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Secondary alcohol metabolites mediate iron delocalization in cytosolic fractions of myocardial biopsies exposed to anticancer anthracyclines Novel linkage between anthracycline metabolism and iron induced cardiotoxicity


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Secondary alcohol metabolites mediate iron

delocalization in cytosolic fractions of

myocardial biopsies exposed to anticancer

anthracyclines. Novel linkage between

anthracycline metabolism and iron-induced


G Minotti, … , R Zamparelli, G Possati

J Clin Invest.






The cardiotoxicity of doxorubicin (DOX) and other quinone-containing antitumor

anthracyclines has been tentatively attributed to the formation of drug semiquinones which

generate superoxide anion and reduce ferritin-bound Fe(III), favoring the release of Fe(II)

and its subsequent involvement in free radical reactions. In the present study NADPH- and

DOX-supplemented cytosolic fractions from human myocardial biopsies are shown to

support a two-step reaction favoring an alternative mechanism of Fe(II) mobilization. The

first step is an enzymatic two-electron reduction of the C-13 carbonyl group in the side chain

of DOX, yielding a secondary alcohol metabolite which is called doxorubicinol (3.9 +/- 0.4

nmoles/mg protein per 4 h, mean +/- SEM). The second step is a nonenzymatic and

superoxide anion-independent redox coupling of a large fraction of doxorubicinol (3.2 +/- 0.4

nmol/mg protein per 4 h) with Fe(III)-binding proteins distinct from ferritin, regenerating

stoichiometric amounts of DOX, and mobilizing a twofold excess of Fe(II) ions (6.1 +/- 0.7

nmol/mg protein per 4 h). The formation of secondary alcohol metabolites decreases

significantly (Pi < 0.01) when DOX is replaced by less cardiotoxic anthracyclines such as

daunorubicin, 4'-epi DOX, and 4-demethoxy daunorubicin (2.1 0.1, 1.2 0.2, and 0.6

+/-0.2 nmol/mg protein per 4 h, respectively). Therefore, daunorubicin, 4'-epi DOX, and

4-demethoxy daunorubicin are significantly (P < 0.01) less effective than DOX in mobilizing


Research Article



Alcohol Metabolites Mediate Iron Delocalization in


Fractions of

Myocardial Biopsies Exposed

to Anticancer


Novel Linkage Between


Metabolism and Iron-induced


Giorgio Minotti,*Anna Franca


AlvaroMordente,5 Marco











andGynecology, §BiologicalChemistry,


and Intensive Care, and 1Cardiac Surgery, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome,Italy



The cardiotoxicity of doxorubicin (DOX) and other

qui-none-containing antitumor anthracyclines has been

tenta-tively attributed to the formation of drug semiquinones

whichgeneratesuperoxide anion and reduce ferritin-bound Fe(III), favoring the release of Fe(II) and its subsequent involvement in free radical reactions. In thepresentstudy

NADPH- and DOX-supplemented cytosolic fractions from

human myocardial biopsies are shown to support a

two-step reaction favoring an alternative mechanism ofFe(II) mobilization. The first step is an enzymatic two-electron reduction of the C-13 carbonyl group in theside chain of

DOX, yielding a secondary alcohol metabolite which is

called doxorubicinol (3.9±0.4 nmoles/mg protein per4 h,

mean±SEM). The secondstepisanonenzymatic and

super-oxideanion-independentredoxcoupling ofalarge fraction

of doxorubicinol (3.2±0.4 nmol/mg protein per 4 h) with

Fe(III) -binding proteins distinct from ferritin, regenerating

stoichiometric amounts ofDOX, and mobilizing a twofold

excess of


ions (6.1±0.7 nmol/mg protein per 4 h).

The formation of secondary alcohol metabolites decreases

significantly(P < 0.01) whenDOX is replaced by less

car-diotoxic anthracyclines suchas daunorubicin, 4'-epi DOX,

and 4-demethoxy daunorubicin


1.2±0.2, and

0.6±0.2 nmol/mgproteinper4 h, respectively).Therefore, daunorubicin, 4 '-epi DOX, and 4-demethoxy daunorubicin

aresignificantly(P< 0.01)lesseffective than DOX in mobi-lizingFe(II) (3.5±0.1, 1.8+0.2, and 0.9±0.3 nmol/mg

pro-teinper4 h,respectively).These results highlight the forma-tion of secondary alcohol metabolites and the availability of nonferritinsourcesofFe(III)asnovel and critical determi-nantsofFe(II)delocalization and cardiac damageby

struc-turally distinct anthracyclines, thus providing alternative routes tothedesign of cardioprotectants for anthracycline-treatedpatients. (J. Clin. Invest. 1995. 95:1595-1605.) Key

words:Doxorubicin *anthracyclines *iron * free



Address correspondence to Dr. Giorgio Minotti, Department of

Pharma-cology,Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome, Italy. Phone: 39-6-301-54367; FAX: 39-6-305-7968.

Receivedfor publication 28 July 1994 and in revisedform 24 Octo-ber 1994.

Theformationof an intracellularpoolof free iron can be viewed as a sortoftwin-edged sword. Some iron is necessary for cell

functions, however,excessironcatalyzesfree radical reactions

thatdegrade polyunsaturated fatty acids, proteins, and nucleic acids ( 1, 2). This dual function of iron is particularly evident

with Fe(II), which is more active than Fe(III) in generating hydroxylradicals,ferryl ions,orequally damagingiron-oxygen

complexes (1). Underphysiologicconditions excess Fe(II) is

sequestered by ferritin, a shell-shaped multisubunit protein

which isplacedin thecytosolicmilieu andincorporates Fe(III) by oxidizing Fe(II) with oxygen (3). Ferritin would

subse-quentlyrelease irononlywhen electron donors accumulate and

reduce Fe(III) to Fe(II) (4, 5). These premises imply that

free radical reactions and tissue damage mustbepreceded by

disturbances of ferritin iron movements, resulting in a patho-logicprocessofFe(II) delocalization. One suchprocess appears tomediatethecardiotoxicity of doxorubicin(DOX),'an anthra-cycline antibiotic which is active against several tumors but

alsocauses apotentially fatalcardiomyopathy when the

cumula-tive dose exceeds 550 mg/M2 (6). Clinical studies indicatethat

theincidence and severity of this cardiotoxicityaresignificantly

lowered by the concurrent administration of dexrazoxane (6, 7), abispiperazinedione which hydrolyzes intracellularly and liberatesadiacid diamidethat chelatesiron(8).The cardiopro-tective effect of dexrazoxane is nevertheless incomplete (9), perhaps becauseacomplex of ironwith thediacid diamidestill generates somefluxes of hydroxyl radicals (10).

Doxorubicin iscomprised ofaquinone-containing

tetracy-clineringandatwo-carbonsidechain, havingacarbonylmoiety

atC-13 andaprimary alcoholatC-14;an aminosugar

(dauno-samine) isattachedby glycosidic bondtothe C-7 ofthe tetracy-clinering (Fig. 1).Invitro studies have shown thatNAD(P)H

oxidoreductases of mitochondrial, nuclear, and microsomal membranes support a oneelectron redoxcycling of DOX that may be relevant to the processes of iron delocalization and

cardiac damage. In fact, one electron addition to the quinone groupformsan unstable semiquinone free radicalthat regener-ates DOXby reducing and releasingiron stored in thefemitin

core(11, 12). Under aerobic conditions, the release ofFe(H)

is preceded by a redox coupling of DOX semiquinone free

radical withoxygen, yielding asuperoxide anion (°2-) as the

ultimate reductant for ferritin-boundFe(III) (11, 12).

