doi:10.1182/blood-2007-07-102335Prepublished online November 1, 2007;2008 111: 1690-1699   

 Yang-Sung Sohn, William Breuer, Arnold Munnich and Z. Ioav Cabantchik therapeutic implicationsRedistribution of accumulated cell iron: a modality of chelation with

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RED CELLS

Redistribution of accumulated cell iron: a modality of chelation withtherapeutic implicationsYang-Sung Sohn,1 William Breuer,1 Arnold Munnich,2 and Z. Ioav Cabantchik1

1Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Safra Campus at Givat Ram, Jerusalem,Israel; and 2Clinical Research Unit, Medical Genetic Clinic and Research Unit, Inserm Unite 781, Hopital Necker-Enfants-Malades and Universite Paris V ReneDescartes, Paris, France

Various pathologies are characterized bythe accumulation of toxic iron in cellcompartments. In anemia of chronic dis-ease, iron is withheld by macrophages,leaving extracellular fluids iron-depleted.In Friedreich ataxia, iron levels rise in themitochondria of excitable cells but de-crease in the cytosol. We explored thepossibility of using deferiprone, amembrane-permeant iron chelator in clini-cal use, to capture labile iron accumu-lated in specific organelles of cardiomyo-cytes and macrophages and convey it toother locations for physiologic reuse. De-

feriprone’s capacity for shuttling iron be-tween cellular organelles was assessedwith organelle-targeted fluorescent ironsensors in conjunction with time-lapsefluorescence microscopy imaging. De-feriprone facilitated transfer of iron fromextracellular media into nuclei and mito-chondria, from nuclei to mitochondria,from endosomes to nuclei, and from intra-cellular compartments to extracellularapotransferrin. Furthermore, it mobilizediron from iron-loaded cells and donated itto preerythroid cells for hemoglobin syn-thesis, both in the presence and in the

absence of transferrin. These uniqueproperties of deferiprone underlie mecha-nistically its capacity to alleviate ironaccumulation in dentate nuclei of Fried-reich ataxia patients and to donate tissue-chelated iron to plasma transferrin inthalassemia intermedia patients. De-feriprone’s shuttling properties could beexploited clinically for treating diseasesinvolving regional iron accumulation.(Blood. 2008;111:1690-1699)

© 2008 by The American Society of Hematology

Introduction

The pathologic effects of iron accumulation in tissue are recog-nized in diseases of systemic iron overload, in which the liver,heart, and endocrine glands are the principal affected organs.1 Atthe cellular level, labile iron begins to rise once the intracellularcapacity for iron storage is surpassed, leading to catalyticformation of reactive oxygen species (ROS) that ultimatelyoverwhelm the cellular antioxidant defense mechanisms andlead to cell damage.2

In recent years, several pathologies have been shown to beassociated with specific defects in cellular iron metabolism that donot give rise to conspicuous systemic iron overload.3 In theneuromuscular disorder Friedreich ataxia, the deficiency of theiron-chaperone protein frataxin is thought to lead to mitochondrialiron accumulation due to improper processing of iron for heme andiron-sulfur-cluster formation.4 In the group of neuromusculardisorders termed NBIA (neurodegeneration with brain iron accumu-lation), a deficiency in pantothenate kinase (PKAN-2), a keyenzyme in coenzyme A synthesis,5 leads to iron deposition andensuing brain damage by still-unresolved mechanisms.6 In heredi-tary X-linked sideroblastic anemia, impaired heme synthesiscauses mitochondrial iron accumulation and siderosis in erythroidcells.3 However, an extreme example of systemic misdistribution ofiron is encountered in anemia of chronic disease (ACD), where ironaccumulates in reticuloendothelial cells responsible for recyclingaged erythrocytes, presumably due to decreased activity of theiron-efflux protein ferroportin.7 The marked down-regulation of

ferroportin in the plasma membranes of macrophages in ACD isthought to be triggered posttranslationally by the circulatingiron-regulatory peptide hepcidin, which is induced by inflamma-tion8 and transcriptionally by certain cytokines.7,8 Thus, macro-phage iron accumulation in ACD not only coexists with anemia butalso is largely responsible for it.9

Here we explore the concept of relocating iron from areas ofaccumulation to areas of deprivation using agents capable ofpermeating membranes, chelating cell labile iron and donating ironto physiologic acceptors. Most agents in clinical use for managingsystemic iron overload1 were designed and/or are applied forcorrecting or preventing hepatic and cardiac siderosis, the majorcauses of premature death in thalassemia.2 Those chelators demon-strably reduce labile iron levels in plasma, but a few, by virtue oftheir membrane-crossing ability, also do so in cells.1,10 However,whether such agents could or should be used in diseases in whichregional iron accumulation is not accompanied by hyperferremia isquestionable. In the present case, the goal of chelation is not merelysystemic depletion, but redistribution of iron.

The selection of the orally active chelator deferiprone (DFP) asa prototype iron-relocating agent is based on several observations.DFP has been shown to shuttle tissue iron into the circulation tohigher-affinity chelators such as desferrioxamine (DFO)11 and toincrease transferrin (Tf) saturation in individuals with unimpairedserum iron–binding capacity.12 Donation of iron by DFP to DFOand to transferrin was also observed in vitro.11 Recently, DFP was

Submitted July 19, 2007; accepted October 25, 2007. Prepublished online asBlood First Edition paper, November 1, 2007; DOI 10.1182/blood-2007-07-102335.

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found to reduce iron accumulation in the dentate nuclei ofFriedreich ataxia patients,13 indicating its ability to cross theblood-brain barrier in humans, which is consistent with previousfindings in animals.14-16

In the present study we assess DFP’s capacity to act as aniron-relocating agent at the cellular level. To trace labile ironmobilized by chelators, we used model cardiac and macrophagecells in culture and fluorescent metal sensors (FMS) targeted tocellular and extracellular compartments. We show that DFP canrelocate iron accumulated in cell compartments within and acrossthe cell, and we demonstrate that iron withdrawn from sites of cellaccumulation can be safely transferred to extracellular transferrinfor physiologic reuse.

