Tài liệu Báo cáo khoa học: Intralysosomal iron chelation protects against oxidative stress-induced cellular damage doc

Intralysosomal iron chelation protects against oxidative
stress-induced cellular damage
Tino Kurz1, Bertil Gustafsson2 and Ulf T. Brunk1
1 Division of Pharmacology, Faculty of Health Sciences, Linkoping University, Sweden
ă
2 Department of Pathology and Cytology, University Hospital, Linkoping, Sweden
ă

Keywords
cell death; lysosomes; mitochondria; redoxactive iron; salicylaldehyde isonicotinoyl
hydrazone
Correspondence
U. T. Brunk, Department of Pharmacology,
University Hospital, SE-581 85 Linkoping,
ă
Sweden
Fax: +46 13 149106
Tel: +46 13 221515
E-mail:
(Received 1 March 2006, revised 28 April
2006, accepted 15 May 2006)
doi:10.1111/j.1742-4658.2006.05321.x

Oxidant-induced cell damage may be initiated by peroxidative injury to
lysosomal membranes, catalyzed by intralysosomal low mass iron that
appears to comprise a major part of cellular redox-active iron. Resulting
relocation of lytic enzymes and low mass iron would result in secondary
harm to various cellular constituents. In an effort to further clarify this
still controversial issue, we tested the protective effects of two potent iron
chelators – the hydrophilic desferrioxamine (dfo) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (sih), using cultured lysosome-rich macrophage-like J774 cells as targets. dfo slowly enters cells via endocytosis,
while the lipophilic sih rapidly distributes throughout the cell. Following

dfo treatment, long-term survival of cells cannot be investigated because
dfo by itself, by remaining inside the lysosomal compartment, induces
apoptosis that probably is due to iron starvation, while sih has no lasting
toxic effects if the exposure time is limited. Following preincubation with
1 mm dfo for 3 h or 10 lm sih for a few minutes, both agents provided
strong protection against an ensuing $LD50 oxidant challenge by preventing lysosomal rupture, ensuing loss of mitochondrial membrane potential,
and apoptotic ⁄ necrotic cell death. It appears that once significant lysosomal
rupture has occurred, the cell is irreversibly committed to death. The
results lend strength to the concept that lysosomal membranes, normally
exposed to redox-active iron in high concentrations, are initial targets of
oxidant damage and support the idea that chelators selectively targeted to
the lysosomal compartment may have therapeutic utility in diminishing
oxidant-mediated cell injury.

Exposing cells in culture to increasing oxidative stress
triggers a range of cellular events. Depending on the
cell type, these may include enhanced proliferation or
growth arrest, DNA damage, protein and lipid oxidation, apoptosis, and finally necrosis [1]. This points to
an important physiological role of redox regulation in
cellular homeostasis [2,3]. While most current studies
suggest a direct effect of oxidative stress on DNA and
mitochondria followed by apoptosis or necrosis, recent
research has established a critical role for lysosomes in

the initiating phase of impairment [4–13]. Because
hydrogen peroxide, added in moderate concentrations
as a bolus, may be consumed within minutes [14] and
most cellular alterations, including apoptosis, do not
occur until hours later, any satisfactory hypothesis on
the mechanisms behind oxidative stress-induced cellular

damage must provide firm and distinct links between
the triggering events and the ultimate cellular injuries.
There are indications that hydrogen peroxide per se
has little harmful effects and that an interaction

Abbreviations
AO, acridine orange base; dfo, desferrioxamine; pHPA, p-hydroxy-phenylacetic acid; PI, propidium iodide; sih, salicylaldehyde isonicotinoyl
hydrazone (N¢-[(1Z)-(2-hydroxyphenyl)methylene]isonicotinohydrazide); SSM, sulfide-silver method; TMRE, tetramethylrhodamine ethyl ester;
Ym, mitochondrial membrane potential.