Notewor-1. Abbreviations used in this paper: CPB, cardiopulmonary bypass;

DNR, daunorubicin; DNRol, daunorubicinol; DOX, doxorubicin;

DOXol, doxorubicinol;H,heavy;L,light;


superoxideanion; rHF, recombinantheart-typeferritin; rLF, recombinant liver-type ferritin. J. Clin. Invest.

©TheAmerican Society for Clinical Investigation, Inc.

0021-9738/95/04/1595/11 $2.00




R= #C-CH0





Figure 1.Structures of DOX and DOXol.

thy, cardiomyocytes haverelatively little SOD activity as

com-pared with other cell types, mostly because ofa selective

de-crease ofthecopper-requiring cytosolic enzyme (13). The

de-ficiencyinSODmight explain how cardiomyocytes become the

targets ofa drug which releases Fe(II) by forming 05-.

In vitro and in vivo studies (14) have also shown that the

side chain C-13 carbonyl group of DOX is liable to a two

electronreduction, yieldingasecondary alcohol which is called doxorubicinol(DOXol) (seealsoFig. 1). This reactionis

cata-lyzed by pancytosolicenzymesthatutilizeNADPH as a source

ofreducing equivalents and have been classified as aldo-keto

reductases [EC] (15) or carbonyl reductases [EC]

(16). Doxorubicinol is the primary circulating

me-tabolite in DOX-treated patients (17) and laboratory animals (18). Perhaps more importantly, the cardiac performance of DOX-treatedanimalsusually decreasesat a time whenDOXol has reached its maximum intramyocardial concentration (19, 20), suggesting that this metabolite might be intimately in-volvedinthe molecular mechanisms of cardiac damage.

Phar-macokinetic studies in DOX-treated animals indicate that car-diac accumulationofDOXolreflectsintramyocardial drug me-tabolism rather than uptake of the metabolite from the blood

stream(14, 20, 21).

Ethical and practicalreasons preclude fine needlebiopsies of sufficient size to evaluate DOXol formation and mecha-nism(s) oftoxicityinthe heart of DOX-treatedpatients.

There-fore, we have studied the cytosolic metabolism of DOX and


under-going open-chestcardiac surgery or obtained atautopsy. The

aims of these studies were to demonstrate that: (a) human

cardiomyocytescanenzymaticallyreduce DOXtoDOXol; (b) this metabolite contributes to cardiac damage by reductively delocalizingiron; and (c) differences insecondaryalcohol for-mation and iron mobilizationcan underlie clinical differences

between DOX andless cardiotoxic anthracyclines. The results indicatethatthecardiotoxicityof DOXand otheranthracyclines strictly reflects intramyocardialformation ofsecondaryalcohol metabolites which reductively delocalize iron from Fe(HI)-binding proteins distinct from ferritin. These findings provide

new clues to thepharmacologic cardioprotection of anthracy-cline-treated


Materials. Lactose-free anthracyclines were kindly provided by Dr. An-tonio Suarato(Pharmacia-FarmitaliaCarlo Erbas,S.p.A.Milan, Italy). Ammonium sulfate (ultrapure grade,Fer < 0.5ppm) was purchased from Schwarz/Mann (Cleveland, OH). EDTA was fromMerck& Co., Inc. (Darmstadt,Germany), whereas FeCl2 and NaBH4 were from Fluka AG (Buchs,Switzerland). Chelex 100 was from Bio-Rad Laboratories (Richmond, CA). Sephadex G-25 and electrophoresis reagents were purchased from Pharmacia LKB Biotechnology Inc. (Uppsala, Swe-den), whereas Sepharose 6B, human holotransferrin, bovine erythrocyte CuZnSOD (EC, and all other chemicals wereobtained from Sigma Chemical Co. (St. Louis, MO). Unless otherwise indicated, all the experiments werecarried out in 0.3 M NaCl, carefully adjusted to pH 7.0 just before use. This was done to avoid ligand-catalyzed interac-tionsof most common buffers with iron (1, 22). Although unbuffered, thepH of the reaction mixtures did not vary throughout the experimental time. Allthe solutions were preparedwithdouble distilled water which had been passedthrough a Synbron Barnstead NANOpure II system

(Idrotecnica S.p.A., Bolzaneto, Italy). Subsequent ion exchange chro-matography on a column of Chelex 100 was performed to remove trace metals.

Patients andmyocardial samples. Small atrial samples (- 0.13 g) were taken from male or female patients undergoing aorto-coronary bypass grafting. Briefly, bioptic fragments of normothermic beating myocardiumweretakenfrom the lateral aspect of excluded right atrium before tying the purse string of atrial cannulation. These samples were collected 15-20 min before or5-10min aftercardiopulmonary bypass (CPB). During CPB, myocardialpreservationwasobtained by infusion ofa St. Thomas Hospital-modified cardioplegic solution (23). The median age ofpatients was58±8 yr (n = 15) or59±7 yr (n = 25) for the pre-orpost-CPB samples, respectively.Thebiopsieswerestored

at -80OCuntilthey formedtwo pools of 2.7or4.4 g for the pre- or

post-CPB samples, respectively.Largersamples (-3g)wereobtained by recovering right auricolas from a 66-yr-old male and a 64-yr-old femalepatient undergoing Maze's correction of drug-unresponsive atrial fibrillation(24). Informed consentwasobtained from allpatients. Ven-tricular postmortem samples (5-10 g) were provided by authorized

pathologists duringthe autopsy of maleorfemale individuals with

mor-phologically normal myocardium and no clinical history of: (a) acute myocardial infarction, severecardiosclerosis,orothercardiomyopathies; (b)inheritedoracquired hemochromatosis; and(c)highly contagious

infectious diseases. Selected samples wereobtained from patients (n

=20,median age=53±3 yr) died of: (a) sudden rupture of abdominal

orintracranial aneurysms (n= 9); (b) unresectableor relapsedbrain

tumors (n = 5); and (c) biliary or pancreatic cancer (n = 6). All

sampleswerecollected 24 h afterdeath. Anthracycline-treated patients

wereroutinely excluded from the studyto avoidsample perturbation by changesindrug-metabolizing enzymesoranomalous modifications of ironcontentanddistribution.

Preparationof cytosols. Thetwopoolsofexvivo pre-orpost-CPB samples and individual 3-5 g samples from postmortem donors or

Maze'spatientswerethawed in 0.3MNaCl, pH 7.0,40C,minced with

scissors,anddisruptedfirstbyfour 15-s bursts inabladehomogenizer (UltraTurrax; Kunkle & Janke GmbH & CokG, Staufen, Germany) and thenbyfour strokes inahomogenizer (Potter-Elvehjem;Thomas

Scientific, Swedesboro, NJ) usingalooseteflonpestle.Tissue

homoge-nates werecentrifugedfor 30 minat16,000g and thesupernatantswere furthercentrifugedfor 30 minat25,000g. Theresultingsupernatants

were eventually ultracentrifuged for 90 min at 105,000 g. Dialyzed

105,000 g supernatants were unstable on freezing and thawing and formed grossparticulates upon incubation with NADPH and/orDOX,

precluding spectrophotometric assays for Fe(II) mobilization.

There-fore, 105,000gsupernatantswereroutinelystirredovernightwith 65% ammonium sulfate. After 30 mincentrifugationat10,000g, the

precipi-tates weredissolved in 1-2 ml of 0.01 Mphosphatebuffer,pH 7.4,and loadedonto a(0.8x 10cm)calciumhydroxyapatitecolumnpreviously equilibrated withthesamebuffer. Thisprocedurewas chosen because


it is known to increase theanthracycline aldo-keto reductase activity of rat liver cytosol (15). The column was washed with a threefold excess(vol/vol)of theequilibrationbuffer and the effluentswerestirred overnight with 65% ammonium sulfate. After 30 mincentrifugationat 10,000 g, the precipitates were dissolved in 3-4 ml of double-distilled water and dialyzed firstagainst two 1-literchanges of 0.3 M NaCl-1 mMEDTA (toremoveadventitiousiron),and thenagainsttwo1-liter changes of 0.3 M NaCl (toremoveEDTA andEDTA-ironcomplexes).