Methods

Materials

Calcein green (CALG) 3,3�-bis[N,N-bis(Carboxymethyl) aminomethyl]fluo-rescein and its acetomethoxy (AM) precursors CALG-AM, lissaminerhodamine sulfonyl chloride, and sulforhodamine were from MolecularProbes (Eugene, OR). Human serum holotransferrins and apotransferrins(aTf) were from Kamada (Rehovot, Israel). Diethylene-triamine-pentaacetic acid (DTPA), nitrilotriacetate (NTA), EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), ferric ammonium citrate (FAC),ferrous ammonium sulfate (FAS), succinyl acetone, HEPES (N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid), 4-morpholine ethanesul-fonic acid (MES), core histone mixture from calf thymus (H), N,N�-hexamethylene-bis-acetamide, and octyl-glucoside were from Sigma-Aldrich (St Louis, MO). Rhodamine isothiocyanate was from Fluka (Buchs,Switzerland). DFP (1,2-dimethyl-3-hydroxypyridin-4-one) was from Apo-Pharma (Toronto, ON). DFO was from Novartis (Basel, Switzerland). Thered fluorescent mitochondrial metal–sensor rhodamine B-[(1, 10-phenanth-rolin-5-yl aminocarbonyl] benzyl ester (RPA), was a kind gift from U.Rauen and R. Sustmann, University of Duisburg-Essen, Essen, Germany.A summary of the probes used and their properties is given in Table 1.

Methods

Complexes of iron with NTA (Fe-NTA) were generated by mixing FAS andNTA (1:3 molar ratio) in water and allowing iron to oxidize in ambientconditions. The DFP-Fe(III) complexes were generated by mixing Fe-NTAwith DFP in water; formation of fully substituted DFP-Fe complexes wasconfirmed by measuring absorbance at 455 nm.19

Cell-culture lines. Rat H9C2 cardiomyocytes, J774 mouse macro-phages, and erythroleukemia (MEL) were grown in 5% CO2 Dulbecco-modified Eagle medium (DMEM) supplemented with 10% fetal calf serum,4.5 g/L D-glucose, glutamine, and antibiotics (Biological Industries,Kibbutz Bet Haemek, Israel). Cells were plated 1 day before experimenta-

tion onto 96-well plates, or onto microscopic slides attached to perforatedtissue culture 3-cm plates.

Probe loading into cells. For cytosolic loading of CALG, cells inDMEM plus 10mM Na-HEPES (DMEM-HEPES) at 37°C were exposedfor 10 minutes to 0.25 �M CALG-AM, washed and incubated inDMEM-HEPES containing 0.5 mM probenecid (to minimize probe leak-age). For CALG-Fe(III) (1:1) complexes and for sulforhodamine loadinginto endosomes, cells were exposed to 50 �M to 100 �M complex inDMEM-HEPES for 30 minutes at 37°C and washed extensively with thesame probe-free medium and subsequently with HEPES-buffered saline(HBS; 20 mM HEPES, 135 mM NaCl; pH 7.4).20,21 RPA was loaded intomitochondria as described in earlier studies.21-23

Histone-CALG. For loading into the nucleus, CALG was coupled tocore histones from calf thymus (H) 7.2 mg/mL, in 50 mM Na-MES,100 mM NaCl, pH 5.5, containing 1.25 mM CALG:Cobalt (Co protectsCALG metal-binding groups). Coupling was initiated by adding 12 mMEDC, followed by incubation for 1 hour at room temperature and overnightat 5°C. After exhaustive dialysis, 11 mM DTPA was added (to removeCo(II)) and histone-CALG (H-CALG) was further dialyzed against HBS.CALG coupling to H was estimated at 1.1:1 by fluorescence (� excitation480 nm, � emission 520 nm). H-CALG-Fe was prepared by titrating CALGwith FAS. Loading 10�M H-CALG or H-CALG-Fe into cells was done inDMEM-HEPES containing 100 �M chloroquine (for improved FMStargeting to cell nuclei),24 followed by HBS washes.

Fluorescein-DFO-histone. The probe was prepared by coupling ofN-(fluorescein-5-thiocarbamoyl)-desferrioxamine (Evrogen, Moscow, Rus-sia) to H using fluorescein-DFO (FlDFO)–Fe (1:1) complex generated byadding 1.1 mM FeSO4 to 1 mM FlDFO in HBS. To 2.64 mL of FlDFO-Fecomplex was added 8.5 mg H, followed by 0.18 mL of 1 M MES (Na form),pH 6.5, and 15 mg of EDC. After overnight agitation at 5°C, the mixturewas dialyzed (3.5 kDa cut-off tubing) against 10 mM acetic acid;1 mM EDTA; 150 mM NaCl, pH 4.5 (to remove bound iron); and finallyHBS. The fluorescein-DFO-histone (H-FlDFO) ratio was 1:1, based onstoichiometric iron-quenching of H-FlDFO fluorescence (� excitation494 nm, � emission 520nm). H-FlDFO, like H-CALG, was loaded into cellsin DMEM-HEPES.

Rhodamine-labeled apotransferrin. Human apotransferrin (4 mg/mLin 25 mM Na2CO3, 75 mM NaHCO3; pH 9.8), was incubated at 5°Covernight with 1 mM lissamine rhodamine sulfonyl chloride and the labeledprotein isolated by gel filtration on Sepharose G25 (Sigma-Aldrich)preequilibrated with 150 mM NaCl, 20 mM MES; pH 5.3.

