Báo cáo Y học: The mechanism of nitrogen monoxide (NO)-mediated iron mobilization from cells NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron from ferritin in a glutathione-dependent manner pot

The mechanism of nitrogen monoxide (NO)-mediated iron mobilization
from cells
NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron
from ferritin in a glutathione-dependent manner
Ralph N. Watts and Des R. Richardson
The Iron Metabolism and Chelation Group, The Heart Research Institute, Camperdown, Sydney, New South Wales, Australia
Nitrogen monoxide (NO) is a cytotoxic effector molecule
produced by macrophages that results in Fe mobilization
from tumour target cells which inhibits DNA synthesis and
mitochondrial respiration. It is well known that NO has a
high affinity for Fe, and we showed that NO-mediated Fe
mobilization is markedly potentiated by glutathione (GSH)
generated by the hexose monophosphate shunt [Watts, R.N.
& Richardson, D.R. (2001) J. Biol. Chem. 276, 4724–4732].
We hypothesized that GSH completes the coordination shell
of an NO–Fe complex that is released from the cell. In this
report we have extended our studies to further characterize
the mechanism of NO-mediated Fe mobilization. Native
PAGE
59
Fe-autoradiography shows that NO decreased
ferritin-
59
Fe levels in cells prelabelled with [
59
Fe]transferrin.
In prelabelled cells, ferritin-
59
Fe levels increased 3.5)fold
when cells were reincubated with control media between 30
and 240 min. In contrast, when cells were reincubated with

NO, ferritin-
59
Fe levels decreased 10-fold compared with
control cells after a 240-min reincubation. However, NO
could not remove Fe from ferritin in cell lysates. Our data
suggest that NO intercepts
59
Fe on route to ferritin, and
indirectly facilitates removal of
59
Fe from the protein.
Studies using the GSH-depleting agent,
L
-buthionine-(S,R)-
sulphoximine, indicated that the reduction in ferritin-
59
Fe
levels via NO was GSH-dependent. Competition experi-
ments with NO and permeable chelators demonstrated that
both bind a similar Fe pool. We suggest that NO requires
cellular metabolism in order to effect Fe mobilization and
this does not occur via passive diffusion down a concentra-
tion gradient. Based on our results, we propose a model of
glucose-dependent NO-mediated Fe mobilization.
Keywords: chelators; ferritin; glutathione; iron; nitrogen
monoxide.
Many of the diverse biological effects of nitrogen monoxide
(NO) are mediated through its binding to iron (Fe) in the
haem prosthetic group of soluble guanylate cyclase [1–3].
Indeed, the high affinity of NO for Fe is a well-characterized

branch of coordination chemistry [2]. Apart from the
regulatory role of NO, its cytotoxic actions are found when
it is produced in large quantities by cells such as activated
macrophages [3]. Interestingly, NO produced by such
systems inhibits the proliferation of intracellular pathogens
and tumour cells [3–5]. These effects can be explained by the
reactivity of NO with Fe in the [Fe–S] centres of critical
proteins, including aconitase and complex I and II of the
electron transport chain [4–6]. The high affinity of NO for
Fe probably results in both the removal of Fe from [Fe–S]
centres and the formation of dinitrosyl Fe species within
[Fe–S] proteins (reviewed in [7]).
It has already been shown that NO forms complexes with
a range of Fe-containing proteins including ferritin [8],
ribonucleotide reductase [9], haem-containing proteins
[10–12], and ferrochelatase [13]. Further, it has been
suggested that ferritin can act as a store of NO [8], and
NO-mediated Fe release from isolated and purified ferritin
has been demonstrated [14]. When activated macrophages
are cocultured with tumour cells, this inhibits target cell
DNA synthesis and results in the release of 64% of cellular
59
Fe within 24 h [15]. This loss of Fe may be due to the
NO-mediated release of Fe from enzymes such as mito-
chondrial aconitase [4,16–18]. Others have suggested that
NO can also target loosely bound pools of nonhaem Fe [19].
Nonetheless, the identification of Fe–nitrosyl complexes
(Fe–dithiol dinitrosyl complexes and haem–nitrosyl com-
plexes) by EPR spectroscopy in activated macrophages and
their tumour cell targets show the importance of the Fe–NO

interaction [17–23].
Apart from the above effects, NO can also increase the
RNA-binding of iron-regulatory protein 1 (IRP1), that plays
an important role in regulating intracellular Fe homeo-
stasis (reviewed in [3,24]). The effect of NO on IRP1-RNA
binding activity occurs via two main mechanisms, a direct
effect on the [4Fe)4S] cluster and Fe mobilization from
Correspondence to D. R. Richardson, The Heart Research Institute,
145 Missenden Road, Camperdown, Sydney, New South Wales,
2050 Australia.
Fax: + 61 2 9550 3302, Tel.: + 61 2 9550 3560,
E-mail:
Abbreviations:BSO,
L
-buthionine-[S,R]-sulphoximine; BSS,
balanced salt solution; DFO, desferrioxamine; GSH, reduced
glutathione; GSNO, S-nitrosoglutathione; HMPS, hexose
monophosphate shunt; IRP1, iron-regulatory protein 1; MEM,
minimum essential medium; NAP, N-acetylpenicillamine; PIH,
pyridoxal isonicotinoyl hydrazone; SNAP, S-nitroso-N-acetylpenic-
illamine; Sper, spermine; SperNO, Spermine-NONOate; Tf,
transferrin; 311, 2-hydroxy-1-naphthylaldehyde isonicotinoyl
hydrazone; DTPA, diethylenetriaminepentaacetic acid.
(Received 14 February 2002, revised 29 April 2002,
accepted 6 May 2002)
Eur. J. Biochem. 269, 3383–3392 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02987.x
cells [25–29]. Our previous studies have shown that a range
of NO-generators [e.g. S-nitroso-N-acetylpenicillamine
(SNAP), S-nitrosoglutathione (GSNO), and spermine
NONOate (SperNO)], could mobilize

59
Fe from prelabelled
cells as, or more, effectively than the clinically used Fe
chelator desferrioxamine (DFO) [29]. In contrast, the
precursor compounds of these latter NO-generators,
namely N-acetylpenicillamine (NAP), glutathione (GSH),
and spermine (Sper), respectively, had no effect [29].
Previous studies have suggested that NO may be released
from cells as a complex composed of NO, Fe, and thiol-
containing ligands such as cysteine or GSH [23,30,31].
Considering this and the other data described above, we
recently examined the energy-dependency of NO-mediated
Fe release from cells [32]. Our investigation showed that
metabolism of
D
-glucose potentiates NO-mediated Fe efflux
from a variety of cell types. Further, we demonstrated that
the metabolism of
D
-glucose by the hexose monophosphate
shunt (HMPS) and the maintenance of GSH levels was
essential for NO-mediated Fe mobilization [32]. However,
we are not proposing a direct coupling between glucose
import/metabolism and NO metabolism. Rather, our ex-
periments suggested that the generation of GSH after
incubation with
D
-glucose could result in GSH acting as a
ligand which together with NO would complete the coordi-
nation shell of Fe [32]. Such a Ômixed Fe complexÕ with both