These "unmodified cytosols" could be stored at -200C and thawed without appreciable formation of gross particulates. Likewise, these cytosols were stable upon reconstitution with NADPH and/or DOX. All preparations were essentially free of mitochondrial cytochrome c oxidase activity, and microsomalornuclearNADPH-cytochromecor P450 reductase activities ( 10,12,25).Inselectedexperiments, unmodi-fied cytosols weresubsequently treated withthioglycolic acid (which reduces Fern to Fe')and bathophenanthroline (which forms a stable

complexwithFe').Briefly,theincubations (2-5mlfinalvol)contained

unmodified cytosols (3 mgprotein/ml), bathophenanthroline disulfo-nate (0.5 mM), andthioglycolicacid (0.5%,vol:vol)in0.3 MNaCl,

pH 7.0. After 1 hstirringonice,themixtureswereloadedonto a(2.5

x 16 cm) Sephadex G-25 column to separatecytosolicproteins from

thioglycolicacid and thebathophenanthroline-Fe(II)complex.Both col-umnequilibrationandsampleelution wereperformedwith0.3MNaCl,

pH 7.0. 5-mlfractionswerecollected at the flowrateof 5 ml/min and monitored forproteins by measuring absorbance at280 nm.

Protein-containing fractions werepooled,concentratedby ultrafiltrationin 10-kD exclusion limit Minicon cells(Amicon Corp., Danvers, MA), and shown nottocontainbathophenanthroline-chelatable Fe(ll)upon rein-cubation withthioglycolic acid. Therefore, these cytosols will be

re-ferred toas"iron-depleted."Inotherexperiments, "iron-reconstituted"

cytosols were preparedby taking advantageof the abilityof iron-de-pleted cytosols toincorporateFe(HI) by oxidizingFe(II).The

incuba-tions(2-5mlfinal vol) containediron-depleted cytosols (3mgprotein/

ml) andFeCl2(0.5 mM) in 0.3 MNaCl,pH7.0,37°C.Thereactions

were terminated at regular timesbythe sequential additionof bathophe-nanthroline disulfonate (2.5 mM)and EDTA(1 mM),tochelate

unre-actedFe(H)and removeloosely bound Fe(III), respectively. After1 h

stirring on ice, themixtures were chromatographed on Sephadex

G-25 to separate cytosolic proteinsfrom bathophenanthroline-Fe(II) and

EDTA-Fe(HI)complexes. Theiron-reconstitutedcytosols with the

high-estFe(III) contentwill bereferredtoas "iron-loaded."

Assays for anthracycline metabolites. Anthracycine metabolism was studied inincubations (0.5-1 ml final vol) containing cytosols (3 mgprotein/ml),drugs(1 mM) and NADPH(1 mM)in 0.3 M NaCl, pH7.0, 37°C. Aftera4-h incubationin a metabolic shaker(Dubnoff; Vismara Associate S.p.A.,Milan, Italy), thereactionswere terminated

bythe addition ofafourfoldexcessofCH30H/CHC13(1:1).After

low-speed centrifugation,polar and apolar metabolites were recovered from ,CH30HorCHC13 phases, lyophilized, and resuspended in aminimum

volume of the appropriate solvent. These samples were analyzed by

two-dimensional TLC on (-0.25/0.5 mm) Silica Gel Plates 60 F-254 (Merck &Co.,Inc.,Darmstadt,Germany).Unmodifiedanthracyclines and.polar C-13 secondary alcohol metabolites were separated using CHC13/CH3OH/CH3COOH/H20 (80:20:14:6) in either dimension

(26). Apolar aglycones were separated using CHCl3/CH30H/ CH3COOH (100:2:5) in the first dimension and


CH3CH2OH/CH3COOH/H20 (80:10:5:5) in the second dimension

(26). Fluorescentspotscorrespondingto the polar or apolar metabolites

were eluted with 100% CH30Hor CHC13, respectively, and assayed fluorimetricallyagainststandards ofunmetabolized anthracyclinesin a

luminescence spectrometer(LS 5;Perkin-ElmerCorp., Norwalk, CT),

asdescribedin detail by Takanashi and Bachur (26).

Enzymatically generated DOXol (referred to as "enzymatic"

DOXol) was identified by cochromatography with authentic DOXol

from Streptomyces peucetius var. caesius, or DOXol prepared by

NaBH4reduction ofDOX, referredto as "chemical" DOXol. The latter

wasprepared by establishedprocedures (26) and purified by two-dimen-sional TLCfor




(4-demethoxy)daunoru-bicinol(DNRol)and 4'-epiDOXolwereidentifiedby cochromatogra-phywith chemical analogs prepared byNaBH4reduction of theparent compounds. Doxorubicin(ol) aglycones were identified vs standards prepared


thermoacidhydrolysisof DOX and chemicalDOXol (26)

andpurified bytwo-dimensional TLC forapolarmetabolites. Doxorubi-cinhydroquinone standards (the productsofatwo-electron reduction of thequinonemoiety)wereprepared enzymatically by incubatingDOX with NADPH andspinachleafNADP+-ferredoxinoxidoreductase(EC (27). Only 7-deoxyDOXhydroquinone aglyconewasformed,

inkeeping with previousreports thata twoelectron reduction of the quinone moietyis accompanied by heterolysisof theglycosidic bond between daunosamine and the tetracycline ring(11, 27). Purification of7-deoxy DOX hydroquinone aglycone was also obtained by

two-dimensional TLC forapolar aglycones, where itmigrated with Rfof 0.01 and 0.24 in the first or second dimension, respectively. These values were lower than those determined for any DOX(ol) oxy- or

deoxy-aglycone (26). 7-deoxyDOXaglyconeand4-demethoxyDOXol aglyconewereidentifiedby 295% agreement of theRfdeterminedby uswith those determinedbyothers undercomparableconditions(26). AssaysforFe(JI)mobilization.Ferrous iron mobilizationwas stud-ied inincubations(1 ml finalvol) containing cytosols (1 mgprotein), anthracyclines (0.25 mM), and bathophenanthroline disulfonate (0.5 mM)in0.3MNaCl, pH 7.0,37°C.The reactionswerestartedby


NADPH(0.25 mM)and the formation ofabathophenanthroline-Fe(II)

complexwasmonitoredspectrophotometricallyat534nmvs areference cuvette containing all the reactants but the chelator (28). Standard curveswith knownamountsofbathophenanthroline-chelatedFeCl2were

used forcalculations,and controlexperimentsshowed that the bathophe-nanthroline-Fe(II) complex producedfromcytosolswasspectrally iden-ticaltoauthenticcomplexes. With theexceptionofthe data inFig. 2,

all valueswerecorrectedfor iron mobilization byNADPHor

anthracy-clinesaddedindividually.Whereindicated,iron mobilizationwas moni-tored undernonenzymaticconditionsby replacingNADPH and anthra-cyclineswithpurifiedC-13 metabolites. The latter had beenextensively washed withmethanol,lyophilized,andeventually suspendedin0.3 M NaCl, pH 7.0, justbeforeuse.