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T. Kurz et al.

between redox-active iron and hydrogen peroxide is
required in order to initiate damage [10,12,15]. The
acidic vacuolar compartment is rich in low mass iron
that would put these organelles in focus as initial targets for oxidative damage [16–19]. Lysosomes, which
together with late endosomes constitute the acidic vacuolar compartment, are the main cellular structures for
normal autophagic turnover of organelles and longlived proteins [20–22]. Autophagic degradation of ferruginous material, such as ferritin and cytochromes, is
responsible for the intralysosomal occurrence of redoxactive low molecular weight iron [23] before it is transported out of the lysosomal compartment for use in a
variety of anabolic processes, e.g. synthesis of ironcontaining macromolecules, while excess iron is stored
in ferritin. This, along with the participation of iron
in Fenton-type reactions producing hydroxyl radicals
(HO•), or similarly reactive iron-centered [oxidoiron(IV)] radicals, would account for the sensitivity of
lysosomes to oxidative stress that, if intense enough,
may result in lysosomal rupture and release to the

cytosol of harmful contents with ensuing cellular damage, including apoptosis and necrosis [4–6,24,25].
Although Christian de Duve, the discoverer of lysosomes, envisaged such a possibility by nicknaming
lysosomes ‘suicide bags’ [26], lysosomes are today
often – although wrongly, we believe – considered to
be sturdy organelles that usually do not rupture until
cells are already dead and necrotic.
We have previously shown that cells exposed for
1–3 h to high (‡ 1 mm) concentrations of desferrioxamine, dfo (either in free form or as a high molecular
weight conjugate to starch, HMW-dfo), are substantially, although not fully, protected against oxidative
stress [10]. dfo (or HMW-dfo), being a strong hydrophilic iron chelator that firmly binds all six coordinates of iron, preventing its iron(II) ⁄ iron(III)
redox-cycling, does not pass membranes but is fluid
phase-endocytosed by cells in culture, passes through
late endosomes and, because of the extensive fusion
and fission activities of the lysosomal compartment, is
transferred to most lysosomes [13,27–30]. As dfo
remains intralysosomal, it will act as a sink for cellular iron in transit through the lysosomal compartment
and, thus, within hours, cells will start to become
affected by iron starvation and finally die [5,12]. Interestingly, dfo-induced apoptosis, and a variety of other
apoptogenic stimuli, involves lysosomal destabilization, suggesting this phenomenon to be related to
apoptosis in general and not only to oxidative stress
[5,6,31,32].
Recently, it was demonstrated that the lipophilic
iron chelator sih (salicylaldehyde isonicotinoyl hydra-

Oxidative stress and intralysosomal iron

zone, systematic name: N¢-[(1Z)-(2-hydroxyphenyl)
methylene]isonicotinohydrazide) fully protects against
oxidative stress-induced mitochondrial and cellular
damage when present in low concentrations during

the oxidative stress period [15,33]. sih was first synthesized in 1953 [34] and its iron binding ability was
demonstrated 20 years ago [35]. Its binding constant
for Fe(III) is 1050 at pH 7.4 [36]. In the present
study, by exposing lysosome-rich macrophage-like
J774 cells to oxidative stress, either in the presence
of sih or following pretreatment with dfo, we find
strong evidence for a primary role of lysosomal
redox-active iron in oxidative stress-induced cell damage and a close correlation between initial lysosomal
rupture and later development of cellular damage
and death.

Results
Hydrogen peroxide-induced mitochondrial injury
and cell death are downstream effects of
lysosomal rupture
In order to confirm and add to earlier findings on the
sequence of events with respect to lysosomal rupture,
mitochondrial injury and apoptosis ⁄ necrosis following
oxidative stress [37], we first assessed lysosomal stability by cytofluorometric evaluation of alterations in
green and red fluorescence, respectively, of cells vitally
stained with acridine orange (AO) before (AO-relocation test [6,10,13,38–40]) and at 6 h after the oxidative
stress period (AO-uptake test [6,10,13,32,40,41]). AO is
a weak base (pKa $10) that, due to proton trapping,
preferentially distributes within the acidic vacuolar
(lysosomal) cellular compartment [4–6,24,25,42–44].
Due to its metachromatic properties, this probe fluoresces red inside lysosomes, where it is highly concentrated, and weakly green in the cytosol and the
nucleus, where it is much less concentrated. When used
as a vital stain at low concentrations, the intercalation
of AO into RNA and DNA is very low and does not
disturb the evaluation of lysosomal stability. Ordinary

photomultipliers are about 10-fold more sensitive to
green than to red photons, making the AO-relocation
test useful for the early detection of a limited number
of ruptured lysosomes.
As shown in Fig. 1, unprotected cells showed lysosomal destabilization that was detectable by the
AO-relocation method as early as 15 min following the
end of the oxidative stress period (peroxidative lysosomal membrane damage develops by time). Using the
AO-uptake technique, unprotected cells showed a substantial increase in ‘pale’ cells (cells with a reduced