NO and GSH ligands bound to Fe may provide an appro-
priate lipophilic balance to allow diffusion through the
membrane and/or transport by a carrier. In fact, we showed
that NO-mediated
59
Fe release was both temperature- and
energy-dependent, suggesting a membrane transport mech-
anism could be involved [32]. However, the intracellular site
of NO-mediated Fe release was not established.
In this investigation we have extended our knowledge of
NO-mediated Fe mobilization. For the first time, we
demonstrate using a cellular system that NO intercepts Fe
before being incorporated into ferritin in a similar manner to
Fe chelators. Further, NO facilitates removal of
59
Fe from
ferritin probably by an indirect mechanism. This process of
depleting ferritin-bound
59
Fe was dependent on cellular
GSH. Our studies also indicate that cellular metabolism was
required for NO-mediated Fe mobilization which appears to
be an active rather than a passive process. These results may
be important in understanding the cytotoxic actions of NO
produced by activated macrophages.
EXPERIMENTAL PROCEDURES
Cell treatments and reagents
The NO-generator SNAP was synthesized by established
techniques [33] from the precursor compound NAP (Sigma
Chemical Co.). Apotransferrin (apoTf),

L
-buthionine-(S,R)-
sulphoximine (BSO), diethylenetriaminepentaacetic acid
(DTPA), E
`
DTA, GSH, GSNO, horse spleen ferritin and
Sper were obtained from Sigma. SperNO was obtained from
Cayman Chemicals. Eagle’s minimum essential medium
(MEM) was obtained from Gibco BRL. DFO was obtained
from Novartis Pharmaceutical Co. Pyridoxal isonicotinoyl
hydrazone (PIH) and its analogue, 2-hydroxy-1-naphthylal-
dehyde isonicotinoyl hydrazone (311), were synthesized by
standard techniques [34]. Both PIH and 311 are strong Fe-
binding ligands [34] and were used as positive Fe chelation
controls. Apolactoferrin was from Calbiochem. A polyclonal
rabbit anti-(human ferritin) Ig was from Roche Diagnostics.
All other chemicals were of analytical reagent quality. The
NO-generators and other compounds were dissolved in
media immediately prior to an experiment [29,35].
Cell culture
Human SK-N-MC neuroepithelioma cells, SK-Mel-28
melanoma cells, and MCF-7 breast cancer cells were from
the American Type Culture Collection. The mouse LMTK
–
fibroblast cell line was from the European Collection of Cell
Cultures. The BE-2 neuroblastoma cell line was a gift from
G. Anderson, Queensland Institute of Medical Research
(Brisbane, Australia). All cell lines were grown in MEM
containing 10% foetal calf serum (Gibco), 1% (v/v) non-
essential amino acids (Gibco), 100 lgÆmL

)1
streptomycin
(Gibco), 100 UÆmL
)1
penicillin (Gibco), and 0.28 lgÆmL
)1
fungizone (Squibb Pharmaceuticals). Cells were grown in an
incubator (Forma Scientific) at 37 °C in a humidified
atmosphere of 5% CO
2
/95% air and subcultured as
described previously [36]. Cellular growth and viability were
assessed by phase contrast microscopy, cell adherence to the
culture substratum, and Trypan blue staining.
Nitrite determination
The accumulation of nitrite in cell culture supernatants is
commonly used as a relative measure of NO production
[25,29,35]. Nitrite was assayed using the Griess reagent that
gives a characteristic spectral peak at 550 nm [37].
Protein preparation and labelling
Apotransferrin was labelled with
59
Fe (Dupont NEN) or
56
Fe to produce Fe
2
-Tf using established procedures [36].
Efflux assay of
59
Fe from prelabelled cells

Standard techniques were used to examine the effect of NO
and other agents on the efflux of
59
Fe from prelabelled cells
[29,32,34]. Briefly, cells were labelled with
59
Fe-Tf (0.75 l
M
)
for 3 h at 37 °C in MEM. After this incubation, the cell
culture dishes were placed on a tray of ice, the medium
aspirated, and the cell monolayer washed four times with
ice-cold balanced salt solution (BSS). The cells were then
reincubated for various incubation times up to 240 min at
37 °C. After this incubation, the overlying supernatant
(efflux medium) was transferred to c-counting tubes. The
cells were removed from the petri dishes after adding 1 mL
BSS and by using a plastic spatula to detach them.
Radioactivity was measured in both the cell pellet and
supernatant using a c-scintillation counter (LKB Wallace
1282 Compugamma, Finland).
Determination of intracellular iron distribution
using native-PAGE-
59
Fe-autoradiography
Native-PAGE-
59
Fe-autoradiography was performed using
standard techniques in our laboratory [38]. Bands on X-ray
film were quantified by scanning densitometry using a Laser

Densitometer and analysed by
BIOMAX I
software (Kodak
Ltd).
3384 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002
Glutathione assay
GSH was measured as described previously [39]. Cellular
GSH levels were reduced using the GSH synthesis inhibitor,
BSO (0.1 m
M
). This latter agent is a potent and selective
inhibitor of the enzyme c-glutamylcysteine synthetase that is
involved in GSH synthesis [40]. A 20-h incubation with BSO
at a concentration of 0.1 m
M
was used, as these conditions
were shown in our previous studies to markedly deplete
GSH levels without affecting cellular viability [32].
Assay for examining the ability of NO or iron chelators
to bind
59
Fe from cell lysates
Cells grown to near confluence in T75 culture flasks were
labelled with
59
Fe-Tf (0.75 l
M
) for 3 h at 37 °C, placed on a
tray of ice, the medium decanted and the cell monolayer
washed six times with ice-cold BSS. The cells were lysed by

one freeze-thaw cycle and then detached from the flask
using a Teflon spatula in the presence of the nonionic
detergent Triton X-100 (1.5%) at 4 °C. These samples were
then centrifuged at 21 300 g for 30 min at 4 °Candthe
cytosol removed and assessed for radioactivity using the
c-counter described above. The cytosolic samples were then
incubated for 3 h at 37 °C with DFO (0.5 m
M
)orGSNO
(0.5 m
M
). The generation of nitrite by GSNO was used as a
control to ensure that the NO-donor was producing NO in
the lysate. After this incubation, the samples were then
subjected to centrifugation at 4 °C through a 5-kDa M
r
exclusion filter (Vivaspin 500, Sartorius AG). After centri-
fugation, the eluent, eluate, and membrane were taken to
estimate
59
Fe levels. Examination of
59
Fe levels on the
membrane were considered important to assess the possi-
bility of adsorption of the
59
Fe-complex.
Statistics
Experimental data were compared using Student’s t-test.
Results were considered statistically significant when