Transfer of iron from DFP-Fe to apotransferrin. DFP-Fe complexeswere generated by incubating Fe-NTA (50:150 �M) with either 150 �M or250 �M DFP in HBS for 20 minutes, and completion of complex formationwas assessed by absorption at 455 nm. The complexes were mixed with50 �M aTf and 25 mM NaHCO3 and incubated for 1.5 hours in a 5% CO2

incubator. Because of the large spectral overlap of DFP-Fe and transferrin-Fe, we added DTPA (10 mM) to selectively scavenge iron from DFP-Fe.Transferrin that was free of low-molecular components was obtained byeither filtering 0.5 mL through 30-kDa cut-off spin filters (Pall, East Hills,NY) via centrifugation (2900g for 20 minutes), or by use of a 5-mLdry-spin–gel filtration-centrifugation at 1100g over precentrifuged Seph-adex G-50 medium columns (Sigma-Aldrich). The complex-free transferrinwas diluted in HBS, pH 7.4. Tf-Fe was analyzed by absorbance at 465 nm,and aTf by tryptophan fluorescence (280 nm excitation, 306 nm emission;Felix spectrofluorimeter station version 2.5; Photon Technology Interna-tional, Lawrenceville, NJ).

Iron-loading of cells was done by overnight incubation of cells with100 �M FAC in culture conditions followed by washing with HBScontaining 100 �M DFO, and with HBS alone. To achieve selectiveaccumulation of iron in mitochondria, cells were treated with 1 mMsuccinyl acetone for 3 hours at 37°C.

Cell hemoglobin synthesis. MEL cells cultured for 2 days in mediasupplemented with 5mM hexamethylene-bis-acetamide were washed andlysed with octyl-glucoside (1.5%). After 2 minutes centrifugation at12 000g, 100 �L of lysate supernatants was transferred to a 96-wellmicroplate for estimating hemoglobin (by measuring absorbance at410 nm). Alternatively, lysates were mixed with 100 �L freshly prepared

Table 1. Iron binding parameters of agents used in this study

Iron ligands (abbreviations) pFe(III)*

Calcein green (CALG) 20.3†

Deferrioxamine (DFO) 26.6

Deferiprone (DFP) 19.3

Diethylene-triamine-pentaacetic acid (DTPA) 28.2

Nitrilotriacetic acid (NTA) 18.1

Transferrin (Tf) 22.3

Rhodamine B-�(1, 10-phenanthrolin-5-yl)

aminocarbonyl� benzyl ester (RPA)

7.7 (11.5 for Fe(II))‡

*pFe(III) values are �log�M�, where M is the concentration of the free metal ionwhen �ligand� � 10�M and �Fe(III)� � 1�M at pH 7.4. Data from Harris.17

†Data from Thomas et al.18

‡Values shown are for 1,10-phenanthroline.

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tetramethylbenzidine (0.5 mg/mL in 10% acetic acid) and finally 8 �L of30% H2O2. Absorption (604 nm) was read after 90 seconds.

Data acquisition and analysis. Epifluorescence imaging (Nikon TE2000 microscope; Melville, NY; and Orca-Era CCD camera; Hamamatsu,Bridgewater, NJ) coupled to a Volocity 4 system (Improvision, Coventry,United Kingdom) was used for image data acquisition and analysis.20 Forhigh-throughput fluorescence monitoring we used fluorescence plate read-ing (Tecan-Safire; Neotec, Mannedorf, Austria) essentially as described.20

Results

To follow DFP-mediated translocation of iron from the medium tocells, within cells, and from cells to the medium, we usedfluorescent acceptors and donors of iron that undergo quenching ordequenching upon metal binding or removal. An additional featureof these probes was their localization in specific cell compartmentsor in the extracellular medium.

Transfer of iron from extracellular DFP-iron complexes tomitochondria

Ingress of ionic iron from the extracellular medium to themitochondria of H9C2 cardiomyocytes was followed with thehigh-affinity Fe(II)–acceptor probe, RPA22,23 (Figure 1). The hydro-phobic cationic rhodamine B is responsible for RPA’s potentiomet-ric partitioning in the mitochondria, and phenanthroline is respon-sible for detection of Fe(II).22 Exposure of cells to Fe(III)-NTAevoked no significant changes in RPA fluorescence relative to thespontaneous decrease (t1⁄2 60 minutes), probably due to binding of

endogenous labile iron and/or to probe photobleaching. On theother hand, addition of preformed DFP-Fe(III) complexes (DFP-Fe, ratio 3:1) evoked a time-dependent (t1⁄2 15 minutes) quenchingof mitochondrial RPA fluorescence (Figure 1E). Consistent withRPA’s selectivity for Fe(II), no quenching of RPA by Fe-NTAoccurred in solution unless ascorbate was added. Transfer of ironfrom DFP-Fe complexes to RPA in the presence of ascorbate wasdetected over a wide range of DFP to Fe ratios (1:1 to 7:1).

Transfer of iron from extracellular DFP-Fe complexes tonuclei. With the view of targeting a fluorescent iron sensor intothe cell nucleus, we constructed a conjugate of H with thehigh-affinity iron chelator and iron sensor H-FlDFO.11 To minimizeuptake via the endocytic pathway, incubation was carried out atroom temperature and in the presence of chloroquine, as describedin “Histone-CALG.” H-FlDFO within nuclei was rapidly(t1⁄2 � 5 minutes) quenched by DFP-Fe (3:1) added to the extracel-lular medium (Figure 2E), consistent with the high cell permeabil-ity of DFP-Fe complexes shown in Figure 1. In solution, H-FlDFOis efficiently and rapidly (t1⁄2 � 1 minute) quenched by DFP-Fecomplexes even at a 9:1 DFP:Fe ratio (Figure 2F), as expected fromDFP’s capacity to shuttle iron to DFO both in vivo and in vitro.11