Ferritinpreparation. Heart ferritin is composedprimarilyofheavy (H)subunits whereas liver andspleenferritins arecomposedprimarily oflight(L)subunits(3).Recombinant human HorLferritin homopoly-mers werekindlyprovidedbyDr.Paolo Arosio(Biotechnology Depart-ment, IstitutoScientificoSanRaffaele, Milan, Italy).Ferritinexpression and purification were as describedby Levi et al. (3). Native HorL samplescontained 65or9atomsFe(Ill)/molecule, respectively. There-fore, apoferritinswereprepared andincubatedwith irontoobtain ferri-tins withhigher Fe(Il): proteinratios. Apoferritins wereobtained by treatingferritinswiththioglycolicacid andbathophenanthroline,as de-scribed for thepreparationofiron-depleted cytosols.Ferriciron incorpo-ration wasobtainedby taking advantage of the ferroxidase activity of

apoferritin orceruloplasmin. Briefly, apoferritins (0.3 jM) andFeCl2

(0.5 mM), plusorminusceruloplasmin (0.3 uM), were incubatedfor

1 h in 0.3 MNaCl, pH 7.0, 37°C. Thereactions were terminated by the

sequential addition of bathophenanthroline and EDTA, as described

for thepreparation ofiron-reconstitutedcytosols. In theseexperiments,

thioglycolic acid, bathophenanthroline-Fe(H), andEDTA-Fe(III)were

removedbyextensivedialysisof the (apo)ferritin samples against 0.3 M

NaCl, pH7.0.The HorLferritinsreconstituted via their ownferroxidase

activityhadiron:proteinratios of 95or32, respectively, whereas H and L ferritins reconstituted via the ferroxidase activity of ceruloplasmin had iron:proteinratios of 110or260, respectively. Ceruloplasminwas

purified fromratblood plasma according to Ryan et al. (29). Ferritins

wereseparated from ceruloplasmin by gel filtration on a (1.8 X27 cm)

Sepharose6B columnequilibratedwith 0.3 M NaCl, pH 7.0, and eluted atthe flowrateof 0.74 ml/min.

Affinity and gelfiltration chromatographies of cytosolic iron and recombinantferritins. 8 mg protein of unmodified or iron-loaded cyto-solsfrom ex vivo Maze's samples (corresponding to 64 or 184nmol

Fer,respectively) weredialyzedovernight against two 1-literchanges






1-- 0-




- Fe(Ill)

NADPH DOX Complete

Figure 2. NADPH- andDOX-dependentFe(ll)mobilization in human heartcytosols.Unmodifiedoriron-depleted cytosols from ex vivo or postmortemsampleswereassayedfor NADPH- and/orDOX-dependent Fe(II) mobilizationasdescribedinMethods. Values are those

deter-minedat4 h andare takenfromrepresentative experiments or four separatedeterminations(mean±1SE).-Fe(HI), iron-depletedcytosols; exvivoopre CPB; * post CPB; Upost mortem.

ultrafiltration, andloaded onto a(1.3 x 2.5 cm)CNBr-Sepharose 4B

columnwhich hadbeenequilibratedwiththesamebuffer.The column

hadbeenconjugated with 1 mgof antirecombinantheart-type ferritin

(rHF) monoclonal RHO2 IgG, (30) and 1 mgof antirecombinant

liver-type ferritin (rLF) monoclonal L03 IgG2b (31). Sampleswereeluted

with 9 ml of theequilibration bufferand theeffluentwas

rechromato-graphed twice. The finaleffluent(referredto as "Triseffluent")was

concentratedto 1 mlby ultrafiltrationandchromatographedon

Sepha-rose6Basalready described for the separation of ferritins from

cerulo-plasmin.Fractions(0.9 ml)were collectedand monitoredfor absorbance at280 nm. Protein-containing fractionsweresubsequently assayed for

nonhemeironby reconstituting 0.3-ml aliquots withthioglycolic acid (0.5% vol:vol),bathophenanthrolinedisulfonate(0.5mM),and0.3M NaClinafinal volume of 0.9 ml. After vigorous mixing, theironcontent

wasdeterminedby measuringtheabsorbance ofthe

bathophenanthro-line-Fe(H) complex at534 nm. In otherexperiments, theantiferritin CNBr-Sepharose4Bcolumnwaschargedwithamixtureof self-recon-stitutedrHF(174


protein -30nmoliron) and ceruloplasmin-recon-stituted rLF (166 ,ug protein -90 nmol of iron). The column was

washed with 9 mlequilibrationbufferand ferritinswereeluted with 4.5 MMgCl2(32) by monitoring theeffluentforabsorbanceat280 nm.

Fractions with absorbance("Mg effluent")werepooled, dialyzed

ex-tensivelyagainst 0.3MNaCl,andconcentratedto1 mlbyultrafiltration

inCentripep 30(Amicon Corp., Danvers, MA).These samples were

eventually chromatographedonSepharose6B andanalyzedfor ironas

describedfor the Tris effluent ofcytosols.

Other assays. Spontaneousorcytosol-induced Fe(ll)oxidationwas

studied in incubations (1 ml final vol) containing FeCl2 (0.5 mM)

±iron-depleted cytosols (3 mgprotein/ml) in 0.3 M NaCl, pH 7.0,

37°C. Aliquots (0.1 ml)weretakenatregular times and reconstituted

withbathophenanthroline disulfonate (0.5 mM)and0.3MNaCl,ina

finalvolume of I ml. After mixing, these samples were assayedfor

absorbanceof thebathophenanthroline-Fe(H)complexat534nm.The

ferricironcontentof allcytosolandferritinpreparationswasroutinely measuredusing thioglycolic acidasthereductantforFe(ll1)and

batho-Table I. Doxorubicinol Formation in NADPH-supplemented

UnmodifiedorIron-depleted Cytosols from Human


Sample Unmodified cytosol Iron-depleted cytosol

nmolDOXol/mgproteinper 4h

Postmortem 0.7±0.2 3.9±0.4t


pre-CPB 0.7 3.9

post-CPB 0.8 3.7

* NADPH- andDOX-supplementedunmodified or iron-depleted

cyto-sols fromex vivo orpostmortemsamples were assayed for DOXol as described inMethods. Values are those determined at 4 h and are means±SE (n=4).Values with ex vivo samples are taken from

repre-sentativeexperiments. t Significantly different from unmodified cytosol (P <0.01).

phenanthrolineas thechromophoreforFe(II).Omission of thioglycolic

acid always preventedtheformation ofthebathophenanthroline-Fe(LI)

complex, indicating that all the assayable iron was in a ferric form.

Unmodified cytosols from postmortem samples contained 8.4±0.33



protein (n = 8). The two preparations from ex vivo

Maze'ssamples contained 8 and 9.2 nmol Fe( 1)/mgprotein, whereas

representative preparations from pools of pre- orpost-CPB samples

contained 7.8 or 8.4 nmol Fe(Il)/mg protein, respectively. Proteins were measured by the bicinchoninic acid method (33). SDS-PAGE

analysisunderreducing conditionswasperformedaccording to Laemmli

(34) using 15% polyacrylamide gels. Unless otherwiseindicated, all

dataareexpressedas the arithmetic mean±SEM. Statistical analyses wereperfonnedby paired Student'sttest.Differenceswereconsidered significant when P < 0.05. All other conditionsare indicated in the

figure legends.


(a) NADPH- andDOX-dependent


mobilization in


oriron-depleted cytosols. NADPH and DOX-supple-mentedcytosolicfractionsfrom humanmyocardiumwerefound



Asshown inFig. 2, comparableresultswere

obtained withcytosols fromselected postmortemsamples and

cytosols from ex vivo pre- orpost-CPB samples. NADPH or

DOX persemobilized very littleFe(II);moreover, the mobili-zation of


was notobservedwhen the



pre-viously been depleted of nonheme Fe(III) (see also

Fig. 2).