P <0.05.
RESULTS
The effects of NO on intracellular iron distribution:
NO decreases ferritin-
59
Fe levels
Considering our previous studies demonstrating that incu-
bation with NO results in intracellular Fe mobilization
[29,32], it was important to determine the source of the
Fe mobilized. For these experiments we used native
PAGE-
59
Fe-autoradiography that has proved useful in
examining the intracellular distribution of
59
Fe in our
previous studies [38,41]. Cells were prelabelled with
59
Fe-Tf
for 3 h at 37 °C, washed on ice, and then reincubated for up
AB
Cellular Iron Released (% Total )
0
10
20
30
40
50
60
Control

GSNO
GSH
SNAP
NAP
SperNO
Sper
DFO
311
Control
GSNO
GSH
SNAP
NAP
SperNO
Sper
DFO
311
59
Fe-Ferritin Levels
Relative Density (% Control)
0
25
50
75
100
125
150
Anti-Ferritin
Antibody
Control

GSNO
GSH
SNAP
NAP
SperNO
Sper
DFO
311
Ferritin-
59
Fe
Low M
r

59
Fe
Anti-Ferritin
Antibody
Supershifted
Band
Fig. 1. A variety of NO-generating agents (GSNO, SNAP, SperNO) and Fe chelators (DFO, 311) increase
59
Fe release from prelabelled cells (A), and
decrease intracellular
59
Fe–ferritin levels (B). (A) SK-N-MC neuroepithelioma cells were labelled for 3 h at 37 °Cwith
59
Fe-transferrin (0.75 l
M
)and

washed four times on ice. The cells were then reincubated for 3 h at 37 °C with control media, GSNO (0.5 m
M
), GSH (0.5 m
M
), SNAP (0.5 m
M
),
NAP (0.5 m
M
), SperNO (0.5 m
M
), Sper (0.5 m
M
), DFO (100 l
M
), 311 (25 l
M
) or polyclonal anti-human ferritin antibody (1 : 10 dilution). The
overlying media and cells were then separated and the
59
Fe levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE-
59
Fe-
autoradiography (see Materials and methods). (B) Native PAGE-
59
Fe-autoradiographs of cellular cytosols treated as described above and
densitometric results of the autoradiograph. The results in (A) are the mean ± SD of three replicates in a typical experiment of three performed.
The data shown in (B) are a representative experiment of three performed.
Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3385
to 240 min at 37 °C in the presence or absence of the agents

to be tested. The cells were then lysed and subjected to
native PAGE-
59
Fe autoradiography.
Cells prelabelled with
59
Fe were incubated with a variety
of NO-generating agents, including GSNO, SNAP and
SperNO at a concentration of 0.5 m
M
(Fig. 1A). The effects
of these NO donors were compared to their respective
control compounds without the NO group, namely GSH,
NAP, and Sper. We also compared NO-mediated
59
Fe
mobilization to the efficacy of the well-characterized Fe
chelators, DFO and 311 [34,41]. Each of the NO-generators
resulted in the release of 18–24% of total cellular
59
Fe,
whereas the relevant control compounds were no more
effective than media alone which released 3 ± 1% of
59
Fe
(Fig. 1A). The NO-generators were more effective at
mobilizing cellular
59
Fe than DFO, but less active than
the potent Fe chelator 311 [34,41] (Fig. 1A).

Examining intracellular
59
Fe distribution in SK-N-MC
cells (Fig. 1B), the most pronounced band identified comi-
grated with purified horse spleen ferritin (data not shown).
Experiments incubating the lysate with an anti-ferritin
antibody demonstrated that only this band could be super-
shifted (Fig. 1B), again indicating that it was ferritin. As
described previously in neoplastic cells [41], a faint and very
diffuse band below ferritin was present which comigrated
with low M
r
Fe complexes (
59
Fe-citrate) (Fig. 1B). We
previously showed that this low M
r
Fe appeared to be
59
Fe
bound from the lysate by the low M
r
chelators in the gel
running buffer e.g. Tris [41]. In the present study, the low M
r
band will not be considered in detail as this component
remains undefined and its relevance uncertain.
In each case, GSNO, SNAP and SperNO, decreased
ferritin-
59

Fe levels to 55–63% of the control while their
respective control compounds (GSH, NAP, and Sper) had
either little effect or increased ferritin-
59
Fe levels (Fig. 1B).
The effect of GSH or NAP at increasing ferritin-
59
Fe was
not a consistent finding in repeat experiments. In addition,
the chelators DFO and 311 decreased ferritin-
59
Fe levels
(Fig. 1B) to 69% and 51% of the control, respectively
(Fig. 1B). As all NO generators had a similar effect,
subsequent studies examining the effect of NO on cellular
Fe metabolism were performed using GSNO because of its
potential physiological importance.
The effect of GSNO concentration and reincubation time
on ferritin-
59
Fe levels: NO intercepts
59
Fe before it
reaches ferritin
To determine the efficacy of NO on
59
Fe mobilization, the
effect of GSNO concentration (0.01–1 m
M
)on

59
Fe release
from prelabelled cells (Fig. 2A) and ferritin-
59
Fe levels
(Fig. 2B) was examined. These experiments showed that
GSNO appreciably increased
59
Fe mobilization from pre-
labelled cells at a GSNO concentration of 0.05 m
M
,and
then plateaued at 0.5 m
M
(Fig. 2A). When assessing the
effect of NO on intracellular
59
Fe distribution, a GSNO
concentration of 0.05 m
M
decreased ferritin-
59
Fe levels to
26% of the control value (Fig. 2B). Higher concentrations
of the NO-donor were no more effective at reducing
ferritin-
59
Fe levels (Fig. 2B).
Studies were performed to determine the effect of
reincubation time in the presence and absence of GSNO

on
59
Fe mobilization (Fig. 3A) and ferritin-
59
Fe levels
(Fig. 3B). As in the studies above, cells were prelabelled with
59
Fe-Tf for 3 h at 37 °C, washed on ice, and then
reincubated in the presence of control media or GSNO
(0.5 m
M
) for 30–240 min at 37 °C. In the control samples
< 3% of total cellular
59
Fe was released, while in GSNO-
treated cells
59
Fe mobilization increased linearly from 30 to
180 min and then plateaued at 240 min (Fig. 3A).
AB
GSNO Concentration (mM)
0.0 0.2 0.4 0.6 0.8
1.0
Cellular Iron Released (% Total)
0
3
6
9
12
15

18
GSNO Concentration (mM)
0.0 0.2 0.4 0.6 0.8 1.0
0
20
40
60
80
100
120
59
Fe-Ferritin Levels
Relative Density (% Control)
Control
0.01 mM
0.05 mM
0.1 mM
0.5 mM
1 mM
GSNO Concentration
Fig. 2. The effect of GSNO concentration on (A) the mobilization of
59
Fe from prelabelled cells and on (B) intracellular ferritin-
59
Fe levels. (A) SK-N-
MC neuroepithelioma cells were labelled for 3 h at 37 °Cwith
59
Fe-transferrin (0.75 l
M
), washed four times on ice and then reincubated for 3 h at