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Fig. 1. Lysosomal rupture is an early event
after oxidative stress. Acridine orange (AO)relocation assay. Cells (106) were preloaded
with the metachromatic fluorophore and
lysosomotropic base AO. Following two
washing steps in culture medium, they
were then exposed for 30 min to 100 lM
H2O2 in 2 mL NaCl ⁄ Pi with ⁄ without 10 lM
sih and returned to standard culture conditions for 15 min. Lysosomal stability was
assayed by the AO-relocation technique
using flow cytofluorometry in the green FL1
channel. Due to release of AO from ruptured lysosomes into the cytoplasm, oxidative stress resulted in an early (15 min)
increase of the mean value for the green

cytoplasmic fluorescence that was
significantly prevented by sih-protection
(mean ± SD; ***P < 0.001; n ¼ 8).
Examples of green fluorescence histograms
are shown above each bar.

Fig. 2. Iron chelation protects against lysosomal rupture by oxidative stress. Acridine
orange (AO)-uptake assay. Cells, either protected by sih or not, were exposed to oxidative stress as described for Fig. 1. Other
cells were initially pretreated for 3 h with
1 mM dfo under otherwise standard culture
conditions before exposure to the same oxidative stress. Following end of the stress
period, cells were returned to standard culture conditions for an additional period of
6 h when lysosomal stability was assessed
using the AO-uptake method. Ruptured
lysosomes do not take up AO resulting in a
population of cells with reduced red fluorescence (‘pale’ cells). The number of ‘pale’
cells was reduced highly significantly in cells
protected by sih or dfo (mean ± SD;
***P < 0.001; n > 6). For each bar a representative histogram of red fluorescence,
with ‘pale cells’ gated, is given.

number of intact lysosomes) after 6 h (Fig. 2), when
these cells started to show apoptotic alterations as described previously [22]. Cells protected by the lipophilic
iron chelator sih, or the hydrophilic iron chelator dfo
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were highly protected against both early and late lysosomal rupture [13].
dfo is known to induce iron starvation and related
cell death and cannot be used in long-term experiments

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T. Kurz et al.

Oxidative stress and intralysosomal iron

Fig. 3. Disruption of mitochondrial membrane potential is a down-stream effect of
lysosomal rupture. Cells, protected against
oxidative stress by sih or not, were exposed
to 100 nM tetramethylrhodamine ethyl ester
(TMRE) for 15 min under standard culture
conditions 1–8 h following the oxidative
stress period. Red fluorescence was analyzed in the FL3 channel by flow cytofluorometry. Damaged mitochondria with
depolarized membranes show reduced
TMRE uptake. In unprotected cultures, significant mitochondrial damage was observed
only 6 and 8 h after end of the oxidative
stress period, while sih-protected cells
showed almost no increase in damaged
mitochondria (mean ± SD; ***P < 0.001;
**P < 0.01; n ¼ 4). At top of the panel,
examples of histograms are given showing
red fluorescence 8 h following end of oxidative stress. Cells with reduced red fluorescence were gated.

[5]. dfo is taken up by endocytosis [27,28], although
this is not generally recognized (often it is just considered to pass membranes very slowly), remains intralysosomal and causes iron starvation [5,12]. Thus, only
the lipophilic sih, which quickly redistributes, was used
in the remaining experiments. As is evident from
Fig. 3, mitochondrial membrane potential was preserved by sih protection, indicating (as suggested
before [6,22,37,40,45]) that mitochondrial damage is

secondary to lysosomal rupture and related to iron-catalyzed intralysosomal peroxidation.
Similarly, sih-protection prevented the oxidative
stress-induced decline in cell numbers (Fig. 4) and postapoptotic necrosis (Fig. 5). Actually, sih-protected
cells multiplied similarly to control cells if exposed to
sih only during the oxidative stress period (Fig. 4),
while cells exposed to >5 lm sih for long periods of
time finally all died by iron-starvation (results not
shown).
Cells degraded hydrogen peroxide as shown before
[22], and pretreatment with dfo or sih did not influence
the rate (results not shown).
In all experiments, cells exposed to sih without oxidative stress behaved like unexposed controls.