These resultsshowedthat themobilizationof


was medi-atedbyDOX metabolites which reduced nonhemeFe(III).

(b) NADPH-dependentDOX metabolism in unmodifiedor

iron-depleted cytosols.



cy-tosols from postmortem orex vivo


werefoundto re-duce DOXto DOXol, however,the


of DOXol increased

dramatically when the cytosols had




of nonheme


(Table I).Omission of NADPHor


abolished DOXolformation, indicatingthatDOX reductionwas mediatedbyanenzymaticmechanism. Doxorubicin-metaboliz-ing enzymesarealso knowntocleave the



be-tween daunosamine and the tetracycline ring

(26), therefore,

DOX- andDOXolaglyconeswere measured inunmodifiedor

iron-depleted cytosols.As shown in TableII,

NADPH-supple-mented unmodified


from postmortem








Table 11. NADPH-dependent DOX Metabolism inUnmodifiedor

Iron-depletedCytosols: ComparisonofDOXol with Aglycone Metabolites

Metabolite Unmodifiedcytosol Iron-depleted cytosol nmol/mgprotein per4 h

DOXol 0.7±0.2 3.9±0.4*

DOX-ag 0.8±0.2 0.8±0.1*

7-deoxyDOX-ag 0.4±0.3 0.5±0.4t

4-demethoxyDOXol-ag 0.3±0.1 n.d.

DOXol-ag n.d. n.d.

7-deoxy DOXhydroquinone-ag n.d. n.d.

NADPH-andDOX-supplementedunmodifiedoriron-depleted cytosols

from postmortem samples wereassayedfor DOXol andapolar aglycones

asdescribed in Methods. Valuesarethose determinedat4 handare

means±SE (n =4). *Significantlydifferent from unmodifiedcytosol

(P<0.01). INS vsunmodified cytosol;ag,aglycone; n.d.,not


however, neither metabolite increased significantly upon iron removal.4-demethoxy DOXolaglyconewasformed by unmod-ified but notbyiron-depleted cytosols, whereas DOXol

agly-cone and 7-deoxy DOX hydroquinone aglycone were not

formedby either cytosolpreparation (see also Table II). Hence,

DOXol was the only DOX metabolite which increased upon

iron removal. Comparable results wereobtained with cytosols from ex vivo pre- orpost-CPB samples (not shown).

(c) Enzymatic and non-enzymaticDOXol-dependent

mobili-zation ofFe(II) in unmodifiedor iron-reconstituted cytosols. Figure 3A shows that90% ofFe(II) could be recovered with bathophenanthroline aftera3-h incubation in unbuffered saline underanair atmosphere. The addition of iron-depleted cytosols decreased the recovery of bathophenanthroline-chelatable Fe(II) and thenetloss of


wasparalleledbyatenacious bindingofFe(IH)tocytosolic proteins (Fig. 3 B). These results showed that cytosols could incorporate Fe(lI) by oxidizing

Fe(II).Taking advantage of this, DOXol formation andFe(ll)

mobilization were measured in NADPH- and DOX-supple-mented cytosols which had been reconstituted with increasing

amounts of Fe(Ill) by incubating iron-depleted cytosols and Fe(II) forvarying lengths of time.AsshowninFig.4A,DOXol decreased with increasing the cytosolic contentof


By contrast, the mobilization of Fe(II) gradually increased with increasing theFe(II )incorporated into the cytosols (Fig. 4 B). These results confirmed that the enzymatic yield of DOXol

was inversely related to the cytosolic content ofFe(IH) and mobilization of


(compare with Fig. 2 and Tables I and

II). Therefore, we hypothesized that DOXol was consumed

during thereductive mobilization of iron. This hypothesis was tested by incubating purified enzymatic DOXol with iron-recon-stituted cytosols and by monitoring the fate of DOXol and the

mobilization of Fe(II). As shown in Fig. 5 A, DOXol was

quantitatively recovered from incubations with iron-depleted

cytosols, but it was extensively consumed by cytosols which had been reconstituted with increasing amounts of Fe(III).The

ferric iron-dependent loss of DOXol was paralleled by a stoi-chiometric recovery of DOX and a twofold mobilization of Fe(II) (Fig. 5 B). Collectively, these experiments provided evidence fora direct interaction of DOXol with Fe(III), and




- 400


0 30 60 90 120 150 130


30 =

01 20

_ ,{ *



10 Fe(IM



0 30 60 90 120 150 180


Figure 3. Fe(II)oxidation andFe(HI) incorporation by iron-depleted

human heartcytosols. In(A)the recoveryof

bathophenanthroline-che-latableFe(ll)wasmonitored in incubationscontainingFeCl2 plusor

minusiron-depletedcytosols from postmortemsamples,asdescribedin

Methods. In (B) the difference betweencytosol-inducedand

spontane-ousoxidationof Fe(II) was determined andcomparedwith the

incorpo-ration ofFe(I).Values are meansof two separate determinations with

>95%experimental agreement.




X 2


l B

t1@I. X

5 o0 IS 20 25

mm.le Fe(Ill)l/ prot

0 5 10 is

amol Fe (111)/ mgpfo.

20 25

Figure4. Doxorubicinol formation andFe(II)mobilization in human heartcytosolswithincreasingFe(III) contents.Iron-depleted cytosols from postmortem samples were incubated withFeCl2for 5,15, 30, and 180 mintopermittheincorporation of the indicated amounts of Fe(II). In(A) DOXol was measured in incubations containing NADPH-supple-mentediron-depletedoriron-reconstitutedcytosols. In (B) cytosols

wereassayed forthemobilization ofFe(II).Values are thosedetermined

at4handare meansof two separate determinations with 91% (A) or 89%(B)experimental agreement.




U o3


a O


0 5 10 15 20 25





10 B



E /:


0 5 10 15 20 25







NADPH+H!f3' 2Fe(H) N+H


StepI Step2

Figure5.Doxorubicinolconsumption, DOXrecovery,andFe(H) mobi-lization iniron-depletedoriron-reconstituted cytosols. Iron-reconstitu-tedsampleswerepreparedasdescribed in thelegendtoFig.3. Incuba-tions(1mlfinal vol) containediron-depletedoriron-reconstituted

cyto-sols(1mgprotein),purified enzymaticDOXol(5 nmol),and

bathophenanthroline disulfonate (0.25 mM)in 0.3 MNaCl, pH 7.0, 37°C.The incubations were monitored for DOXolconsumption (A)and DOX formation or


mobilization(B).Valuesarethose determined at1hand are meansoftwoseparatedeterminationswith>95% experi-mentalagreements. (C) Two-step mechanismfor the mobilizationof Fe(II)in NADPH-andDOX-supplemented humanheartcytosols.

suggestedthatthe overall mechanism ofNADPH- and

DOX-dependent Fe(II)mobilizationwasmediatedbyatwostep

reac-tion. Step 1 consisted ofan enzymatic twoelectron reduction of DOXtoDOXol,whereas step 2 consisted ofanonenzymatic

twoelectron reoxidation of DOXoltoDOX,


with the reductive mobilization oftwoiron ions(Fig.5C).This mecha-nism would adequately explain how the enzymatic yield of DOXolincreasedordecreased upon iron removalor reincorpo-ration, respectively.