37 °C with increasing concentrations of GSNO (0.01–1 m
M
). The media and cells were separated and the
59
Fe levels in each assessed. Cells were
lysed and the cytosols subjected to native PAGE-
59
Fe-autoradiography (see Materials and methods). (B) Native PAGE-
59
Fe-autoradiographs of
cellular cytosols treated as described above and densitometric results of the autoradiograph. The results in (A) are the mean ± SD of three
replicates in a typical experiment of four performed. The data shown in (B) are from a representative experiment of three performed.
3386 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002
Examining the intracellular distribution of
59
Fe in control
cells, ferritin-
59
Fe increased as a function of reincubation
time (Fig. 3B). In fact, after a reincubation time of 240 min
in control media, ferritin-
59
Fe levels were 3.5-fold that
found after 30 min (Fig. 3B). These results in control cells
demonstrated that there was some redistribution of
59
Fe
between compartments, with
59
Fe incorporation into

ferritin gradually increasing as a function of reincubation
time (Fig. 3B). In contrast, in cells treated with GSNO,
ferritin-
59
Fe levels decreased after 90 min of reincubation.
In fact, after 240 min, ferritin-
59
Fe levels were 10-fold less
than those of control cells at the same reincubation time
(Fig. 3B). These data suggest that NO directly or indirectly
results in
59
Fe mobilization from ferritin and also intercepts
59
Fe before it is deposited within this protein.
NO reduces ferritin-
59
Fe levels in a variety of cell types
TheeffectofNOatreducingferritin-
59
Fe levels was found
in a number of cell types including SK-N-MC neuroepi-
thelioma cells, MCF-7 breast cancer cells, LMTK
–
fibro-
blasts, BE-2 neuroblastoma cells and SK-Mel-28 melanoma
cells. However, there was marked variation in the effect of
NO between cell types, with a 15–74% decrease in
ferritin-
59

Fe being observed (data not shown).
Depletion of intracellular GSH prevents the NO-mediated
decrease in ferritin-
59
Fe levels
Our previous studies showed that NO-mediated
59
Fe efflux
was GSH-dependent [32]. Considering this, we examined
the effect of a 20-h incubation of both LMTK
–
and SK-N-
MC cells with the specific GSH synthesis inhibitor, BSO
(0.1 m
M
) [40], on the ability of GSNO to increase
59
Fe
mobilization (Fig. 4A) and reduce ferritin-
59
Fe levels
(Fig. 4B). As shown previously, preincubation with BSO
markedly decreased NO-mediated
59
Fe mobilization from
both cell types (Fig. 4A). Examining intracellular
59
Fe
distribution, NO decreased ferritin-
59

Fe levels, while BSO
treatment totally prevented this decrease (Fig. 4B). These
results indicate that GSH is required for the effect of NO at
decreasing ferritin-
59
Fe levels.
It is of interest that incubation of BSO-treated LMTK
–
and SK-N-MC cells with GSNO resulted in an increase in
the amount of ferritin-
59
Fe (Fig. 4B). These data were in
contrast to the decrease observed after treatment of control
cells with GSNO (Fig. 4B).
Cell membrane-impermeable or -permeable iron
chelators do not increase NO-mediated
59
Fe efflux
It was possible that passive diffusion may be involved in
NO-mediated
59
Fe release from cells. Previous studies have
shown that Fe mobilization in the absence of NO is
increased by incubation with apoTf and extracellular
chelators, presumably due to the ability of these agents to
act as an extracellular Fe ÔsinkÕ to increase the concentration
gradient across the cell membrane [42–44]. Considering this,
and the fact that a NO–Fe–GSH complex may be released
from cells [32], experiments were designed to investigate the
effects of 0.1 mgÆmL

)1
of apoTf, apolactoferrin, or BSA (as
a protein control) on
59
Fe mobilization during incubation
with increasing concentrations of GSNO (0.025–0.5 m
M
).
However, apoTf, apolactoferrin and BSA only very slightly
increased
59
Fe release from prelabelled cells at a GSNO
concentration of 0.5 m
M
(Fig. 5A). However, compared
with the BSA protein control, there was no significant effect
of apoTf or apolactoferrin on
59
Fe mobilization from
Control
GSNO
Control
GSNO
Control
GSNO
Control
GSNO
30 90 180 240
Time (min):
AB

Time (min)
0 60 120 180
240
Cellular Iron Released (% Total)
0
2
4
6
8
10
12
14
Control
GSNO
Time (min)
0 60 120 180 240
0
2
4
6
8
10
Control
GSNO
59
Fe-Ferritin Levels
Relative Density (Arbitrary Units)
Fig. 3. The effect of GSNO on (A) the mobilization of
59
Fe from cells, and (B) ferritin-

59
Fe levels in prelabelled cells as a function of reincubation time.
(A) SK-N-MC neuroepithelioma cells were prelabelled for 3 h at 37 °Cwith
59
Fe-transferrin (0.75 l
M
), washed four times on ice, and then
reincubated with control media or media containing GSNO (0.5 m
M
) for 30–240 min at 37 °C. The media and cells were separated and the
59
Fe
levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE-
59
Fe-autoradiography (see Materials and methods). (B) Native
PAGE-
59
Fe-autoradiographs of cellular cytosols treated as described above and densitometric results of the autoradiograph. The results in (A) are
the mean ± SD of three replicates in a typical experiment of two performed. The data shown in (B) are a representative experiment of three
performed.
Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3387
SK-N-MC cells (Fig. 5A). Studies combining GSNO
(0.5 m
M
) with increasing concentrations (0.03–1 m
M
)of
the extracellular chelators, EDTA or DTPA, also demon-
strated no potentiation of
59

Fe mobilization from prela-
belled cells (Fig. 5B).
As NO acted like an Fe chelator to mobilize
59
Fe from
prelabelled cells [29,32], studies were performed to deter-
mine if the same Fe pool bound by the permeable chelators,
DFO (Fig. 6A) or PIH (Fig. 6B), was bound by NO. In
these studies, cells were prelabelled with
59
Fe-Tf for 3 h at
37 °C, washed, and then reincubated for 3 h at 37 °Cwith
either increasing concentrations of DFO (0.05–1 m
M
)or
PIH (1–50 l
M
) or these chelators combined with GSNO
(0.5 m
M
). The addition of increasing concentrations of PIH
or DFO to GSNO had no significant effect on cellular
59
Fe
mobilization (Fig. 6A and B). These results suggested that
(A) (B)
Cellular Iron Released (% Total)
0
5
10

