ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 283, No. 2, December, pp. 537-541, 1990

COMMUNICATION
Nitric Oxide Mediates Iron Release from Ferritin
David W. Reif’ and Roy D. Simmons
Biology Department, Fisons Pharmaceuticals, 755 Jefferson Road, Rochester, New York 14603

Received July 16, 1990, and in revised form September 16, 1990

and mitochondrial (13) iron as a result of cocultivation with

Nitric oxide (NO) synthesis by cytotoxic activated cytotoxic activated macrophages. Granger and Lehninger (14)
macrophages has been postulated to result in a progres- demonstrated that activated macrophages also cause a selective
sive loss of iron from tumor target cells as well as inhi- inhibition of the iron-dependent, mitochondrial enzymes NADH
bition of mitochondrial respiration and DNA synthesis. coenzyme Q reductase and succinate coenzyme Q reductase. Ad-
In the present study, the addition of an NO-generating ditionally, activated macrophages inhibit aconitase in the target
agent, sodium nitroprusside, to the iron storage protein cells as a result of the loss of an iron atom from the 4Fe-4S
cluster within the aconitase active site (15). Aconitase activity
ferritin resulted in the release of iron from ferritin and
could be recovered if the damaged target cells were subsequently
the released iron-catalyzed lipid peroxidation. Hemoglo-
incubated in the presence of ferrous, suggesting that the cells
bin, which binds NO, and superoxide anion, which reacts
had lost the ability to utilize intracellular stores of iron.
with NO, inhibited nitroprusside-dependent iron release The ability of activated macrophages to inhibit non-heme
from ferritin, thereby providing evidence that NO can iron-dependent enzymes as well as to synthesize NO, led Lan-
mobilize iron from ferritin. These results suggest that caster and Hibbs (4) to speculate that mitochondrial inhibition
NO generation in uivo could lead to the mobilization of may be a result of a direct interaction between NO and the Fe-
iron from ferritin disrupting intracellular iron homeo- S centers of these enzymes. Indeed, using ESR spectroscopy,
stasis and increasing the level of reactive oxygen species. they observed iron-nitrosyl complexes in cytotoxic activated
0 1990 Academic Press, Inc. macrophage effector cells. The source of this iron is unknown,
but these results provide a direct link between NO generation
and a disruption in intracellular iron utilization. The relatively
large amount of iron that is lost from target cells led Wharton
It has been known for many years that macrophages are cy- et al. (13) to suggest that some of this iron may arise from the
totoxic to tumor cells in culture when the macrophages are ac- iron storage protein ferritin.
tivated by tumor necrosis factor or interferon-y, in combination Ferritin stores iron as ferric and can store up to 4500 atoms
with a second signal such as Escherichia coli lipopolysaccharide of Fe/molecule (16). The physiological reductant(s) is unknown,
(l-3). One aspect of this cytotoxicity is a selective inhibition of but in recent years the ability of numerous toxicological reduc-
certain metabolic functions, an effect which is also observed in tants to mobilize ferritin iron has been reported (17, 18). Once
the activated macrophage effector cells (4). The mechanism of released, the iron can react with cellular oxidants and result in
this metabolic inhibition is unknown, but recent evidence sug- the oxidation of critical cellular constituents (19). Since NO
gests that nitric oxide (NO) production by the activated mac- interacts with nonheme iron and the majority of intracellular
rophages may contribute to the cytotoxic effects. nonheme iron is stored within ferritin, we investigated the ability
Stuehr and Marletta (5-7) first reported that activated mac- of NO to mobilize iron from ferritin using sodium nitroprusside
rophages produced nitrite and nitrate and subsequently dem- as the source of NO. We report here that NO releases iron from
onstrated that NO was an intermediate in their production (8). ferritin in uitro and the released iron catalyzes lipid peroxidation.
L-Arginine is required for NO production as well as the bio- These results suggest an additional effect of NO generation in
chemical changes associated with activated macrophage cyto- uiuo and may provide a partial mechanism that explains the loss
toxicity (9,lO). These effects can be blocked by NG-monomethyl- of intracellular iron reported by Hibbs et al. (12).
L-arginine (g-11), which inhibits the NO synthetase enzyme.
Target cells previously loaded with radiolabeled iron lose a
major fraction (64% of labeled iron) of their cellular (12, 13) MATERIALS AND METHODS
Chemicals. Xanthine, glucose, sodium hydrosulfite (dithionite), 3-
(2-pyridylj-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine), 4-
1 To whom correspondence should be addressed. (2.hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), and Sephadex