The mechanism of iron mobilization was further investi-gated byincubatingincreasingamountsof DOXol with

unmodi-fiedcytosols.As shown inTable m,purifiedenzymaticDOXol

wascapable of mobilizing Fe(II)inaconcentration-dependent

manner. Again,the release ofFe(II) wastwice theconcurrent formation ofDOX,in


with theoxidation ofoneDOXol

Table III. Fe(II) Mobilization and DOX Fonnation inDOXol-Supplemented Unmodified Cytosols

DOXol Fe(ll) release DOXformation DOXol:Fe(m) DOXol oxidation

nmol/mg protein %

1 2.0 1.0 0.11 100

2 3.9 2.0 0.23 100

4 7.8 4.0 0.46 100

6 8.6 4.3 0.68 72

8 8.7 4.4 0.91 55

10 8.5 4.2 1.10 42

Theincubations (1 ml final vol) contained unmodified cytosols from postmortemsamples(1 mgproteinl8.8nmolFe'),bathophenanthroline disulfonate(0.25mM), and increasing amounts of purified enzymatic

DOXolin0.3MNaCl, pH 7.0,370C.After spectrophotometric

determi-nation ofFe(ll)release, the incubations were extracted with

CH30H:CHC13 (1:1)toassayfor DOXas anindexof DOXol oxidation. Inthelast two columns, DOXol oxidation is given as a function of the DOXol:Fe(llI)ratio in each individual incubation. Values are those determined at1 hand are means of two separate determinations with

>95% experimentalagreement.

at the expense of two Fe(EI). This 0.5



DOXoloxidation vsFe(I)reduction wasconfirmed by mea-suring DOXol oxidation as a function of the molar ratio with cytosolicFe(EI).Infact, DOXol oxidation decreased when the


ratio exceeded 0.5, i.e., when the electron donor (DOXol) was in excess of the electron acceptor


(see also TableHI). Similar studies with chemical DOXol gave

a .90% experimental agreement (not shown). At this time,

evidence for a 0.5


of DOXol oxidation vs

Fe(III) reduction can also be inferred from previous

experi-mentswithNADPH-and/or DOX-supplemented,unmodified,

oriron-depleted cytosols. For example, the experiments with

postmortemcytosolsshowed that NADPHorDOX perse mobi-lized0.7±0.1or0.7±0.3 nmolFe(fl)/mg protein, respectively,

whereas DOXplus NADPH mobilized 7.5±0.2 nmol Fe(H)/

mg protein, according to the net increase of 6.1±0.7 nmol

Fe(II)/mgprotein(compare withFig.2).Consideringthat iron removalabolished Fe(H) mobilization but increased theyield of DOXol from 0.7±0.2to3.9±0.4nmol/mgprotein (compare with Table I),one cancalculate that 3.2±0.4 nmol of DOXol

wereinvolved in the mobilization of6.1±0.7 nmolFe(II), in

keeping with a median stoichiometry of DOXol oxidation vs


reduction of 0.54±0.04. Similar calculations with ex vivo pre-orpost-CPB samples gave stoichiometries of 0.54or


In otherexperimentswithDOX-supplementedpostmortem

cytosols, the effects ofreplacing NADPH with NADH were

studied. The results showed that: (a) iron release decreased from 6.1±1.7 to 1.8±0.3nmol/mg proteinper4 h(n = 4, P

<0.01); and(b)DOXolformationwasapparentlyabolished in unmodifiedcytosolsandsignificantlydecreased iniron-depleted

cytosols (from3.9±0.4to0.9±0.2nmol/mg proteinper 4h; n

=4,P<0.01).Consideringthat iron removal didnotincrease theyield of othermetabolites, one cancalculate that 0.9±0.2 nmol of DOXolwereinvolved inthe reductive mobilization of 1.8±0.3 nmol ofFe(II),according to amedian stoichiometry of DOXol oxidation vsFe(M)reduction of 0.51±0.03.


Table IV. NADPH-andAnthracycline-dependentFe(II) Mobilizationin UnmodifiedCytosols: Comparison ofDOX with DNR, 4'-epiDOX, and4-demethoxyDNR*

Anthracycline nmolFe(II)/mg protein per4h

DOX 6.1±0.7

DNR 3.5±0.6*

4'-epiDOX 1.8±0.2t§

4-demethoxy DNR 0.9±0.3*11

*NADPH- and


unmodifiedcytosolsfrom postmortemsamples wereassayedforFe(fl)mobilizationasdescribed inMethods. Values are those determined at 4 h and are means±SE (n =4). Significantly different from DOX(P<0.01). § P <0.01 vs DNR. P <0.01 vs4'-epiDOX.

fore, the NADH-dependent mechanism of iron mobilizationwas

stoichiometrically similarto that promoted by NADPH,

how-ever, the overallreaction waslimited byadecrease of DOXol formation. This is in keeping with previous demonstration that

NADH cannotsubstitute forNADPH as a source of reducing equivalents for anthracycline C-13 reductases (15, 16).

Doxorubicinol-dependent iron reduction is mediated by a

direct electron transfer mechanism rather than byaredox

cou-pling with oxygen and formation of02--astheultimate reduc-tant. Infact, DOXol oxidation did not occur upon aerobic incu-bation with iron-depleted cytosols (compare with Fig. 5 A), nor did it occur when the


ratio exceeded an optimum value of - 0.5 (compare with Table


This means

that DOXol redox couples with iron, not with oxygen. More-over, experiments with unmodified cytosols from postmortem

samples have shown thatNADPH-and DOX-dependent Fe(II)

mobilization occurs independently of the presence orabsence of 100 U/ml of CuZnSOD (5.9±1 vs6.3±0.8, respectively; n

= 4,P > 0.05). Similar results have been obtained ina

dupli-cate experiment withan exvivo Maze's sample (6.2 [+SOD] vs 6.1 [-SOD]).

(d) Comparisons ofDOXwith other anthracyclines. Having demonstrated the formation of DOXol and its redox coupling with iron, we performed comparative experiments with other anthracyclines of clinical interest. Studies with NADPH- sup-plemented unmodified cytosols showed that DNR, 4 '-epi DOX,

and4-demethoxyDNRreleased - 57,30, and 15% of the iron

released by DOX, respectively (Table IV). According to Fig.

5 C, iron release might be affected by adecreased formation

ofC-13 metabolites (Step 1) and/or by an altered interaction

ofC- 13 metabolites with iron (Step 2). Therefore, the two steps

were examined under separate experimental conditions. Step 1 was studied in iron-depleted cytosols, to abrogate redox cou-plings with Fe(II) anddetermine the net reduction of DNR,

4'-epi DOX, and 4-demethoxy DNR to the corresponding

sec-ondary alcohols. As shown in Fig. 6 A, DNR, 4 '-epi DOX, and

4-demethoxyDNR were46, 69, and 84% less susceptible than

DOX toan enzymatic reduction at C-13. Step 2 was studied by reconstituting purified enzymatic DNRol, 4'-epi DOXol, and

4-demethoxyDNRol with unmodified cytosols, to evaluate how

effectivelythey redox coupled with cytosolicFe(III).As shown

in Fig. 6 B, DNRol, 4'-epi DOXol, and 4-demethoxy DNRol

were as effective as DOXol in redox coupling with Fe(III), inasmuch as they oxidized back to the parent compounds by

5 A

jI -r [\



iX. 1R ;.i1tX -.;,,..



metabo-lite formation and redox couplingwithiron: com-parisonsofDOX with


4-demethoxyDNR. In

(A)NADPH- and an- thracycline-supple-mentediron-depleted cytosolsfrom


as-sayedfor C-13secondary alcohol formation. Val-° DOXol ues aretnose


cE 4- 03 DNRol at 4 handaremeans±1

o £~~~~~~~~~4'-epi DOXol

3 F 4-demethoxyDNRol SE (n = 4). DNRol,

4'-E 3 Fe~ll) release / epi DOXol, and


0 significantly different

from DOXol(P< 0.01).4'-epiDOXolwas

alcohol oxidation

00 (P<0.01)vsDNRol,

O 1 2 3 and4-demethoxyDNRol

nmol C-13 alcohol / mg protein


4'-epiDOXol. In(B)the in-cubations(1 ml finalvol) containedtwodifferentconcentrationsof

purified enzymatic alcohols, unmodified cytosols frompostmortem

sam-ples (1 mgprotein), and bathophenanthroline disulfonate (0.25 mM),

in 0.3 MNaCl,pH 7.0, 370C. These incubationswereassayed for the

releaseof Fe(II)andsubsequentlyextractedwithCH30H:CHCl3 ( 1:1 )

todeterminethereoxidation of alcohol metabolites backtothe parent compounds. Values arethosedetenrnined at1 handare meansoftwo

separatedeterminations with>95%experimental agreement.

reductively mobilizingatwofold excess ofFe(H). Thus,a

spe-cific modification of Step 1 affectediron release by DNR, 4'-epi DOX, and 4-demethoxy DNR.