Fig. 4. sih-protected cells retain normal proliferation capacity following oxidative stress. Cells were seeded (500 000 ⁄ well) and 24 h
later exposed to oxidative stress, with or without sih-protection as
described before. Directly after end of the oxidative stress period
(0 h) and again after return to standard culture conditions for
another 12 and 24 h, cells were fixed in 4% formaldehyde in
NaCl ⁄ Pi and counted in five predefined areas per dish (mean ± SD;
***P < 0.001; **P < 0.01; *P < 0.05; n.s., nonsignificant; n ¼ 4).
After a short lag-phase, sih-protected cells continued to proliferate
normally, while about half of the unprotected cells underwent apoptotic ⁄ necrotic cell death.

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Fig. 5. sih-protected cells show no decrease
in viability following oxidative stress. Cells
were seeded (500 000 ⁄ well) and 24 h later
exposed protected or unprotected to oxidative stress as described before and were
then returned to standard culture conditions
for another 24 h. The cells were then
scraped, exposed to 40 lgỈmL)1 propidium
iodide for 90 min, centrifuged and washed
in NaCl ⁄ Pi ⁄ centrifuged twice. Red fluorescence of PI-stained nuclei was measured by
flow cytofluorometry in the FL3 channel
(mean ± SD; ***P < 0.001; n ¼ 3). sih
strongly protected cells against oxidative
stress-induced postapoptotic necrosis.
Examples of red fluorescence histograms
are given. PI-positive cells were gated.

Cellular labile iron is located predominantly
inside lysosomes
To prove that cellular labile iron is located predominantly inside lysosomes, we utilized the sulfide-silver
method (SSM), which is a very sensitive cytochemical
technique to demonstrate heavy metals [18,41,46]. As
iron is the dominating heavy metal in most normal
cells (the exceptions being some zinc-containing neurons and endocrine cells), it can be considered specific
for this transition metal. As shown in Fig. 6 (and our
previous publications, reviewed in [45]), the outcome
of the reaction is a mainly granular staining with a distinct lysosomal pattern when the SSM is applied to
normal J774 cells in culture. The SSM is an auto-catalytic procedure and a short development time results in
few stained lysosomes, while a longer development

results in a more general staining of lysosomes (Fig. 6,
compare parts A, B and C). This shows that the
amount of redox-active iron varies between lysosomes,
probably depending on the extent of recent engagement in autophagic degradation of ferruginous material [22,47], explaining why there is a pronounced
heterogeneity between lysosomes with respect to sensitivity to oxidative stress [47].
The fact that some cells and individual lysosomes
resist oxidative stress better than others is considered
an important and not well understood phenomenon
[48]. We have previously suggested that the difference
in cellular and lysosomal amount of redox-active iron
could be a major cause [47].
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In order to show that the SSM does indeed demonstrate iron, we exposed some cell cultures to 30 lm
FeCl3 for 3 h before the SSM. In the culture medium,
iron ions complex with phosphate groups under the
formation of a hydrated iron phosphate complex,
which is endocytosed by cells and transported to the
lysosomal compartment [22,41]. Following such iron
uptake, a lysosomal staining pattern was obvious
already after 30 min development time, while the control cells showed no labeling (Fig. 6A and D).

Discussion
Using the cytochemical SSM and the calcein technique
in combination with induction of lysosomal rupture,
we have here and previously demonstrated that lysosomes contain a major part of the cellular redox-active
low mass iron, making the lysosomal compartment
notably vulnerable to oxidative stress [12,18,22,41].
The lysosomal concentration of low mass iron differs
between the lysosomes of individual cells as well as

between different cells, probably reflecting the participation of individual lysosomes in the autophagic degradation of ferruginous material. These variations may
explain the obvious differences in the sensitivity to oxidative stress of individual lysosomes of the same cell
and of different cells of the same population [47,48].
Lysosomes are a heterogeneous group of vesicular
structures and, at a given point in time, some lysosomes are performing degradation, while others are
‘resting’ [20–22,47]. Those engaged in the degradation

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Oxidative stress and intralysosomal iron

Fig. 6. Cytochemical demonstration of iron by the sulfide-silver method (SSM). With increasing development time (30–60 min) control cells
(A–C) show an increasingly intense lysosomal pattern of black granular silver precipitates, indicating the presence of lysosomal low molecular
weight iron. While there was no granular staining after a 30-min period of development (A), occasional granules were evident in some cells
(examples are indicated by arrow heads) after 40 min (B) and a distinctly granular staining with a lysosomal pattern was found in all cells
after 60 min of development (C). Cells exposed for 3 h to a hydrated iron phosphate complex (obtained by adding FeCl3 to the culture medium to a final concentration of 30 lM) showed many stained lysosomes after development for 30 min (D) when the control cells (A) were
still empty. This finding reflects the fluid phase endocytosis of the iron phosphate complex, as well as the capacity of the method to demonstrate iron.