0003-9861/90 $3.00 537

Copyright 0 1990 by Academic Press, Inc.
All rights of reproduction in any form reserved.
538 REIF AND SIMMONS

G-25 and G-100 were from Sigma. Sodium nitroferricyanide (nitro-

prusside) was from Baker, Inc. (Phillipsburg, NJ). Chelex 100 was ob-
tained from Bio-Rad (Richmond, CA). Distilled deionized water was
1.6
purified with a Milli Q Water Purification System (resistivity 2 16
Mohm/cm). All reagents were prepared in 50 mM Hepes, pH 7, which 2
had been chromatographed over Chelex 100 to remove trace metal im-
purities.
Proteins. Glucose oxidase (EC 1.1.3.4), xanthine oxidase (EC
1.1.3.22) grade III from buttermilk, bovine erythrocyte superoxide dis-
mutase (EC 1.15.1.1), catalase (EC 1.11.1.6), human hemoglobin, and
horse spleen ferritin were purchased from Sigma. Glucose oxidase and
ferritin were incubated with 10 mM EDTA for 1 h on ice followed by
chromatography over a Sephadex G-25 column equilibrated in 50 mM
Hepes, pH 7, prior to use (17). Human hemoglobin was treated with
dithionite to obtain reduced hemoglobin as described (20) and quantified
as described (21). Xanthine oxidase was purified by chromatography 10 20 30
over a G-100 column equilibrated in 50 mM Hepes, pH 7, and assayed
according to McCord and Fridovich (22). Superoxide dismutase was TIME (minutes)
assayed according to McCord and Fridovich (22) and catalase according FIG. 1. Effect of sodium nitroprusside on iron release from ferritin.
to Beers and Sizer (23). Reaction mixtures (1 ml final volume) in 50 mM Hepes, pH 7.0, contained
Iron release assay. Iron release from ferritin was measured utilizing 500 pM ferrozine, 200 pM Fe as horse spleen ferritin iron (0.1 pM protein),
the ferrous chelator ferrozine and the amount of iron released calculated and sodium nitroprusside (0,O.l mM; n , 1.0 mM; 0,lO mM). The reaction
using cr,62 = 27.9 mM-‘cm-’ (24). Reaction mixtures (1.0 ml final volume) was initiated with nitroprusside and the amount of iron released deter-
in 50 mM Hepes, pH 7.0, contained 500 ~.LM ferrozine and other reactants mined as described under Material and Methods. Values presented are
as indicated in the figure and table legends.. Sodium nitroprusside (SNP)’ the means + SE of five replicate incubations from two experiments.
was prepared in argon-purged 50 mM Hepes, pH 7.0, stored on ice, and
used within 4 h. All reactions were performed at 37”C, utilized a reference
cuvette that contained all reactants except ferritin, and were initiated
Since the reaction of NO with O2 proceeds rapidly, the ability
by the addition of SNP. The increase in absorbance at 562 nm was
continuously monitored using a Beckman Model 35 spectrophotometer. of NO to release iron from ferritin should be attenuated by the
For the anaerobic experiments, all solutions were purged with argon O2 present in the system. Accordingly, when the reactions were
and glucose and glucose oxidase were included to scavenge any re- performed in an anaerobic environment, an enhanced (P < 0.01)
maining Op. rate of SNP-dependent iron release from ferritin was detected
Lipid peronidation assay. Rat liver microsomal phospholipid was (Table I). The rate of iron release from ferritin anaerobically