15
20
25
Control
Control
GSNO
BSO
Control
GSNO
LMTK
–
Control
Control
GSNO
BSO
Control
GSNO
SK-N-MC
Control
GSNO
Control
GSNO
Control
BSO
LMTK
–
Control
GSNO
Control
GSNO

Control
BSO
SK-N-MC
59
Fe-Ferritin Levels
Relative Density (% Control)
0
40
80
120
160
Fig. 4. The depletion of GSH prevents (A) NO-mediated
59
Fe mobilization from prelabelled cells, and (B) the decrease in intracellular ferritin-
59
Fe
levels seen in the presence of GSNO. (A) SK-N-MC neuroepithelioma cells and LMTK
–
fibroblasts were pretreated for 20 h at 37 °C in the presence
or absence of the specific GSH inhibitor BSO (0.1 m
M
). The cells were then prelabelled for 3 h at 37 °Cwith
59
Fe-transferrin (0.75 l
M
), washed four
times on ice, and then reincubated with control media or media containing GSNO (0.5 m
M
)for3hat37°C. The media and cells were separated
and the

59
Fe levels in each assessed. Cells were lysed and the cytosols subjected to native PAGE-
59
Fe-autoradiography (see Materials and methods).
(B) Native PAGE-
59
Fe-autoradiographs of cellular cytosols treated as described above and densitometric results of the autoradiograph. The results
in (A) are the mean ± SD of three replicates in a typical experiment of seven performed. The data shown in (B) are a representative experiment of
five performed.
Fig. 5. The extracellular high affinity Fe-binding proteins, apoTf and apolactoferrin, and the extracellular Fe chelators, DTPA and EDTA, do not
promote NO-mediated
59
Fe mobilization from prelabelled cells. (A) SK-N-MC neuroepithelioma cells were prelabelled for 3 h at 37 °Cwith
59
Fe-
transferrin (0.75 l
M
), and washed four times on ice. The cells were then reincubated with: control media or media containing GSNO (0.025–
0.5 m
M
) and either apoTf (0.1 mgÆmL
)1
), apolactoferrin (0.1 mgÆmL
)1
)orBSA(0.1mgÆmL
)1
)for3hat37°C. (B) SK-N-MC cells labelled and
washed as in (A) were then incubated for 3 h at 37 °C with control media or media containing EDTA or DTPA (0.03–1 m
M
). The overlying media

and cells were then separated and the
59
Fe levels in each assessed (see Materials and methods). These results are mean ± SD (three replicates) in a
representative experiment of three performed.
3388 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002
GSNO and the chelators were acting on the same intracel-
lular compartment of
59
Fe. In contrast, when added without
GSNO, increasing concentrations of DFO or particularly
PIH, resulted in enhanced
59
Fe mobilization from cells
(Fig. 6A,B).
Examination of the direct effect of NO
and iron chelators on iron pools in cytosolic lysates:
comparison with intact cells
As NO could form an intracellular low M
r
Fe–dithiol
dinitrosyl complex [23], experiments were performed to
determine if NO or DFO could mobilize
59
Fe from lysates
prepared from cells labelled with
59
Fe-Tf for 3 h at 37 °C
(Fig. 7A). The lysates were centrifuged to obtain cytosols
and then incubated for 3 h at 37 °C with DFO (0.5 m
M

)or
GSNO (0.5 m
M
). The cytosol was then subjected to
centrifugation at 4 °C through a 5-kDa M
r
exclusion filter.
In five experiments, only DFO significantly (P <0.009)
increased the amount of
59
Fe that was passing through the
membrane, while GSNO had no significant effect (Fig. 7A).
In contrast with the lysates, when cells were prelabelled with
59
Fe-Tf and reincubated with DFO or GSNO under the
same conditions, GSNO was significantly (P < 0.00001)
more effective than DFO or media at mobilizing cellular
59
Fe (Fig. 7B). These results suggest that intact cellular
metabolism was required for NO-mediated
59
Fe mobiliza-
tion.
Fig. 6. There is no potentiation on NO-mediated
59
Fe mobilization from prelabelled cells upon combination of GSNO with permeable Fe chelators.
SK-N-MC neuroepithelioma cells were prelabelled for 3 h at 37 °Cwith
59
Fe-transferrin (0.75 l
M

), washed four times on ice, and then reincubated
with increasing concentrations of desferrioxamine (DFO; 0.05–1 m
M
) or pyridoxal isonicotinoyl hydrazone (PIH; 1–50 l
M
) either alone or in the
presence of GSNO (0.5 m
M
) for 3 h at 37 °C. The overlying media and cells were then separated and the
59
Fe levels in each assessed (see Materials
and methods). These results are mean ± SD (three replicates) in a representative experiment of three performed.
Fig. 7. GSNO, in contrast to DFO, does not mobilize
59
Fe from (A) cytosolic lysates, derived from prelabelled cells, while (B) GSNO is more effective
than DFO at mobilizing
59
Fe from prelabelled intact cells. (A) Cells were labelled with
59
Fe-Tf for 3 h at 37 °C and washed four times on ice and
lysates prepared. The lysates were centrifuged to obtain cytosols and then incubated for 3 h at 37 °C with DFO (0.5 m
M
)orGSNO(0.5m
M
). The
cytosols were then subjected to ultracentrifugation through a 5-kDa cut-off filter. (B) Cells were prelabelled with
59
Fe-Tf (0.75 l
M
)for3hat37 °C,

washed four times on ice, and then reincubated for 3 h at 37 °C with DFO (0.5 m
M
)orGSNO(0.5m
M
). The overlying media and cells were then
separated and the
59
Fe levels in each assessed (see Materials and methods). The results in (A) are mean ± SD of five experiments. The results in (B)
are mean ± SD (three replicates) in a representative experiment of three performed.
Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3389
Further experiments examined the effect of incubation of
lysates derived from
59
Fe-labelled cells with either DFO,
311, SNAP, NAP, GSNO, GSH, or GSH in the presence
of GSNO. The lysates were then subjected to native
PAGE-
59
Fe-autoradiography (Fig. 8). In these studies
examining the direct effect of the chelators and NO on the
lysate, no significant effect was observed on ferritin-
59
Fe
levels. In addition, in the presence of both GSH and GSNO
no effect was apparent (Fig. 8). This indicated that intact
cellular metabolism was required for the NO-mediated
effect on this molecule, and that NO did not directly remove
substantial
59
Fe from the protein (Fig. 8).