DFP-mediated redistribution of iron between cellular compart-ments: from nuclei to mitochondria. DFP’s facilitation of ironmovement between discrete cell compartments was followed inH9C2 cells sequentially labeled in cell nuclei with the iron-donorprobe H-CALG-Fe(III) and in mitochondria with the Fe(II)-acceptor probe RPA. H-CALG-Fe penetrates the plasma membraneand accumulates initially in the cytosol and subsequently in the

Figure 1. Transfer of iron from extracellular DFP-ironcomplexes to mitochondria. H9C2 cells loaded with themitochondrial iron-sensor RPA were incubated with orwithout DFP-Fe (15:5 �M) and epifluorescence micro-scopic images were recorded every 5 minutes undersettings for rhodamine. Representative fields of initial cellfluorescence at time 0 (A,C) and after incubation for 1 hourin the presence (B) or absence (D) of 5 �M DFP-Fecomplex. Magnification was 600; oil objective was a(Plan Apo) 60/1.40 NA. (E) Mean fluorescence values inrelative units (r.u.) plus or minus SD of 5 cells per fieldcalculated for each time-point image and normalized to theinitial fluorescence, representing cells incubated without(None) and with 5 �M Fe-complex (DFP-Fe or NTA-Fe);the lines denoted as DFP-Fe and NTA-Fe were cor-rected for spontaneous decay given by the control (None).Arrow indicates time the Fe complex was added. (F) Effectof various iron chelates on the fluorescence (f normalizedto initial value, f0, in r.u.) of 0.5 �M RPA in solution (HBSbuffer). The iron chelates all contained 5�M Fe complexedto NTA (1:3 ratio) in the absence (f) and presence (�) of100 �M ascorbate (Fe:NTA, Fe:NTA � ASC) or to DFP,with 100 �M ascorbate, at DFP:Fe ratios of 3:1 (F), 5:1 (E),and 7:1 (‚). The values of fluorescence intensity aremeans of triplicate samples run in parallel, plus orminus SEM.

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nucleus, particularly in nucleolus-like sites.21 H-CALG-Fe wasused subsaturated with iron so that its accumulation in the nucleicould be followed via changes in the residual fluorescence of theunquenched fraction of the probe (revealed by chelation; Figure3A). Loading of RPA into mitochondria was apparent from thecharacteristic perinuclear staining pattern (Figure 3C), from colo-calization with other potentiometric probes and sensitivity toprotonophores.22,23 Addition of 50�M DFP produced an increase inthe fluorescence of H-CALG-Fe in the nuclei and a concomitantdecrease in the fluorescence of RPA in the mitochondria, both ofwhich occurred within 5 to 8 minutes and were complete within20 minutes (Figure 3E). The changes are attributable to dequench-ing of H-CALG-Fe due to removal of iron by DFP and toquenching of RPA due to transfer of iron from DFP-Fe. Only minorchanges in fluorescence were observed in untreated control cells. Itis likely that nuclear H-CALG-Fe was not the only source of irondelivered to RPA by DFP, because the addition of DFP toRPA-loaded cells also led to quenching of mitochondrial probe(data not shown), although to a much lesser extent than in thepresence of H-CALG-Fe.

DFP-mediated relocation of iron between cellular compart-ments: from endosomes to nuclei. H9C2 cells were labeledsequentially in the nuclei with the iron-acceptor probe H-FlDFO and inthe endosomes with the iron-donor probe CALG-Fe (Figure 4). Weshowed the bulk-phase pinocytic uptake of the iron-quenched probe

CALG-Fe into endosome/pinosomes and its accessibility to DFP, asevidenced by dequenching, in a previous study.20 Moreover, CALG-Fecoendocytosed with the classical fluid phase markers sulforhodamineand rhodaminated dextran, both of which showed less than 65% cellcolocalization as obtained with the Volocity program (not shown).H-FlDFO was detectable in the nuclear area, while CALG-Fe wasconcentrated in punctuate, perinuclear spots, attributable to endosome-trapped CALG-Fe (Figure 4). However, after incubation with 50 �MDFP, the fluorescence in the endosomes increased significantly, whilethat in the nuclei decreased relative to the respective untreated controls.The kinetics of the changes in the 2 compartments followed similarpatterns, although in opposite directions. To ascertain whether the ironacquired by DFP was exclusively intracellular, the cells were washedwith 0.1 mM DFO to remove all extracellular iron before the addition ofDFP. Furthermore, as the addition of DFP to cells loaded with H-FlDFOalone failed to produce a decrease in its fluorescence (data not shown),the quenching of H-FlDFO in cells coloaded with CALG-Fe is mostlikely due to DFP-mediated mobilization of iron from endosomes.However, the possibility that in the course of the experiment someiron might have spontaneously leaked from endosomes to cytosoland rendered it transferable to mitochondria by added DFP cannotbe ignored.

Figure 2. Transfer of iron from extracellular DFP-iron complexes to nuclei.H9C2 cells loaded with the nuclear iron sensor H-FlDFO were incubated with orwithout 5�M DFP-Fe complex (DFP:Fe ratio 3:1) and epifluorescent microscopicimages were recorded every 5 minutes under settings for fluorescein. Representativefields of initial cell fluorescence at time 0 (A,C) and after incubation for 1 hour in theabsence (B) or presence (D) of 5�M DFP-Fe complex. (E) Mean fluorescence valuesin r.u. plus or minus SD of 5 cells per field, calculated for each time-point image andnormalized to the initial fluorescence (f/f0), representing cells incubated without(None) and with 5�M DFP-Fe complex (DFP-Fe). Arrow indicates time the Fecomplex was added. (F) Effect of DFP-Fe chelates (DFP:Fe at ratios of 1:1, 3:1, 5:1,9:1) on the fluorescence (normalized to initial value, f0, in r.u.) of 0.5�M H-FlDFO insolution (HBS buffer). The values of fluorescence intensity are means of triplicatesamples run in parallel, plus or minus SEM.