isolated and liposomes were prepared anaerobically as described (19). was consistently between 20 and 40% greater than the rate aer-
Reaction mixtures (2.5 ml final volume) in 50 mM Hepes, pH 7.0, con- obically (P < 0.01). Increasing the amount of ferritin iron present
tained phospholipid liposomes (1 pmol/ml) and other reactants as de- (25-200 FM) also resulted in an increase in the amount of iron
scribed in the legend to Table III. The extent of lipid peroxidation fol- released from ferritin (Table I).
lowing a 20-min incubation at 37°C was determined as the amount of Hemoglobin binds NO and has been employed as a functional
malondialdehyde (MDA) formed as assayed by the thiobarbituric acid
antagonist to confirm the involvement of NO (27). Thus, the
assay (25).
addition of hemoglobin (l-25 FM) to incubations containing 1
Statistical analysis. The data in Tables II and III and Figs. 1 and 2
mM SNP and 200 PM ferritin iron resulted in the inhibition (P
were analyzed by ANOVA and the data in Table I by Analysis of Co-
variance. Significance was designated at the P < 0.01 level. < 0.01) of iron release from ferritin (Fig. 2). In contrast to NO,
neither sodium nitrite (1 mM) nor sodium nitrate (1 mM) was
capable of releasing iron from ferritin (data not shown). These
RESULTS results strongly suggest that either NO or some species derived
Sodium nitroprusside releases NO spontaneously and provides from NO directly mobilizes iron from ferritin.
a continuous flux of NO (26). The addition of increasing con- Another criterion for establishing the involvement of NO is
centrations of SNP (0.1-10 mM) to horse spleen ferritin in the inactivation by superoxide anion (27). As shown in Table II, the
presence of ferrozine (an iron chelator) resulted in a corre- combination of SNP and xanthine/xanthine oxidase decreased
sponding increase in the formation of the Fe(II):(ferrozine), the iron released as compared to either the SNP-dependent sys-
complex, indicating that iron is being released from ferritin (Fig. tem or the superoxide-dependent system (P < 0.01). Since su-
1). The rate of iron release from ferritin is dependent upon the peroxide also mobilizes iron from ferritin (19, 28), then the
concentration of SNP and is linear over a 30-min incubation products of the reaction of NO and superoxide, N204 and Nz03,
period (P < 0.01). At 1 mM SNP, NO is released at a rate of 110 as well as their hydrolysis products NO*- and NOBe (as mentioned
nM min-’ (26). Thus, assuming that a mole of NO could mobilize above), do not appear to release iron from ferritin. SNP (1 mM)
a mole of iron from ferritin, then NO exhibits an efficiency of did not affect the activity of xanthine oxidase as assayed by
approximately 32% in this test system. urate production (data not shown). The inclusion of superoxide
dismutase (100 U/ml) to scavenge superoxide increased iron
release in the presence of both SNP and xanthine/xanthine ox-
2 Abbreviations used: SNP, sodium nitroprusside; ANOVA, analysis idase to a level approximating the iron release observed with
of variance; MDA, malondialdehyde; EDRF, endothelium-derived re- SNP alone. Superoxide dismutase completely inhibited iron re-
laxation factor. lease in the xanthine/xanthine oxidase system (Table II), but
 NITRIC OXIDE MEDIATES IRON RELEASE FROM FERRITIN 539