of iron-containing macromolecules may contain a high
concentration of low-mass iron, while a ‘resting’ lysosome would contain almost none [21,49,50], explaining
our finding (Fig. 6) that some lysosomes contain much
more iron than others.
Although iron is an essential transition metal
required for many vital functions, including electron
transport, it is potentially dangerous because of
its capacity to participate in Fenton-type reactions
(Eqn 1).

Fe2ỵ ỵ H2 O2 ! Fe3ỵ ỵ HO ỵ OH

1ị

Hydroxyl radicals (HOã) are short lived ($10)9 s),
extremely reactive, and able to bring about oxidative
injury to a variety of biomolecules. Consequently, cells
and organisms handle iron with great care and usually
hide it within stable metallo-organic complexes,
thereby preventing hydrogen peroxide from encountering redox-active iron; an exception being low mass
iron in late endosomes and lysosomes as well as, perhaps, a small amount of low mass iron in transit from
these structures for storage in ferritin or use in the synthesis of iron-containing macromolecules [10–12].

As lysosomes break under oxidative stress, as proven
here and earlier with the AO-relocation and -uptake
tests [6,10,13,32,38–41], we may assume that a certain
fraction of lysosomal low mass iron exists in iron(II)
form. Most probably this is related to the low lysosomal pH and presence of reducing equivalents, such
as cysteine [51–53]. At pH 5, cysteine reduces iron(III)
to iron(II), as has been shown before [22]. This reduction of iron (Eqn 2) is driven by the removal of Cys-S•
(Eqn 3) and the subsequent reduction of dioxygen
(Eqn 4) [54,55].
Cys SH ỵ Fe3ỵ ! Cys S ỵ Fe2ỵ ỵ Hỵ

2ị

Cys S ỵ Cys À SH ! ðCys À S À S À CysÞÀ þ Hþ ð3Þ
À

ðCys À S À S À CysÞÀ þ O2 ! Cys S S Cys ỵ O2

ð4Þ

Autophagy is a normal and continuously ongoing
process that allows a fine-tuned turnover of organelles
and most long-lived macromolecules being of major
importance for intracellular iron turnover [21,49,50,56].
Due to intralysosomal degradation of a large variety of
biomolecules, including many ferruginous materials,

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such as ferritin, mitochondrial complexes and various
other metalloproteins, low-mass iron is set free intralysosomally. As an effect, a substantial part of such iron
seems to be temporarily harbored within these organelles before being transported, by not yet well characterized carrier systems, to the cytosol and used for
anabolic purposes or stored in ferritin [11,12,22,23,57].
Differences between cells of different origin with
respect to lysosomal stability to oxidative stress may
reflect a divergence in their capacity to degrade hydrogen peroxide, while differences in amounts of intralysosomal redox-active iron may explain intra- and
intercellular variation [47,58].
Here we exposed 106 cells in 2 mL NaCl ⁄ Pi to a
bolus dose of 100 lm hydrogen peroxide that is rapidly and exponentially degraded under these conditions. Due to efficient intracellular degradation of
hydrogen peroxide, a steep gradient across the plasma

membrane is established and the actual concentration
of hydrogen peroxide sensed by the lysosomes would
probably not exceed $15 lm soon after the start of
the oxidative stress [14]. As lysosomes do not contain
any catalase or glutathione peroxidase, this initial
magnitude of oxidative stress proved sufficient to
induce lysosomal rupture that initially was of limited
scale. Lysosomal rupture accelerated by time through
a lipid peroxidation chain reaction until it initiated
apoptosis, probably by a direct or indirect effect on
mitochondrial stability [37,59,60]. This lysosomal rupture results in release to the cytosol of powerful
hydrolytic enzymes and redox-active iron that,
depending on the magnitude of the rupture, may initiate a variety of cellular injuries, including reparative
autophagocytosis, apoptosis and necrosis [45]. Clearly,
lysosomes are not the sturdy organelles they once
were believed to be, breaking only late during necrotic
(accidental) cell death.
Oxidative stress-induced lysosomal rupture, secondary mitochondrial injury and final cell death were
almost fully prevented by a pre-exposure to the potent
iron chelators sih and dfo, as was also shown previously [37,45], suggesting that the damaging effect of
hydrogen peroxide per se is of minor importance in
comparison to its iron-mediated effect on lysosomal
stability. Even if the importance of lysosomal rupture
for oxidative stress-induced damage, including apoptosis and DNA damage, has been shown before [5,10–
13], this view is still controversial. A major reason for
the present lack of general acceptance of an initiating
role of lysosomal break in oxidative stress-induced
injury may be that dfo, which often has been used to
chelate intralysosomal iron, works like a double-edged
sword. It does prevent early lysosomal damage under