Figure 3. DFP-mediated relocation of iron between cellular compartments:from nuclei to mitochondria. H9C2 cells were loaded with the nuclear iron sensorH-CALG that had been precomplexed to iron (H-CALG-Fe), followed by loading withthe mitochondrial iron sensor RPA. The double-labeled cells were then exposed to50�M DFP and epifluorescence images were recorded every 5 minutes. Representa-tive fields of cell fluorescence observed under settings for fluorescein (A,B) andrhodamine (C,D). Images are shown at time 0 (A,C) and after incubation for 1 hour inthe presence of 50�M DFP (B,D). (E) Mean fluorescence values plus or minus SD inr.u. of 5 cells per field, calculated for each time-point image and normalized to theinitial fluorescence (f/f0), representing cells incubated with 50�M DFP (circles) andwith no addition (squares). Fluorescence of H-CALG is indicated by filled symbols(F and f), and fluorescence of RPA is indicated by open symbols (E and �). Thescheme illustrates entry of DFP into the cytosol (C), nuclei (N) containing H-CALG-Fe(iron donor) and mitochondria (M) containing RPA (iron acceptor), and transfer of ironfrom nuclei to mitochondria. E refers to endosomes.

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Transfer of iron from DFP-Fe complexes to transferrin. Wefollowed iron complexation by transferrin using DFP-Fe com-plexes (3:1 and 5:1, 50 �M in Fe) and NTA-Fe complexes (3:1,50 �M in Fe) as iron sources and apotransferrin (50 �M) asacceptor. To remove from the reaction mixture any traces of ironassociated with low molecular weight complexes or free ligands,we added DTPA after 1 hour of reaction and followed it by sizefiltration. Filtration and differential chelation allowed the assess-ment of DFP-Fe transfer of iron to aTf both by light absorption ofTf-Fe complexes at 465 nm (Figure 5) and by tryptophan fluores-cence of aTf that is quenched after specific binding of iron25

(Figure 5). While the 2 analytical methods monitored binding ofiron to transferrin, the optical changes are mirror images of eachother. Transfer of iron from DFP-Fe to transferrin ensued at both3:1 and 5:1 stoichiometries of DFP:Fe and was of a stable nature: itpersisted after addition of chelators that differentially dissociateDFP-Fe complexes (but not Fe-transferrin). On the other hand,DFP alone and NTA alone evoked only minor changes that wereeliminated by addition of DTPA before the reaction with aTf,indicating that the changes were associated with traces of ironpresent in the medium. Essentially similar results were obtained byassessing the transfer of iron to apotransferrin using fluorescein-labeled aTf (not shown). However, with lissamine rhodamine-labeled aTf (R-aTf), the acquisition of iron evoked insignificantchanges in fluorescence.

DFP-mediated mobilization of iron from H9C2 cells toextracellular transferrin: receptor-mediated uptake ofholotransferrin as an index of iron shuttling

Mobilization of cellular iron by DFP and its transfer to extracellulartransferrin are critical features of DFP’s potential function as aniron shuttle. This capability was assessed using H9C2 cellsincubated with lissamine R-aTf in the presence of DFP (Figure 6).

The rationale was that intracellular iron mobilized to the extracellu-lar medium by DFP would be transferred to the fluorescent Tf,which, upon binding iron, would then bind to cell-surface trans-ferrin, and after receptor-mediated endocytosis, would becomeconcentrated in endosomes. To increase the level of mitochondriallabile iron, the cells were pretreated with the heme-synthesisinhibitor succinylacetone, which causes selective accumulation ofunused, chelatable iron in the mitochondria.22 After 1 hour ofincubation with DFP, the level of cell-bound fluorescent aTf roseby 2.3-fold over controls without DFP (Figure 6A,B). The bindingwas specific and iron dependent, as it was fully blocked by excessholotransferrin (Figure 6C,D) and enhanced by addition of saturat-ing concentrations of Fe-NTA (Figure 6E,F), both in the presenceand in the absence of DFP. In control cells not treated withsuccinylacetone, DFP failed to enhance fluorescent-Tf binding(data not shown), indicating that under normal conditions whereiron incorporation into heme is not blocked, the concentration ofDFP-mobilized iron is insufficient to be detectable in this system.

DFP-mediated iron delivery for hemoglobin synthesis. Thepotential of DFP-Fe complexes as donors of iron for physiologicuse was assessed using a model system of cellular iron incorpora-tion, synthesis of hemoglobin (Hb) by differentiating preerythroidcells. The well-documented induction of Hb synthesis in MEL cellsby hexamethylene bisacetamide (HMBA) is highly dependent onthe supply of iron.26 It is repressed in serum-free medium lackingtransferrin, but it is significantly enhanced by the addition ofFe-NTA irrespective of the presence of Tf (Figure 7). DFP-Fe (3:1)supported Hb synthesis to a degree similar to that seen withFe-NTA, both in the absence and presence of added Tf. Fullysaturated holotransferrin (15 �M) was equally effective as Fe-NTAand DFP-Fe (data not shown), indicating that various forms of ironcan equally support Hb synthesis in this experimental system. Onthe other hand, DFP without added iron depressed Hb synthesis

Figure 4. DFP-mediated relocation of iron betweencellular compartments: from endosomes to nuclei.H9C2 cells were loaded with the nuclear iron-sensorH-FlDFO and subsequently exposed to preformedcomplexes of CALG and iron (CALG-Fe, 1:1 ratio) thatwere taken up by adsorptive pinocytosis into endo-somes. The cells containing both labels were thenexposed to 50 �M DFP and epifluorescent imageswere recorded every 5 minutes under fluorescein set-tings. Representative fields of cell fluorescence areshown at time 0 (A) and after incubation for 20 minutes(B) in the presence of 50 �M DFP. (C) The nuclei(N) and endosomes (E) are indicated by arrows.(D) Mean fluorescence values in r.u. within selectedsubcellular areas corresponding to endosomes(Di) and nuclei (Dii), using 5 cells per field, calculatedfor each time-point image and normalized to the initialfluorescence (f/f0).