DISCUSSION
Previous studies have clearly demonstrated that NO has a
marked effect on cellular Fe metabolism [25–27,32]. Indeed,
NO-mediated Fe depletion of tumour target cells by
activated macrophages could play an important role in
immune surveillance [3–5,15,16,45]. Our previous studies
have shown that NO-mediated Fe mobilization is potenti-
ated by incubating cells with
D
-glucose due to the
subsequent generation of GSH [32]. In the present study
we have significantly extended our knowledge of this
process. For the first time, we demonstrate in a cellular
system that NO intercepts Fe before it is incorporated into
ferritin and appeared to indirectly mobilize Fe from this
protein.
NO could remove Fe from ferritin by two possible
mechanisms: (a) by directly chelating ferritin-bound Fe, or
(b) by chelating a cellular Fe pool which leads to ferritin
releasing its Fe. Of these two possibilities our evidence
favours the second mechanism, as NO could not remove
59
Fe from ferritin in cellular lysates (Fig. 8). Furthermore,
the processes resulting in cellular Fe mobilization and Fe
release from ferritin were dependent on cellular metabolism
(Fig. 7) and the generation of GSH (Fig. 4 and [32]). These
latter observations indicate that active cellular metabolism
was required for Fe mobilization rather than direct chela-
tion of ferritin-Fe by NO.
A previous in vitro study by Reif & Simmons [14] using

isolated horse spleen ferritin showed that NO generated by
sodium nitroprusside could remove some Fe from this
protein. Our current data are obviously different, and these
inconsistent results may relate to the very different experi-
mental systems being used. Lee and colleagues [8] have
reported, using isolated ferritin, that NO forms a complex
with Fe in its core, and have suggested that ferritin could act
as a store of NO. Again, it is difficult to compare this latter
study to our present experiments, as we have examined the
effect of NO using intact cells or cellular lysates. It is
significant that we have shown that NO not only releases Fe
from ferritin indirectly (Figs 1B, 2B, 4), but can also
intercept Fe on route to this molecule (Fig. 3B). At present
the precise molecular mechanism(s) involved in the intra-
cellular trafficking and delivery of Fe to ferritin remain
unclear, although intermediates of low M
r
[46] or high M
r
(e.g. metal-binding chaperones) [47] could be involved.
Nevertheless, our results demonstrate that both permeable
Fe chelators (e.g. PIH and DFO) and NO can intercept the
same intermediary pool of Fe (Fig. 6).
It is of interest that the ability of NO to induce Fe
mobilization is dependent on GSH while that for chelators is
independent of GSH [32]. This suggests that NO by itself
does not have the capacity to remove Fe from intermediates
and could require the reducing capacity of GSH. Alternat-
ively, or in combination with this latter mechanism, GSH
may form a mixed Fe complex with NO that has the

appropriate lipophilicity and charge to diffuse or be
transported from the cell. Previous studies using EPR
spectroscopy have demonstrated the presence of dithiol
dinitrosyl–Fe complexes within cells [23,30]. Further, Rogers
and Ding [48] have shown that
L
-cysteine is necessary for the
removal of dinitrosyl–Fe complexes from [Fe–S]-containing
proteins in Escherichia coli. Interestingly, these authors
showed that GSH was able to perform the same function but
Con
DFO
311
SNAP
NAP
GSNO
GSH
GSH + GSNO
Fig. 8. The direct effect of incubating GSNO and Fe chelators on
59
Fe-containing molecules in cytosolic lysates derived from prelabelled
cells. SK-N-MC neuroepithelioma cells were labelled for 3 h at 37 °C
with
59
Fe-transferrin (0.75 l
M
) and washed four times on ice. Cells
were then lysed and the cytosols incubated with DFO (0.5 m
M
), 311

(50 l
M
), SNAP (0.5 m
M
), NAP (0.5 m
M
), GSNO (0.5 m
M
), GSH
(0.5 m
M
), or GSNO (0.5 m
M
)andGSH(0.5m
M
) for 3 h at 37 °C.
These samples were then subjected to native PAGE-
59
Fe-autoradio-
graphy (see Materials and methods). The results are a representative
experiment of three performed.
Cell
Membrane
ADP
GS-Fe-NO
Transporter ?
?
GS-Fe-NO
Protein
?

ATP
TCA
HMPS
Glucose
G-6-P
GSH
Glucose
Transporter
Fe-Protein
NO
NO
NO-Fe-Protein
Transporter
Diffusion
Fig. 9. Hypothetical model of
D
-glucose-dependent NO-mediated Fe
mobilization from cells.
D
-Glucose is transported into cells and is used
by the tricarboxylic acid cycle for the production of ATP and by the
HMPS for the generation of reduced GSH. Nitrogen monoxide (NO)
either diffuses or is transported into cells where it intercepts and binds
Fe bound to proteins or Fe on route to ferritin. The high affinity of NO
for Fe results in the formation of an NO–Fe complex and GSH may
either be involved as a reductant to remove Fe from endogenous lig-
ands or may complete the Fe coordination shell along with the NO
ligand(s). This complex may then be released from the cell by an active
process requiring a transporter (see text for details).
3390 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002

not as efficiently as
L
-cysteine [48]. Hence, these results
provide support for the possible mechanism of action of
GSH in our experimental system. However, at present, we
cannot exclude that GSH has other roles. Indeed, recently
the S-nitrosylated form of GSH has been suggested to act
as a transport molecule for NO which increases its half-life
and allows effective biological activity [49,50].
Considering that a low M
r
Fe–NO–GSH complex may
be released from cells and that a concentration gradient
across a membrane can facilitate diffusion [44], we examined
whether NO-mediated Fe release could be potentiated by
strong extracellular Fe chelators (Fig. 5). In these studies,
high concentrations of both physiological chelators
(apolactoferrin and apoTf) and synthetic chelators (EDTA
and DTPA) were used, and for all ligands no significant
potentiation of
59
Fe mobilization was observed upon
combination with NO. These studies suggest that enhance-
ment of the concentration gradient across the cell membrane
did not alter NO-mediated Fe release. Considering this, it is
of note that intact cellular metabolism was required for Fe
mobilization by NO (Fig. 7), and NO-mediated Fe release
was prevented by metabolic inhibitors and at 4 °C [32].
Collectively, these data suggest that an energy-dependent
mechanism was required to enable efflux of the NO–Fe

complex.
Based upon the results presented in this and our previous
study [32], we suggest in Fig. 9 a hypothetical model of
D
-glucose-dependent NO-mediated Fe mobilization from
cells.
D
-Glucose is transported into cells and is used by the
tricarboxylic acid cycle (TCA) for the production of ATP
and by the HMPS for the generation of GSH. NO diffuses or
is transported [51] into cells where it intercepts Fe on route to
ferritin and binds Fe bound to proteins (Fig. 9). The high
affinity of NO for Fe [2] results in the formation of an NO-Fe
complex and GSH may either be involved as a reductant to
remove Fe from endogenous ligands [48] or may complete
the Fe coordination shell along with NO [17,20,23,30]. This
complex may then be transported out of the cell by an energy-
dependent transporter such as ferroportin 1 [52], or
alternatively, the ATP-binding cassette (ABC) transporter
family (e.g. glutathione-S-conjugate export pump), which
are known to mediate the efflux of glutathione-conjugates
[53,54] (Fig. 9). Further studies aimed at identifying the exact
molecular nature of the Fe released by NO and the
transporter involved are underway. Finally, out current
results may be important in understanding the cytotoxic
actions of NO produced by activated macrophages.
ACKNOWLEDGEMENTS
The authors thank J. Kwok for her excellent suggestions on this
manuscript prior to submission. This work was supported by an
Australian Research Council Large Grant and Grants 970360 and