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oxidative stress, but after some time dfo itself induces
apoptosis [5]. The reason for this phenomenon seems
to be that dfo following endocytotic uptake remains
inside the lysosomal compartment where it scavenges and acts as a sink for iron that is in transit through the compartment as a result of autophagy
[11,12,21,49,50].
The lipophilic iron chelator sih does not, however,
have this disadvantage. sih swiftly penetrates membranes and binds iron very strongly throughout the cell
in a non redox-active form, also at lysosomal pH [36].
In a recent study, it was shown that in the presence of
sih, cells show no mitochondrial damage following oxidative stress, indicating the need of free iron for such
damage [15]. Here we add the information that the
defense is mediated by lysosomal stabilization and that
cells protected by sih during oxidative stress and then
brought back to normal culture conditions continue to
proliferate normally. Because of its lipophilicity, sih
enters and leaves cells rapidly and, in contrast to dfo,
it can be used to chelate intracellular redox-active iron
for a limited period of time without causing long-lasting effects.
A key aspect of oxidative regulation of physiological processes is the disparity of the time-scales
involved. The apoptotic ⁄ necrotic process takes several
hours to fully develop, although cells need to be
exposed to hydrogen peroxide for only a short period of time to be committed to apoptosis ⁄ necrosis.
The very sensitive AO-relocation technique to detect
early lysosomal rupture allowed us to observe a
strong correlation between a hydrogen peroxideinduced cellular modification that occurred rapidly,
by induction of partial lysosomal rupture, and the
signs of apoptosis ⁄ necrosis hours later. Both processes showed the same dose-dependent response to
hydrogen peroxide and were inhibited by dfo- and

sih-mediated iron chelation during the period of oxidative stress.
Release of lysosomal contents initiates a process that
results in mitochondrial destabilization as well as further lysosomal rupture [37]. In this context, it is worth
mentioning that the change in mitochondrial membrane potential that was demonstrated, as well as
release of cytochrome c from mitochondria under similar conditions [37,61] does not occur until 1–3 h after
the end of exposure to hydrogen peroxide and long
after the lysosomal rupture is observed. Interestingly,
we observed a progressive decrease in the number of
intact lysosomes over time when the cells were no longer under oxidative stress, which is in accordance with
a self-amplifying loop and cross-talk between lysosomes and mitochondria (Fig. 7), as previously pointed

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Oxidative stress and intralysosomal iron

Fig. 7. The lysosomal-mitochondrial pathway of cell death; a tentative scheme. Slightly modified from Zhao et al. [37], the scheme shows
intralysosomal Fenton-type reactions resulting from oxidative stress. Lysosomal contents are released to the cytosol following lysosomal
destabilization and may activate pro-apoptotic proteins, such as Bid [59,60], and ⁄ or attack mitochondria with release of cytochrome c and
enhanced production of superoxide. Released lysosomal enzymes (LE) may also activate cytosolic phospholipases, which in turn may attack
mitochondria and lysosomes, inducing a self-amplifying loop. Released redox-active iron may bind to nuclear and mitochondrial DNA and
induce site-specific damage under continuous oxidative stress [10,12].

out as the foundation of the lysosomal-mitochondrial
axis theory of apoptosis [37].
Taken together, the findings of this study suggest
that the major effect of harmful oxidative stress, rather
than being a direct effect on targets such as DNA and

mitochondria, is a result of lysosomal rupture due to
intralysosomal peroxidative events, with ensuing relocalization of lysosomal hydrolytic enzymes and lowmass iron.