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below control levels, indicating that the chelator can have aniron-withdrawing effect under conditions of limited iron supply.In the serum-free medium used in these experiments, DFP(� 100 �M) without added iron, as well as DFO (100 �M),prevented differentiation and ultimately caused massive cell death

(data not shown), an effect similar to DFO-induced iron depriva-tion in other hematopoietic cells.27 To demonstrate that transfer ofiron from DFP-Fe to aTf gave rise to functionally active holotrans-ferrin, DFP-Fe was allowed to interact with aTf for 1 hour, and thenthe mixture was exhaustively dialyzed (cut-off 12 kDa) against

Figure 5. Transfer of iron from DFP-Fe complexesto transferrin. DFP-Fe complexes (1:3 and 1:5 ratios;50 �M Fe(III)) were mixed with apotransferrin aTf(50 �M) and incubated for 1.5 hours in a humidified 5%CO2 incubator at 37°C. At the conclusion of the reac-tion, 10 mM DTPA was added and the low–molecularweight material was removed by filtration as describedin “Histone-CALG.” The tryptophan (trp) fluorescenceat 280 nm excitation/306 nm emission (top graph) wasobtained from the emission spectra (inset). The high–molecular weight fractions were diluted in HBS, pH 7.4,and the absorbance was read at 465nm (bottom graph).Data are given as means plus or minus SD of3 independent experiments. The labels below the barsindicate the complexes tested and their concentrations.The values above the bars represent the percentagechange in absorbance or fluorescence.

Figure 6. DFP-mediated mobilization of iron fromH9C2 cells to extracellular apotransferrin: receptor-mediated uptake of holotransferrin as an index of irontransfer. H9C2 cells were pretreated with succinylac-etone as described in “Methods” to increase mitochon-drial labile iron levels. They were then incubated at37°C in DMEM-HEPES medium containing 20 �Mlissamine R-aTf with various additions, and epifluores-cence microscopy images were obtained under rhodaminesettings after 60 minutes of incubation. Representativeimages of cells with no addition (A), 30 �M DFP (B),100 �M unlabeled holotransferrin (C), 100 �M unlabeledholotransferrin with 30 �M DFP (D), 20 �M Fe-NTA, 1:3ratio (E), and 20 �M Fe-NTA with 30 �M DFP (F). (G) Theillustration represents entry of DFP into cells, mobilizationof iron from the cytosol (C), nuclei (N) and mitochondria(M), followed by exit of DFP-Fe complexes from the cells(step 1). This is followed by transfer of iron from DFP-Fe toR*-aTf to form R*-Tf-Fe (step 2), which then binds totransferrin receptors on the cell surface (step 3), concen-trates in the endosomes, and is detected by fluorescencemicroscopy as punctuate fluorescence typical of mi-crovesicles. (H) Mean cell-associated fluorescence valuesin r.u. of 5 cells per field ( SD, from 3 separate experi-ments), calculated from snapshots such as shown inpanelsA through F, obtained after 1 hour of incubation.

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buffer containing 80 mg/L aTf to remove DFP-Fe without gainingcontaminant iron from the solution. When added to differentiatingMEL cells, this preparation, generated from DFP-Fe and aTf, fullysupported Hb synthesis (Figure 7B).

Finally, we assessed DFP’s ability to shuttle iron from cell tocell (Figure 7C). In vivo, direct intercellular transfer of iron viaDFP is not likely, due to the presence of more than 50 �M due tothe presence of more than 25 �M transferrin in plasma.28 However,intercellular transfer of iron via DFP could occur in the brain wherethe iron-binding capacity of transferrin in cerebrospinal fluid (CSF)is estimated to be in the submicromolar range.29,30 In the experi-ment illustrated in Figure 7A, J774 mouse macrophages werechosen as the iron-donating cells because of their high capacity foriron accumulation.31 DFP at a concentration of 30 �M, achieved invivo with moderate oral doses,1,32 mobilized sufficient iron fromiron-loaded J774 cells, but not from untreated ones, to significantlyenhance Hb synthesis in MEL cells (Figure 7C). Spontaneousefflux of iron from iron-loaded J774 cells took place, as evidencedby the increased MEL cell Hb levels even in the absence ofDFP (although increased, these levels were significantly lower inthe absence of DFP than in its presence). This result provides

support for the proposed use of DFP as an iron shuttle even withoutthe mediation of transferrin, such as might occur in the centralnervous system.

Discussion

The recognition that intracellular iron accumulation is one of theunderlying causes of some clinical syndromes has led to thesuggestion that it might be amenable to treatment with ironchelators.3,33,34 These syndromes include Parkinson disease, neuro-degeneration with NBIA, and Friedreich ataxia, where iron depos-its in specific areas of the brain have been identified by histologic orimaging techniques34 and are thought to be a central factor in thedevelopment of the diseases.35 Similarly, in anemia of chronicdisease, iron is retained within erythrocyte-phagocytosing macro-phages, presumably to prevent access by invading pathogens toiron from the circulation.7-9,31 While regional iron mobilizationmight be essential for stabilizing or reversing the toxic effects oflabile iron, in some pathologies it might be equally important torender the mobilized iron available for metabolic reuse. This is