(e) Comparisons of cytosolic iron with recombinant human

ferritins. Three lines of evidence- showed that cytosols were

essentially free of ferritin and interactedwith ironby virtueof

alternateFe(III)-binding proteins. First, all theFe(III) ions of unmodified oriron-loaded cytosols could easily pass through

anaffinity chromatography column whichimmobilized human

rHFandrLF(Table V). Second, the iron bound to either cytosol

Table V.AffinityChromatography of Ferritins andUnmodifiedor

Iron-loaded Cytosols

Sourcesofiron Sample Triseffluent Mg effluent



Unmodified 64 63 n.d.

Iron-loaded 184 185 n.d.

rHF + rLF 120 n.d. 105

ACNBr-Sepharose4B column with anti-rHF and anti-rLF monoclonal antibodieswas chargedwith (a)anunmodified or iron-loaded cytosol fromexvivo Maze's samples, or (b) a mixture of self-reconstituted rHF andceruloplasmin-reconstituted rLF, corresponding to the indicated

amountsofiron.Sampleswereelutedwith theequilibrationbuffer (Tris

effluent)or 4.5 MMgCl2 (Mg effluent). Other conditions were as







I--3 30





ironloaded cyto9o01i

/ unmodified



H-rHF- rH.F


Elution volume (ml)

Figure7. Sepharose 6B chromatography of cytosolic ' ferritin Mgeffluents. The Tris effluent of unmodified

cytosolsand the Mgeffluentof rHF plus rLF werechi

on aSepharose6B column and analyzed for nonheme asdescribedin Methods. Fractions with iron were pool

by ultrafiltration,andsubjectedtoSDS-PAGE analysis. anelectrophoretogram where lane a was loaded with '

tions containingrHFplus rLF, and lane b was loaded the ironfractionsfrom unmodifiedcytosol. Lane c was

followingM,markers: phosphorylaseB (94,000);bov min(67,000);ovalbumin(43,000);carbonicanhydras beantrypsininhibitor(20,100);and a-lactalbumin(1 werevisualizedwithCoomassie brilliantblue.V0,voic mined with blue dextran); HoSF, horsespleenferritin

preparation was excluded by a Sepharose 6B included rHF, rLF, and horse spleen ferritin w

elution volume (Fig. 7). And finally, SDS-P showed that theSepharose 6B fractions with the lacked the M - 20,000 and23,000 bands of fe

subunits, respectively (Fig.7, inset). Itis possit had been immobilizedby the calcium hydroxyl used for increasing the stability of cytosols or

thawing or reconstitution with NADPH and D

with Methods). This would bealikely consequ

complexation and cluster formation between the

of the resin and the acidic H subunits which I

heart ferritin. In the light of these results we i

DOXol could have reacted with cardiac isoferr subunits.Therefore,westudied DOXformation; bilization in 1-ml incubations containing 5 nn

enzymatic DOXol and 10 nmol of Fe(III) bot




protein, respectively).Under theseconditions,ne

Fe(II)couldbeassayed, suggestingthatDOXo]

couplewith rHF-boundFe(III). Negativeresultf

tainedbyreplacingrHFwithequimolariron bou







respectively); (b) commercially


transferrin (2 atoms


corresl nmol


mgprotein);or(c) partially ( transferrin (0.76atoms


corre: nmol


mgprotein).The latter had be

removing iron from holotransferrin under redu(



with anitrilot complex,



procedures (3'

the formation of 4.8 or4.9

nmol DOX/h,


a b c I of 9.5 or 9.8 nmol


were observed by incubating 5

94000 nmol of DOXol with 10 nmol of


6B-chromato-67,000 graphed Fe(III) from unmodified or iron-loaded cytosols of ex 43,000 vivo Maze's samples (corresponding to 0.83 or 0.29 mg protein, respectively). These results showed that the redox coupling of one DOXol with two Fe(HI) was entirely contingent on the




of alternativesources of iron.

14,400 Discussion

In this study we have demonstrated, for the first time, that

human cardiomyocytes possess the NADPH-dependent


matic machinery which reduces DOX to DOXol. Evidence for

45 this activity has been obtained by reconstituting NADPH and

DOX with cytosolic fractions of myocardial samples from se-Triseffluents or lected postmortem donors or patients undergoing open-chest

riron-loaded cardiac surgery. This in vitro evidence provides the conceptual


framework to discuss the potential role of DOXol in the clinical

ed,concentrated settings of DOX toxicity.

The insetshows

Previous studies


the molecular mechanisms of DOXol



of thefrac- toxicity have emphasized that it inhibits the ATPases of

sarco-with 10


of lemma, sarcoplasmic reticulum, or mitochondrial membranes loadedwith the from


orrabbit heart

(36, 37).

This inhibitionwas shown


serumalbu- tooccurirrespectiveofasimultaneous addition ofiron, hence,

e(30,000); soy- a free radical-independent mechanism was envisioned (36). 4,400). Proteins However, DOXol-inhibitable enzymes, such as the





ATPase, have been subsequently recognizedtocontain loosely bound iron which catalyzes an in situ formation of hydroxyl radicals or equally reactive oxidants (38). It should also be considered that laboratory buffers (39) and membrane

column which preparations (40) are themselves contaminated by iron ions

ithin the same which are liable to participate in free radical reactions upon 'AGE analysis reduction from the ferric to the ferrous form. Therefore, one cytosolic iron can suspect that enzyme inhibition was mediated by an unrecog-rritin L andH nized free radical chemistry, involving the redox coupling of ble that ferritin DOXol withcontaminating iron. In ourstudies,unmodified or

apatite column iron-reconstituted cytosols were prepared in NaCl and treated

n freezing and extensively with chelators to minimize metal contaminations, )OX (compare yet,they tenaciouslyboundaniron



tenceof strong reductive mobilization by DOXol.Inlightof theseresults,we

e calcium sites propose that thecardiotoxicityof DOX


be the reflection


in ofaredoxcouplingbetween DOXol andtightlybound


wondered how iron.

itins rich ofH Enzymatic experimentswith NADPHandDOX,aswellas

andFe(II)mo- nonenzymatic experiments with


DOXol, indicatethat


of purified 1 mol of this metabolite releases 2molof



and to self- or electrons needed forreducingtheC- 13

carbonyl moiety

of DOX

to58or50 fig are returned to two independent Fe(III)

ions, according

to a zither DOXnor - 0.5stoichiometry ofDOXol oxidation vs


reduction. I didnotredox These resultspointto avery efficient and novel



swerealsoob- DOX bioactivation and iron


inasmuchas the DOX ndto:(a) self- semiquinonefree




would reduce< 1


ngto 150or18 (12). Moreover, we have shown that the redox



available holo- DOXol with iron doesnotinvolve





ponding to 10 section C), suggesting that SOD


not be the first-line 38%)saturated defenseagainstDOX


This could very wellaccountfor

sponding to 10 thefindingthat


diets decrease the tissue levels

enprepared by of SOD activity, but fail to exacerbate the


of a

cing conditions cardiomyopathy in DOX-treated animals





feature of the


antibiotics is that 5). By contrast, minor chemical modifications


result in rather


he mobilization changes of




DNR is in all similar


to DOX except that its side chain terminates with a methyl group in place of a primary alcohol. Nevertheless, DNR is

reportedlyless toxic than DOX(42, 43). Similarly,apositional changein thehydroxylgroup at the C-4' of daunosamine (com-pare with Fig. 1) is the sole difference between DOXand