Experimental procedures
Chemicals
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum, penicillin and streptomycin were from Gibco (Paisley,
UK). Acridine orange base was from Gurr (Poole, UK),
while silver lactate was from Fluka AG (Buchs, Switzerland).
Glutaraldehyde was from Bio-Rad (Cambridge, MA, USA),
and ammonium sulfide and hydroquinone were from BDH
Ltd (Poole, UK). dfo was from Ciba-Geigy (Basel, Switzerland). sih was a kind gift from D. Richardson (University of
New South Wales, Sydney, Australia). All other chemicals
were from Sigma (St Louis, MO, USA).

Cell culture and exposure to hydrogen peroxide
with ⁄ without iron chelator protection
Murine macrophage-like J774 cells (ATCC, Manassas, VA,
USA) were grown in DMEM supplemented with 10% fetal

bovine serum, 2 mm l-glutamine, 100 ImL)1 penicillin
and 100 lgỈmL)1 streptomycin, at 37 °C in humidified air
with 5% CO2. The cells were subcultivated twice a week,
plated at a concentration of 1 · 106 cells per well in six-well
plates, with or without cover-slips, and typically subjected
to oxidative stress after another 24 h.
Concentrations of hydrogen peroxide and exposure times
(in relation to cell density) and exposure to sih and dfo
were established in preliminary experiments. In final experiments, control and chelator-protected cells were oxidatively
stressed (or not) for 30 min by exposure to a bolus dose of

100 lm H2O2 in 2 mL NaCl ⁄ Pi at 37 °C. Note that under
these conditions the H2O2 concentration declines quickly
(t1 ⁄ 2 $15 min) to < 20 lm after 30 min (see below). dfo
(1 mm) was added to the culture medium under otherwise
standard conditions 3 h before oxidative stress. sih was prepared as a 10 mm solution in dimethyl sulfoxide and then
diluted to a 1 mm stock solution in absolute ethanol. To
produce a final concentration of 10 lm sih during the oxidative stress exposure, some of the stock was added to the
NaCl ⁄ Pi immediately prior to the addition of hydrogen peroxide. No sih pretreatment was found necessary. After the
oxidative stress period, cells were directly analyzed or
returned to standard culture conditions and assayed at indicated periods of time.
In some experiments, cells were incubated for 3 h in
complete medium with FeCl3 added to a concentration of
30 lm (resulting in the formation of a nonsoluble iron
phosphate complex that is endocytosed and transported
into the lysosomal compartment).

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Degradation of hydrogen peroxide
To ensure that the observed resistance to oxidative stress
was not an effect of enhanced H2O2 catabolism induced by
the iron-chelating drugs, the rate of H2O2 clearance was
determined. Control cells and cells protected by the iron

chelators (in concentrations described above) were exposed
to a bolus dose of 100 lm H2O2 in 2 mL NaCl ⁄ Pi at 37 °C.
During a 60-min period, aliquots (50 lL) were sampled for
H2O2 analysis by the horseradish peroxidase-mediated
H2O2-dependent p-hydroxy-phenylacetic acid (pHPA) oxidation technique [62]. Fluorescence intensity was read
(kex315 nm; kem410 nm) using a RF-540 spectrofluorometer (Shimadzu, Kyoto, Japan) connected to a DR-3 data
recorder.

Lysosomal membrane stability assay
Six hours after the oxidative stress period (see above), cells
were exposed to 10 lgỈmL)1 acridine orange (AO) in complete medium at 37 °C for 15 min, detached by scraping and
collected for flow cytofluorometric assessment of lysosomal
AO-uptake. AO is a metachromatic fluorophore and a lysosomotropic base (pKa ¼ 10.3), which becomes charged
(AOH+) and retained by proton trapping within acidic compartments, mainly secondary lysosomes (pH 4.5–5.5). When
normal cells are excited by blue light, highly concentrated
lysosomal AO emits an intense red fluorescence, while nuclei
and cytosol show weak diffuse green fluorescence. In AOuptake experiments, red fluorescence was measured (FL3
channel) using a Becton-Dickinson FACScan (Becton-Dickinson, Mountain View, CA, USA) equipped with a 488 nm
argon laser. Cells with a reduced number of intact,
AO-accumulating lysosomes (here termed ‘pale’ cells) were
detected as described earlier [6,10,13,32,40,41].
The AO-relocation technique [6,10,13,38–40] was used to
measure early lysosomal damage. For this assay, cells were
preloaded with AO (10 lgỈmL)1) for 15 min in complete
culture medium, rinsed with culture medium and kept
under standard culture conditions for a further 15 min
before being exposed to oxidative stress. After the oxidative
stress period cells were returned to standard culture conditions for 15 min and then scraped and assayed by flow
cytofluorometry. The increase in green cytoplasmic fluorescence, due to the release of AO from ruptured lysosomes,
was measured in the FL1 channel. cellquest software (BD

Biosciences, Franklin Lakes, NJ, USA) was used for acquisition and analyses.