Figure 7. Stimulation of Hb synthesis in murine erythroleukemia cells. (A) DFP-mediated iron delivery to murine erythroleukemia (MEL) cells synthesizing hemoglobin isschematically depicted. (B) MEL cells suspended in serum-free DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA were supplemented with various ironcomplexes (step 1 in panel A) and cultured for 48 hours. Hemoglobin synthesis (step 2 in panel A) was assessed in terms of hemoglobin content in cell lysates as described in“Cell hemoglobin synthesis” and is expressed relative to the hemoglobin content in control cells with no additions (set as 100%). The additions included 10 �M Fe complexed to30 �M NTA without (Fe-NTA) and with 15 �M human apotransferrin (Fe:NTA � aTf); 10 �M Fe complexed to 30 �M DFP without (DFP-Fe) and with 15 �M humanapotransferrin (DFP-Fe � aTf); 30 �M DFP alone (DFP); 15 �M human apotransferrin alone (aTf). Generation of holotransferrin from DFP and apotransferrin (DFP-Fe � aTfDial.): 10 �M Fe:30 �M DFP complex was preincubated with 15 �M human for 1 hour and dialyzed. Results shown are averages of 3 separate experiments plus or minus SD;the average Abs604 of control cells was 0.39. (C) DFP-mediated mobilization of iron from J774 (Fe donor; step 1a in panel A) cells to the extracellular medium (step 1b in panelA), followed by entry of DFP-Fe complexes into MEL (Fe acceptor) cells (step 1c in panel A) and intracellular donation of iron for hemoglobin (Hb) synthesis (step 2 in panel A).MEL cells were cultured for 48 hours in DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA without (Con) or with 10 �M Fe-NTA (NTA-Fe), or with varioussupernatants of J774 cell lysates that were supplemented with 5 mM HMBA, and lysates were assayed for hemoglobin content. To obtain J774 mouse macrophagesupernatants, J774 cells were cultured overnight without (untreated J774) or with 100 �M FAC (Fe-loaded J774), washed with 100 �M DFO to remove all extracellular iron, andincubated for 2 hours at 37°C in serum-free DMEM containing 1.25 mg/mL bovine serum albumin, without (�DFP) or with 30 �M DFP (� DFP). The cell supernatants werecollected and centrifuged to remove detached cells, and HMBA (5 mM) was added to MEL cells. Shown are values of hemoglobin (Hb; from a representative experiment)obtained in MEL cells exposed for 48 hours to the various conditions.

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especially important in those conditions in which regional ironaccumulation is accompanied by regional or systemic deprivation.All the chelators in clinical use have been designed for massiveremoval and excretion of iron from organs of iron-overloadedpatients.2,10,33 Here we assessed the potential application of an ironchelator in clinical use (DFP) as a shuttling agent capable ofredistributing iron within and among cells, while satisfying certainbasic requirements.

Permeability across cell membranes in the free and iron-bound form. The high membrane permeability of the iron-freeform of DFP is well documented, as shown by its capacity torapidly access and deplete intracellular labile iron pools.36,37 On theother hand, the ability of DFP-Fe complexes to traverse intracellu-lar and plasma membranes of cells has not been studied indetail.20,21,36,37 One indication is the capacity of orally administeredDFP to increase serum transferrin saturation12 and labile iron11

within an hour after intake in thalassemia patients, which isattributable to the rapid exit of DFP-Fe complexes from cells intothe circulation. At the intracellular level, it is shown in this workthat DFP can facilitate the removal of labile iron from nuclei(Figure 3), from endosomes (Figure 4), and from mitochondria(Figure 6), and can transfer iron to acceptors in the mitochondria(Figure 3), nuclei (Figure 4), and extracellular medium (Figure 6).The transfer might involve dissociation of the DFP-Fe complexesand/or formation of ternary complexes with other ligands.

Ability to compete effectively for intracellular labile iron withother intermediate affinity species. Labile iron that is likely to beaccessible to DFP is assumed to be bound to several possibleligands (eg, citrate, ATP) and to be in dynamic equilibrium betweenFe(II) and Fe(III) as dictated by the redox status of the intracellularenvironment.38,39 DFP competes effectively for labile cell iron bothin vitro and in vivo,32 and in individuals with normal iron balance itraises serum iron levels, indicating mobilization of tissue iron.12

This was also observed with isolated cells (Figures 3,4), whereDFP readily chelated iron bound to the EDTA analog CALG inendosomes and nuclei and was indicated in other systems.40 Anundesirable property of a chelating molecule would be interferencewith normal cellular iron metabolism by overchelation. In thisrespect, DFP ought to be used at restricted doses, as at high levels itappears to inhibit iron-requiring tyrosine and tryptophanhydroxylases.10,14

Ability to donate iron to physiologic acceptors. The rationalebehind DFP-mediated redistribution of iron leans on DFP’s abilityto transfer the metal to extracellular transferrin, thereby minimizingundesired iron loss by excretion via the urinary or biliary pathways.We surmised that under physiologic conditions iron transfer to aTfis likely to occur (1) spontaneously, as the pFe value of Fe(III) fortransferrin is 3 orders of magnitude higher than for DFP41 (Table 1),and (2) directly as Fe(III), because the thermodynamically favoredDFP-Fe complexes are (DFP)2-Fe(III) and (DFP)3-Fe(III).10,25,42 Invitro studies based on an indirect analytical method11 corroboratedearlier observations that a single oral dose of 3 g DFP raised within6 hours the transferrin saturation (determined by urea-gel electro-phoresis) of a healthy individual from a normal level of 20% to80%.12 In this work we provided direct demonstration of irontransfer from DFP-Fe to aTf at physiologic concentrations (Figure5). Using spectrophotometric and fluorimetric methods, we showedthat efficient transfer occurs at various DFP:Fe ratios and within1 to 2 hours in culture conditions. The resulting holotransferrinformed from DFP-Fe was biologically active, as it was recognizedby the transferrin receptor (Figure 6) and provided iron to cells for

hemoglobin synthesis when presented together with DFP or afterDFP’s removal (Figure 7).