981826 from the National Health and Medical Research Council of
Australia.
REFERENCES
1. Moncada, S., Palmer, R.M.J. & Higgs, E.A. (1991) Nitric oxide:
physiology, pathophysiology and pharmacology. Pharmacol. Rev.
43, 109–142.
2. Stamler, J., Singel, D.J. & Loscalzo, J. (1992) Biochemistry of
nitric oxide and its redox-activated forms. Science 258, 1898–1902.
3. Richardson, D.R. & Ponka, P. (1997) The molecular mechanisms
of the metabolism and transport of iron in normal and neoplastic
cells. Biochim. Biophys. Acta 1331, 1–40.
4. Drapier, J C. & Hibbs, J.B. Jr (1986) Murine cytotoxic activated
macrophages inhibit aconitase in tumor cells. Inhibition involves
the iron-sulfur prosthetic group and is reversible. J. Clin. Invest.
78, 790–797.
5. Drapier, J C. & Hibbs, J.B. Jr (1988) Differentiation of murine
macrophages to express non-specific cytotoxicity for tumor cells
results in
L
-arginine-dependent inhibition of mitochondrial iron-
sulfur enzymes in the macrophage effector cells. J. Immunol. 140,
2829–2838.
6. Henry, Y., Ducrocq, C., Drapier, J C., Pellat, C. & Guissani, A.
(1991) Nitric oxide as a biological effector molecule. Electron
paramagnetic resonance detection of nitrosyl-iron-protein com-
plexes in whole cells. Eur. Biophys. J. 20, 1–15.
7. Drapier, J C. (1997) Interplay between NO and [Fe-S] clusters:
relevance to biological systems. Methods 11, 319–329.
8. Lee, M., Arosio, P., Cozzi, A. & Chasteen, N.D. (1994) Identifi-
cation of the EPR-active iron-nitrosyl complexes in mammalian

ferritins. Biochemistry 33, 3679–3687.
9. Lepoivre, M., Fieschi, F., Coves, J., Thelander, L. & Fontecave, M.
(1991) Inactivation of ribonucleotide reductase by nitric oxide.
Biochem. Biophys. Res. Commun. 179, 442–448.
10. Ignarro, L.J. (1991) Heme-dependent activation of guanylate
cyclase by nitric oxide: a novel signal transduction mechanism.
Blood Vessels 28, 67–73.
11. Khatsenko, O.G., Gross, S.S., Rifkind, R.R. & Vane, J.R. (1993)
Nitric oxide is a mediator of the decrease in cytochrome P450-
dependent metabolism caused by immunostimulants. Proc. Natl
Acad. Sci. USA 90, 11147–11151.
12. Griscavage, J.M., Fukuto, J.M., Komori, Y. & Ignarro, L.J.
(1994) Nitric oxide inhibits neuronal nitric oxide synthase by
interacting with the heme prosthetic group. Role of tetra-
hydrobiopterin in modulating the inhibitory action of nitric oxide.
J. Biol. Chem. 269, 21644–21649.
13. Kim, Y M., Bergonia, H.A., Muller, C., Pitt, B.R., Watkins,
W.D. & Lancaster, J.R. Jr (1995) Loss and degradation of
enzyme-bound heme induced by cellular nitric oxide synthesis.
J. Biol. Chem. 270, 5710–5713.
14. Reif, D.W. & Simmons, R.D. (1990) Nitric oxide mediates iron
release from ferritin. Arch. Biochem. Biophys. 283, 537–541.
15. Hibbs, J.B. Jr, Taintor, R.R. & Vavrin, Z. (1984) Iron depletion:
possible cause of tumor cell cytotoxicity induced by activated
macrophages. Biochem. Biophys. Res. Commun. 123, 716–723.
16. Hibbs, J.B. Jr, Taintor, R.R., Vavrin, Z. & Rachlin, E.M. (1988)
Nitric oxide: a cytotoxic activated macrophage effector molecule.
Biochem. Biophys. Res. Commun. 157, 87–94.
17. Lancaster, J.R. & Hibbs, J.B. Jr (1990) EPR demonstration of
iron-nitrosyl complex formation by cytotoxic activated macro-

phages. Proc. Natl Acad. Sci. USA 87, 1223–1229.
18. Pellat, C., Henry, Y. & Drapier, J C. (1990) IFN-gamma-
activated macrophages: detection by electron paramagnetic
resonance of complexes between
L
-arginine-derived nitric oxide
and non-heme iron proteins. Biochem. Biophys. Res. Commun.
166, 119–125.
19. Vanin, A.F., Men’shikov, G.B., Moroz, I.A., Mordvintcev, P.I.,
Serezhenkov, V.A. & Burbaev, D.S. (1992) The source of non-
heme iron that binds nitric oxide in cultivated macrophages.
Biochim. Biophys. Acta 1135, 275–279.
20. Drapier, J C., Pellat, C. & Henry, Y. (1991) Generation of
EPR-detectable nitrosyl-iron complexes in tumor target cells
co-cultured with activated macrophages. J. Biol. Chem. 266,
10162–10167.
21. Bastian, N.R., Yim, C.Y., Hibbs, J.B. Jr & Samlowski, W.E.
(1994) Induction of iron-derived EPR signals in murine cancers by
Ó FEBS 2002 NO-mediated iron mobilization from cells (Eur. J. Biochem. 269) 3391
nitric oxide. Evidence for multiple intracellular targets. J. Biol.
Chem. 269, 5127–5131.
22. Commoner, B., Woolum, J.C., Senturia, B.H. Jr & Ternberg, J.L.
(1970) The effects of 2-acetylaminofluorene and nitrite on free
radicals and carcinogenesis in rat liver. Cancer Res. 30, 2091–2097.
23. Vanin, A.F. (1991) Endothelium-derived relaxing factor is a
nitrosyl iron complex with thiol ligands. FEBS Lett. 289, 1–3.
24. Theil, E.C. (1998) The iron responsive element (IRE) family of
mRNA regulators. Regulation of iron transport and uptake
compared in animals, plants and microorganisms. Met. Ions Biol.
Syst. 35, 403–434.