Mitochondrial membrane potential assay
Mitochondrial membrane potential (Ym) was measured by
flow cytofluorometry, using the cationic and lipophilic
dye tetramethylrhodamine ethyl ester (TMRE), which

3114

accumulates in the mitochondrial matrix. Decreased Ym is
indicated by a reduction of the TMRE-induced red fluorescence. At different points of time (1–8 h) following the end
of oxidative stress (see above), cells were incubated with
TMRE in complete culture medium (100 nm; 15 min;
37 °C) and assayed by flow cytofluorometry. Red (FL3
channel) fluorescence was recorded in a log scale and analyzed using the cellquest software. Cells with reduced red
fluorescence were gated.

Assessment of cell proliferation
Five hundred thousand cells were seeded per well and
exposed for 30 min to oxidative stress (or not) 24 h later
with 10 lm sih present (or not). Directly after oxidative
stress, and after another 12 and 24 h under standard culture conditions, cells were washed in NaCl ⁄ Pi and fixed in
4% formaldehyde in NaCl ⁄ Pi. For each condition cells
were counted in five predefined areas of two separate
dishes. The cell proliferation experiments were done twice.

Assessment of postapoptotic necrotic cells
Cells were seeded and treated as described above for the
cell proliferation assay. The magnitude of oxidative stress
applied is known to induce apoptosis but little direct necrosis [22]. After 24 h under standard conditions following the

oxidative stress, cells were scraped and exposed in the dark
for 90 min to 40 lgỈmL)1 propidium iodide in complete
culture medium at 22 °C. Cells were then centrifuged and
washed in NaCl ⁄ Pi twice before red fluorescence was analyzed by flow cytofluorometry using the FL3 channel.
Propidium iodide does not cross the plasma membrane of
normal or early apoptotic cells, while it penetrates into postapoptotic necrotic cells and binds to nuclear DNA.

Cytochemical assay of lysosomal reactive iron
For evaluation of cellular low-mass iron, we used the autometallographic sulfide-silver method as previously described
[18], modified (high pH; high S2–) from Timm [46]. Cells
were grown on cover-slips and exposed, or not, for 3 h to
an insoluble hydrated iron phosphate complex, obtained by
addition of FeCl3 to complete culture medium to a final
concentration of 30 lm. Cells were rinsed briefly in NaCl ⁄ Pi
(22 °C) prior to fixation with 2% glutaraldehyde in 0.1 m
sodium cacodylate buffer with 0.1 m sucrose (pH 7.2) for
2 h at 22 °C. The fixation was followed by five short rinses
in glass-distilled water at 22 °C. Cells were then sulfidated
at pH $9 with 1% (w ⁄ v) ammonium sulfide in 70% (v ⁄ v)
ethanol for 15 min. Following careful rinsing in glassdistilled water for 10 min at 22 °C, development was performed using a physical, colloid-protected developer
containing silver-lactate and hydroquinon (the method is an

FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS


T. Kurz et al.

autocatalytic one and the precipitation of metallic silver on
the FeS core is dependent on time and the amount of initiating FeS). The reaction was performed in the dark at
26 °C for various periods of time (30–60 min). Following

dehydration in a graded series of ethanol solutions and
mounting in Canada balsam, the cells were examined and
photographed, using transmitted light, under an Axioscope
microscope (Zeiss, Oberkochen, Germany) connected to a
Zeiss ZVS-47E digital camera. easy image measurement
2000 software (version 2.3, Bergstrom Instruments AB,
ă
Solna, Sweden) was used for image acquisition.

Oxidative stress and intralysosomal iron

8

9

10

Statistical analysis
Results are given as mean ± SD. Statistical comparisons
were made using analysis of variance (anova), whereby
pair-wise multiple comparisons were made using Tukey’s
adjustment. For comparison of two means, Student’s t-test
was used. P<0.05 (*), P<0.01 (**), P<0.001(***).

11

12

Acknowledgements
This study was supported by Linkoping University

ă
Hospital Funds to BG and UTB. sih was a kind gift
from Professor Des Richardson, University of Sydney,
NSW, Australia.

13

14

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