The direct bioavailability of DFP-bound iron was investigatedusing hemoglobin synthesis by cells in culture as a model. DFP-Fesupported hemoglobin synthesis even in the absence of transferrin(Figure 7), consistent with a similar activity of 1:1 complexes ofiron with salicylaldehyde isonicotinoyl hydrazone.43 More impor-tant, DFP enhanced the transfer of iron from iron-loaded macro-phages to preerythroid cells for hemoglobin synthesis, without theintermediary presence of transferrin (Figure 7). Such direct dona-tion to intracellular iron-requiring or iron-metabolizing enzymesmay not be a necessary feature if iron is efficiently transferred fromthe shuttle to circulating transferrin. However, it may be desirableor even essential in the brain, where the iron-binding capacity oftransferrin in the CSF is negligible and has been estimated in thesubmicromolar range.29,30

Low toxic potential of iron-shuttling agent complexes.A major concern in the use of chelators is the formation of ironcomplexes that undergo redox cycling and thereby catalyze ROSgeneration. This tendency is present in chelators with relatively lowredox potentials, such as those based on amine- and carboxyl-liganding groups but not with the bidentate 3:1 complexinghydroxypyridone DFP.20,21,44,45 However, speciation studies10 andrecent examination of ROS production42 indicated that completeabolition of labile iron by DFP is attained only at DFP:Fe(III) ratiosclose to 5:1. Whether DFP levels reached therapeutically areconsistently high enough to avoid formation of substoichiometricredox-active complexes is difficult to predict. This is particularlyimportant in susceptible, DNA-containing organelles such asnuclei and mitochondria. The same applies to intracellular signal-ing pathways activated by oxidative stress that could be generatedby DFP-mediated redistribution of cell iron.

Permeability across the blood-brain barrier (BBB) for thera-peutic applications to neurodegenerative diseases. The capacityof an iron-shuttling agent to redistribute iron in the central nervoussystem (CNS) may be an essential requirement for iron redistribu-tion in Parkinson disease, Friedreich ataxia, or NBIA. A high rate ofBBB penetration is expected for free DFP, based on its lowmolecular weight (below 300) and lipophilicity. This was con-firmed by direct measurement of DFP accumulation in the perfusedrat brain.15,16 In rats given DFP doses several times higher thananalogous doses given to thalassemia patients, brain tyrosine andtryptophan hydroxylase activities were significantly inhibited, asmeasured by the accumulation of 3,4-dihydroxyphenylalanine(DOPA) and 5-hydroxytryptophan, respectively, presumably viacoordination to iron bound by these enzymes.14 Yet, despiteDFP’s brain accessibility, major effects on CNS function have notbeen reported.32,44

Whether DFP can alter the iron balance in the brain by shuttlingiron across the BBB remains an open question. The absence of ironloading of the CNS in iron-overload conditions indicates thatneither transferrin-bound nor non–transferrin-bound iron is able tofreely cross the BBB.35 Because DFP treatment has not beenreported to be associated with increased iron loading of the CNS inthalassemia patients, it is conceivable that DFP-Fe complexes donot readily permeate the BBB. The permeabilities of free andiron-bound DFP may differ due to differences in their molecularweights (473 for 3DFP:1Fe compared with 139 for DFP). In arecent study, 6 months of treatment with DFP was shown to resultin a decrease in the iron-associated magnetic resonance imaging(MRI) transverse relaxation rate (R2*) signals in dentate nuclei ofFriedreich ataxia patients.13 Whether this decrease was caused by

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egress of DFP-Fe complexes from the brain or by redistribution ofiron from an area of accumulation to areas of lower concentrationwithin the brain is unclear.

We show that redistribution of iron in multiple directions and tomultiple recipients is experimentally feasible, particularly fromareas of accumulation to areas of iron need, via donation totransferrin or by direct metal donation. A modality of iron chelationbased on iron redistribution has several therapeutic implications. Inthe CNS, it could potentially be used to relocate local iron depositsthat may be an important factor in the etiology of severalneurodegenerative diseases. In ACD, it could be used to releaseiron trapped within macrophages. Current experimental approachesto alleviation of ACD tend to be directed at depressing the levels ofhepcidin, the iron-regulator protein responsible for macrophageiron retention. However, recent evidence for hepcidin-independentdown-regulation of the iron exporter ferroportin by the inflamma-tory mediators46 could obviate such an approach. As shown in thiswork, an alternate or complementary approach could be based oniron-shuttling agents that relocate misdistributed iron and safelybypass impaired internal routes of iron trafficking. Early pilotstudies with iron chelators in rheumatoid arthritis patients withACD47 provided clinical evidence that agents like DFP might beadopted as part of a short-term strategy of regional metal detoxifi-cation and systemic relocation that generates no significant urinary

iron loss.48,49 Optimization of such strategies with novel chelatorsfor treating the various conditions of regional iron accumulationdemand further laboratory and clinical work.

Acknowledgments

This work was supported by the Association Française contre lesMyopathies, the Israel Science Foundation (ISF), the EEC Frame-work 6 (LSHM-CT-2006-037296 Euroiron1) and French-IsraeliOrganization for Research in Neuroscience (AFIRN). Z.I.C. is theSergio and Adelina Della Pergolla Professor of Life Sciences.

Authorship

Contribution: Y.-S.S. and W.B. performed experimentation andanalysis; A.M. and Z.I.C. designed and supervised the research. Allauthors contributed to the writing of the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Z. I. Cabantchik, Alexander Silberman Insti-tute of Life Sciences, Hebrew University of Jerusalem, SafraCampus at Givat Ram, Jerusalem, Israel 91904; e-mail:ioav@cc.huji.ac.il.

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