25. Drapier, J C., Hirling, H., Wietzerbin, J., Kaldy, P. & Ku
¨
hn, L.C.
(1993) Biosynthesis of nitric oxide activates iron regulatory factor
in macrophages. EMBO J. 12, 3643–3649.
26. Weiss, G., Goossen, B., Doppler, W., Fuchs, D., Pantopoulos, K.,
Werner-Felmayer,G.,Wachter,H.&Hentze,M.W.(1993)
Translational regulation via iron-responsive elements by the nitric
oxide/NO-synthase pathway. EMBO J. 12, 3651–3657.
27. Richardson, D.R., Neumannova, V., Nagy, E. & Ponka, P. (1995)
The effect of redox-related species of nitrogen monoxide on
transferrin and iron uptake and cellular proliferation of erythro-
leukemia (K562) cells. Blood 86, 3211–3219.
28. Kennedy, M.C., Antholine, W.E. & Beinert, H. (1997) An EPR
investigation of the products of the reaction of cytosolic and
mitochondrial aconitases with nitric oxide. J. Biol. Chem. 272,
20340–20347.
29. Wardrop, S.L., Watts, R.N. & Richardson, D.R. (2000)
Nitrogen monoxide activates iron regulatory protein 1 RNA-
binding activity by two possible mechanisms: effect on the [4Fe-4S]
cluster and iron mobilization from cells. Biochemistry 39,
2748–2758.
30. Vanin, A.F., Bliumenfel’d, L.A. & Chetverikov, A.G. (1967) EPR
study of non-heme iron complexes in cells and tissues. Biofizika
[Russian] 12, 829–841.
31. Woolum, J.C., Tiezzi, E. & Commoner, B. (1968) Electron spin
resonance of iron-nitric oxide complexes with amino acids, pep-
tides and proteins. Biochim. Biophys. Acta 160, 311–320.
32. Watts, R.N. & Richardson, D.R. (2001) Nitrogen monoxide (NO)
and glucose: unexpected links between energy metabolism and

NO-mediated iron mobilization from cells. J. Biol. Chem. 276,
4724–4732.
33. Field, L., Dilts, R.V., Ravichandran, R., Lenhert, P.G. &
Carnahan, G.E. (1978) An unusually stable thionitrite from
N-acetyl-
D
,
L
-penicillamine; X-ray crystal and molecular structure
of 2-(acetylamino)-2-carboxy-1,1-dimethylethyl thionitrite. J. Chem.
Soc. Chem. Commun. 249, 249–250.
34. Richardson, D.R., Tran, E. & Ponka, P. (1995) The potential of
iron chelators of the pyridoxal isonicotinoyl hydrazone class as
effective antiproliferative agents. Blood 86, 4295–4306.
35. Pantopoulos, K., Weiss, G. & Hentze, M.W. (1996) Nitric oxide
and oxidative stress (H
2
O
2
) control mammalian iron metabolism
by different pathways. Mol. Cell. Biol. 16, 3781–3788.
36. Richardson, D.R. & Baker, E. (1992) Two mechanisms of iron
uptake from transferrin by melanoma cells. The effect of desfer-
rioxamine and ferric ammonium citrate. J. Biol. Chem. 267,
13972–13979.
37. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L.,
Wishnok, J.S. & Tannenbaum, S.R. (1982) Analysis of nitrate,
nitrite and [
15
N]nitrate in biological fluids. Anal. Biochem. 126,

131–138.
38. Richardson, D.R., Ponka, P. & Vyoral, D. (1996) Distribution of
iron in reticulocytes after inhibition of heme synthesis with suc-
cinylacetone. Examination of the intermediates involved in iron
metabolism. Blood 87, 3477–3488.
39. Sedlack, J. & Lindsay, R. (1968) Estimation of total protein-
bound and nonprotein sulfhydryl groups in tissue with Ellman’s
reagent. Anal. Biochem. 25, 192–205.
40. Griffith, O.W. & Meister, A. (1979) Potent and specific inhibition
of glutathione synthesis by buthionine sulfoximine (S-n-butyl
homocysteine sulfoximine). J. Biol. Chem. 254, 7558–7560.
41. Richardson, D.R. & Milnes, K. (1997) The potential of iron
chelators of the pyridoxal isonicotinoyl hydrazone class as
effective antiproliferative agents II. The mechanism of action of
ligands derived from salicylaldehyde benzoyl hydrazone and
2-hydroxy-1-naphthylaldehyde benzoyl hydrazone. Blood 89,
3025–3038.
42. Baker, E., Page, M. & Morgan, E.H. (1985) Transferrin and iron
release from rat hepatocytes in culture. Am. J. Physiol. 248,
G93–G97.
43. Baker, E., Morton, A.G. & Tavill, A.S. (1980) The regulation of
iron release from the perfused rat liver. Br. J. Haematol. 45,
607–620.
44. Young, S.P., Fahmy, M. & Golding, S. (1997) Ceruloplasmin,
transferrin and apotransferrin facilitate iron release from human
liver cells. FEBS Lett. 411, 93–96.
45. Nestle, F.P., Greene, R.N., Kichian, K., Ponka, P. & Lapp, W.S.
(2000) Activation of macrophage cytostatic effector mechanisms
during acute graft-versus-host disease: release of intracellular iron
and nitric oxide-mediated cytostasis. Blood 96, 1836–1843.

46. Jacobs, A. (1977) Low molecular weight intracellular iron trans-
port compounds. Blood 50, 433–439.
47. Harrison, M.D., Jones, C.E. & Dameron, C.T. (1999) Copper
chaperones: function, structure and copper-binding properties.
J. Biol. Inorg. Chem. 4, 145–153.
48. Rogers, P.A. & Ding, H. (2001)
L
-Cysteine-mediated destabiliza-
tion of dinitrosyl iron complexes in proteins. J. Biol. Chem. 276,
30980–30986.
49. Lipton, A.J., Johnson, M.A., Macdonald, T., Lieberman, M.W.,
Gozal, D. & Gaston, B. (2001) S-Nitrosothiols signal the venti-
latory response to hypoxia. Nature 413, 171–174.
50. Lipton, S.A. (2001) Nitric oxide and respiration. Nature 413,
118–121.
51. Zai, A., Rudd, M.A., Scribner, A.W. & Loscalzo, J. (1999) Cell-
surface protein disulphide isomerase catalyzes transnitrosation
and regulates intracellular transfer of nitric oxide. J. Clin. Invest.
103, 393–399.
52. Donovan,A.,Brownlie,A.,Zhou,Y.,Shepard,J.,Pratt,S.J.,
Moynihan,J.,Paw,B.H.,Drejer,A.,Barut,B.,Zapata,A.et al.
(2000) Positional cloning of zebrafish ferroportin 1 identifies a
conserved vertebrate iron exporter. Nature 403, 776–781.
53. Suzuki, T., Nishio, K. & Tanabe, S. (2001) The MRP family
and anticancer drug metabolism. Curr. Drug Metab. 2,367–
377.
54. Dietrich, C.G., Ottenhoff, R., de Waart, D.R. & Oude Elferink,
R.P. (2001) Role of MRP2 and GSH in intrahepatic cycling of
toxins. Toxicology 167, 73–81.
3392 R. N. Watts and D. R. Richardson (Eur. J. Biochem. 269) Ó FEBS 2002