabolize hemin. HO-1 expression in astrocytes was
also found to be greatly upregulated following hemin
accumulation. Despite this upregulation and the slowing
rate of hemin accumulation, the rate of hemin degradation did not appear to increase over the 24 h period,
such that the ratio of cellular hemin-iron to heminderived non-hemin-iron remained steady throughout the
24 h period. There are several potential explanations for
this stable ratio. One possibility is that the hemin
accumulated by astrocytes is inaccessible to the newly
synthesized HO-1, due to compartmentalization, precipitation or adsorbance of hemin to membranes or biomolecules (Tsiftsoglou et al., 2006). It is also possible that
the HO-1 is inactivated by peroxynitrite (Kinobe et al.,
2004), given that astrocytes increase their production of
peroxynitrite in response to hemin (Laird et al., 2008).
However, this option is inconsistent with the fact that at
all time points investigated, the ratio of hemin-iron to
non-hemin-iron remained steady; if HO-1 was progressively inactivated by peroxynitrite, the proportion of
non-hemin-iron should have decreased with time. A
more likely reason for the steady rate of metabolism
may be the export of hemin from the cells. Feline leukemia virus subgroup C receptor (FLVCR) and the multidrug resistant protein Bcrp/Abcg2 are transport proteins
involved in the export of hemin (Krishnamurthy et al.,
2007), however it is not known whether these proteins
are expressed in astrocytes or whether they are involved
in hemin export in the brain. It is possible that hemin
was released concomitantly with LDH, leading to underestimates of the intracellular concentrations of this compound. Since hemin remained in the culture medium
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1548
throughout the incubation period, it was not possible to
quantify hemin export in the present study.
Incubation with hemin resulted in the induction of
ferritin expression, confirming previous observations
(Regan et al., 2002). In this study however, we also
examined the presence and location of the iron released
from hemin, and found that the distribution of non-hemin-iron is very similar to that of ferritin. Both non-hemin-iron and ferritin labeling are first evident as granular staining in the soma and perinuclear region of the
cell. The iron and ferritin started to appear within the
processes of astrocytes at similar time points. This correspondence suggests that the iron released from hemin
metabolism is stored within ferritin, which would prevent the iron from participating in the Fenton reaction
and from contributing to the toxicity observed from incubation with hemin.
The current study found that 30 lM hemin is toxic to
astrocytes, which is consistent with previous studies
(e.g., Dang et al., 2010; Laird et al., 2008; Regan et al.,
2001, 2002). However, we found that PHEN did not
attenuate hemin toxicity, suggesting that iron released
from hemin metabolism does not mediate cell damage.
This finding is in contrast to reports that deferoxamine
protects mouse astrocytes from hemin toxicity (Laird et
al., 2008; Regan et al., 2001). Furthermore, contrary to
the findings of Laird et al. (2008), Trolox and NAC failed
to attenuate hemin toxicity in this study. These discrepancies may be attributable to species differences (rats vs.
mice) or to phenotypic differences that result from different cell culture harvesting procedures. This is evident by
the observation that in astrocyte cultures derived from
mice, hemin produced substantial cell death at a much
earlier time point (Laird et al., 2008; Regan et al., 2001)
than was observed in this study. In fact, the substantial
level of cell death observed in previous studies was not
achieved in this study, even after 24 h incubation with
equivalent concentrations of hemin.
Since iron is unlikely to contribute to hemin toxicity in
this study, the remaining 80% of hemin that was not
metabolized by HO-1 may be the cause of toxicity.
Recently, it has been suggested that intact hemin can
interact with H2O2 to mediate toxicity in cells (Robinson
et al., 2009). Intact hemin can engage directly in Fenton
reactions with H2O2 and generate hydroxyl radicals
(Huffman et al., 2000), and hemin can also initiate lipid
peroxidation in the presence of H2O2 (Gutteridge and
Smith, 1988; Klouche et al., 2004). This study demonstrated that exogenous H2O2 potentiated hemin toxicity,
and this additional cell loss was prevented by PHEN.