TABLE I TABLE II
Effect of Ferritin Iron Concentration and O2 on Sodium Effect of Superoxide on Sodium Nitroprusside-Dependent
Nitroprusside-Dependent Iron Release Iron Release from Ferritin

Iron released (PM) System” Iron released (PM)

Ferritin iron (PM) Aerobic Anaerobic Nitroprusside 0.67 f 0.03

Xanthine oxidase 0.83 f 0.02
25 0.18 + 0.02 0.29 f 0.02 Nitroprusside and xanthine oxidase 0.44 f 0.03
50 0.30 + 0.01 0.44 * 0.02 Nitroprusside, xanthine oxidase, and SOD 0.60 + 0.02
100 0.49 f 0.01 0.58 + 0.02 Xanthine oxidase plus SOD ND*
200 0.75 I!z 0.03 0.90 +_ 0.04
’ The reactions in 50 mM Hepes, pH 7.0, contained 200 PM ferritin
Note. The aerobic system was identical to those described in the legend iron, 0.5 mM ferrozine, and where indicated, 1 mM sodium nitroprusside,
for Fig. 1 and utilized 1 mM sodium nitroprusside with the indicated 0.33 mM xanthine, 1 mU xanthine oxidase, and 100 U/ml superoxide
concentrations of ferritin iron. Anaerobiosis was achieved by purging dismutase (SOD). Iron release from ferritin was determined as described
the solutions in the cuvette with argon for 5 min. The anaerobic reaction under Materials and Methods following a 20-min incubation and the
mixture also contained catalase (100 U/ml) to prevent iron oxidation values presented are the means + SE of five replicate incubations of
by hydrogen peroxide, and glucose (5 mM) and glucose oxidase (15 U/ two experiments.
ml) to scavenge traces of Or. Iron release was determined for 20 min as * Rate not detectable.
described under Materials and Methods and the values presented are
the means f SE of replicate (n = 5) incubations of two experiments.
ments utilizing xanthine oxidase and xanthine to generate su-
peroxide at a rate resulting in an equivalent rate of iron release
had no effect on the SNP-dependent system (data not shown). from ferritin as the SNP system resulted in similar extents of
These results provide additional evidence that No is capable of lipid peroxidation (Table III) (P < 0.01). The strikingly similar
results between SNP-dependent and superoxide-dependent lipid
mobilizing iron from ferritin and support the finding that No
does not appear to complex with the Type 2 copper of SOD (29). peroxidation support the premise that NO-dependent iron re-
To evaluate the potential biological significance of NO-de- lease from ferritin could be significantly damaging to biological
pendent iron release from ferritin, the effect of SNP-dependent membranes.
iron release from ferritin in a model lipid peroxidation system
was investigated. As shown in Table III, SNP-dependent iron DISCUSSION
release is sufficient to catalyze lipid peroxidation. The extent of Cytotoxic activated macrophages produce NO, which results
lipid peroxidation detected increased more than 16-fold in the in a loss of iron from tumor target cells (12, 13) and inhibition
presence of both ferritin and SNP (P < 0.01). Parallel experi-

TABLE III
Sodium Nitroprusside- and Ferritin-Dependent

z
0.8
L
1 Lipid Peroxidation

MDA (PM)”
2

Ei 0.6 Additions Nitroprusside b Superoxide’

I1
2 None 0.08 + 0.01 0.07 k 0.01
Y
2 0.4 Complete 1.34 f 0.05 1.50 f 0.05
1,
-Ferritin 0.19 Zk 0.03 0.21 2 0.02
8 -Nitroprusside
fi 0.2 or superoxide 0.09 + 0.03 0.13 2 0.01

o All values presented are the means + SE of five replicate incubations

- - I 1 from two experiments.
0 1 10 25 * Reaction mixtures (2.5 ml final volume) in 50 mM Hepes, pH 7.0,
HEMOGLOBIN contained 1 pmol/ml rat liver microsomal phospholipid liposomes, horse
(PM)
spleen ferritin (200 PM Fe), and sodium nitroprusside (1 mM) as indicated.
FIG. 2. Inhibition of sodium nitroprusside-dependent iron release from MDA formation at 20 min was assayed as described under Materials
ferritin by hemoglobin. The reaction mixtures were identical to those and Methods.
described in the legend to Fig. 1 and utilized 1 mM sodium nitroprusside. ’ These incubations were identical to the nitroprusside system with
Oxhemoglobin (tetramer) was added to the reaction at the indicated the omission of the nitroprusside and the inclusion of 0.33 mM xanthine
concentrations. Iron release was determined for 20 min as described and 1 mu/ml xanthine oxidase to generate superoxide. Catalase (100
under Materials and Methods and the values presented are the means U/ml) was also included to eliminate iron oxidation by hydrogen per-
i SE of five replicate incubations from two experiments. oxide.
540 REIF AND SIMMONS

of iron-dependent enzymes (14, 15). The mechanism by which that NO has the capacity to catalyze deleterious reactions due
iron is lost from target cells is presently unknown. However, to the affinity of NO for transition metals (29). Therefore, the
the amount of iron lost from these target cells is not accounted biological effect(s) of NO will depend upon the site as well as
for simply by losses from iron-dependent enzymes and may result the rate of NO generation. This may explain why NO generation,
from iron release from ferritin (13). Supporting a role for NO like intracellular calcium concentration, appears to be so tightly
in this process, Lancaster and Hibbs (4), utilizing ESR spec- regulated (27).
troscopy, recently demonstrated an iron:nitrosyl complex in cy-
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