These results indicate that the iron released from hemin
only becomes toxic when supraphysiological levels of
H2O2 are present. Interestingly, hemin was more toxic
than iron from FAC in the presence of H2O2; when taken
together with the observation that Trolox was only able to
attenuate toxicity in cells preloaded with FAC, this suggests that the iron released from hemin may be in a different form to the iron accumulated from FAC.
The lipophilic nature of hemin may position the iron
closer to the membranes of organelles, making it more
1549
likely that hemin-iron induces lipid peroxidation. However, we consider this possibility unlikely since Trolox,
which is able to attenuate lipid peroxidation (Balla et
al., 1991; Klouche et al., 2004), did not protect astrocytes
from hemin toxicity in the absence of excess H2O2. Alternately, hemin may aggregate in regions of the cell that
are more likely to induce cell death when damaged. This
possibility is supported by a previous study demonstrating HO-1 expression in the perinuclear region, nucleus
and nucleoli of astrocytes (Li Volti et al., 2004), in addition to the results of this study showing that HO-1 and
the iron liberated from hemin are concentrated in the
perinuclear cytoplasm. Although the presence of exogenous H2O2 potentiates hemin toxicity, direct interactions
between H2O2 and intact hemin are unlikely to be a significant cause of hemin toxicity in astrocytes, since
PHEN was able to attenuate hemin toxicity in the presence of exogenous H2O2, but could not attenuate hemin
toxicity when no H2O2 was added. Therefore other
mechanisms of hemin toxicity must be considered. Chou
and Fitch (1981) found that free radical scavengers
were unable to protect mouse erythrocytes from hemin
toxicity. Instead, they found that hemin toxicity was
attributable to membrane cation leakage via unidentified mechanisms. Given that antioxidants were also
unable to protect astrocytes from hemin toxicity in this
study, it is possible that a similar mechanism of toxicity
may exist in astrocytes, although future studies are
needed to clarify this possibility.
An alternative mechanism may involve bilirubin and/
or carbon monoxide (CO), since these byproducts of hemin metabolism can exert toxic effects. Micromolar concentrations of bilirubin can inhibit the uptake of glutamate into brain cells, and can induce inflammation, oxidative stress and apoptosis in rat astrocytes and
neurons (Brito et al., 2008; Fernandes et al., 2007; Silva
et al., 2002). However further studies are needed to
elucidate how much bilirubin is accumulated within
astrocytes following incubation with hemin. In addition,
the role of CO needs to be investigated as it has a strong
affinity for heme groups, and when present in excess,
CO can bind to cytochrome c oxidase in the electron
transport chain and block cellular respiration (Alonso
et al., 2003; D’Amico et al., 2006). The preceding
observations make it possible that the high production
of bilirubin and CO, may compromise cell viability and
contribute to the toxicity observed following incubation
with hemin.
This study has demonstrated that intact hemin, or
non-hemin-iron produced by hemin metabolism, is
unlikely to cause toxicity through interactions with
endogeneous H2O2. This novel finding suggests that hemin toxicity is due to other mechanisms such as membrane cation leakage, or to the breakdown products of
hemin. A deeper understanding of hemin metabolism,
and the roles of bilirubin and CO may help to elucidate
why some studies find the actions of HO beneficial to
cells (Benvenisti-Zarom and Regan, 2007; Chen-Roetling
and Regan, 2006) while others find it detrimental
(Koeppen et al., 2004; Wang and Dore, 2007). InvestigaGLIA
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HEMIN METABOLISM AND TOXICITY IN ASTROCYTES
DANG ET AL.
tions of whether CO or bilirubin mediate hemin toxicity
may aid the development of new therapeutic interventions for hemorrhagic stroke.
ACKNOWLEDGMENTS
The authors thank Dr Alfons Lawen from the Department of Biochemistry and Molecular Biology, Monash
University for the use of his spectrophotometer.
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