4'-epi DOX, yet, experimentaland clinical evidencesuggeststhat 4'-epi DOXisless cardiotoxic than either DOX or DNR (44-46). Andfinally,deoxydemethylationof the C-4 in the tetracy-clinering (compare withFig. 1)converts DNRintoa newdrug which appears to be the least cardiotoxic among all clinically tested anthracyclines(6, 47). Structure activity studiesin vitro

have failed to demonstratesignificant differences between

equi-molar DOX, 4'-epi DOX, DNR, and 4-demethoxy DNR with

respecttothe redoxcyclingof thequinone moiety ( 12, 48)and

iron release from ferritin (12, 49). Some differences might nevertheless surface in vivo ifanthracyclines exhibit variable

pharmacokinetics and tissue distribution. For example, 4'-epi DOX isunusuallysusceptibletoglucuronidationand isexcreted morerapidly thanDOX(50). The decreased cardiotoxicity of

4'-epi DOX could therefore be the obvious consequence ofan

acceleratedelimination and reduced availability to thecardiac reductases that support one electron redox cycling of the

qui-nonemoiety and iron release from ferritin. On the otherhand,

4-demethoxy DNR is less toxic than DOX, DNR, or 4'-epi

DOX when assessed in vivo, however, it can be more toxic whentested in vitro on neonatal rat heart myocytes (43). Col-lectively, these observations wouldcastdoubts on the

signifi-cance of in vitro models of myocardial damage, suggesting that theclinical cardiotoxicity ofagivenanthracycline depends predominantly on pharmacokinetic factors. In contrast to this picture,ourstudies demonstrate that the reductive mobilization of cytosolic iron decreases gradually and significantly upon replacing DOX with equimolar DNR, 4'-epi DOX, and 4-de-methoxy DNR, suggesting that a stringent relationship does exist between the severity ofcardiac damage in vivo and the

efficiencyof iron releaseinvitro (compare with Table IV). By dissecting the two-step mechanism leadingto thereduction of cytosolicFe(HI),wehavealsodemonstrated that DNR,4'-epi DOX, and4-demethoxyDNRmobilize less


because they

are less susceptible than DOX to an enzymatic formation of secondary alcohol metabolites (compare with Fig. 6 A).

Daun-orubicinol, 4'-epi DOXol, and 4-demethoxy DNRol per se

wouldbe as effective asDOXolin redox coupling withFe(III) (Fig. 6 B). Collectively, these observations indicate that a two electron reduction of the side chain C- 13 moiety overshadows the one-electron reduction of the quinone group as a critical determinant of anthracycline metabolism and iron mobilization, irrespective of pharmacokinetic differences that may exist

be-tweenvarious analogs.

In anattempttoelucidatethemolecular mechanisms under-lying the different toxicity of DOX vs DNR, Zweier et al. (51 ) have suggested that a key role might be played by the side chain C-14 moiety. According to these authors, the C-14 pri-mary alcohol of DOX oxidizes at the expense of Fe(III), yield-ing an aldehyde plus Fe(H)ions which subsequently catalyze free radical reactions (51). This would explain how DOX is moretoxic than DNR or any DNR analog having a C-14 methyl moiety in place ofaprimary alcohol. Our results do not provide any evidence for the involvement of the C-14 moiety in the reductive mobilization of cytosolic iron. In fact, DOX per se

couldmobilizeaslittleasonetenth of the iron mobilized upon simultaneous addition ofNADPHand



DOXol(comparewithFig. 2). Moreover,wehave shown that

cytosolic Fe(III) can be reduced to a comparable extent by

DOXol, DNRol,4'-epi DOXol,and 4-demethoxy DNRol, i.e., the C-13 metabolites of anthracyclines which either lack or containaprimary alcoholatC-14.We havenoexplanation for the exclusive role of the C-13 moiety in Fe(III) reduction. We canonlynotethat theprimaryalcohol of DOX has been shown

to reduce a low-mol-wt acetohydroxamate-Fe(III) complex

(51 ), whereasourstudiesindicate that C-13 secondary alcohols reduceaniron species which is tightly bound toproteins. It is possible that the access to protein-bound iron is limited by sterical requirements thatare metbyasecondary alcoholat

C-13, butnotbyaprimary alcoholatC-14. We should also empha-size that the intracellularconcentration of low molecularweight iron is extremely low and difficulttoquantify ( 1, 2, 52). Hence, areaction between secondary alcohols and themore abundant

protein-boundiron should predominateas amechanism of


The iron-detoxifying activity of ferritin is the result of a functional cooperation between H andL subunits. The

oxygen-dependent oxidation of Fe(II)occurspredominantlyatdiscrete sites of the H subunits, whereas the size of the cavity which

accommodates Fe(III) usually increases with the number of L subunits(3). Heart ferritin is composed primarily of H subunits, hence, it is particularlywell suited for the rapid sequestration of iron. By contrast, liver and spleen ferritins are composed primarily ofLsubunits andare moresuitable forthelong-term storage of copious amounts of iron (3, 4). We haveconfirmed

the existence of thesedistinct functions for H or L subunits. In fact, H-type recombinant homopolymers were found to oxida-tively incorporategreater amounts of iron than didL-type homo-polymers devoid of ferroxidase activity (95 vs32 atomsFe"'/ molecule per h). However, this difference was reverted by

re-constituting ferritins in the presence of ceruloplasmin, which also oxidizes Fe(II) and is frequentlyused in vitro to

incorpo-rateFe(III) into L-type apoferritins with little ferroxidase activ-ity (2, 53,54).Inthepresence of ceruloplasmin, L-type homo-polymers could incorporate much greater amounts of Fe(III) than did H-type homopolymers (260 vs 110atoms

Fe'm/mole-culeperh), in keeping withamajor role forLsubunits in the formationof an iron core. Inourstudy, iron-depleted cytosols were shown to oxidize Fe(II) and incorporate Fe(III) in a large

excess of that recovered in unmodified samples, in apparent

agreement with an H-type ferroxidase activity (compare with Figs. 3, A andBand7). Nevertheless, we have provided analyti-cal and functional evidence that: (a) iron was oxidatively incor-porated by proteins distinct from ferritin (compare with Fig. 7

andTable V); and (b)ferritin did not redox couple with DOXol, irrespective of the subunit composition, the iron:protein ratio, and the presenceorabsence of ceruloplasminatthe timeof iron incorporation into apoferritin (compare with Results, section e). Purified DOXol was also shown not to redox couple with transferrin, irrespective of the degree of iron saturation (see Results, section e). Hence, the DOXol-releasable iron was not accountedfor by transferrin which had been released from endo-cytic vesicles during tissue manipulation and cytosol prepara-tion. These results indicate that the availability of nonferritin sources of Fe(III) represents an additional determinant of the molecularlinkage between DOX metabolism and Fe(II) mobili-zation. The existence of "nonferritin" pools of iron, having variable mol wt and intracellular distribution, has been


precise nature and biologic significance of these sources of iron have remained unknown, mostly because of technical difficult-ies in achieving purification and molecular characterization (55, 60). It has been tentatively proposed that thenonfemtin pools of iron may serve, upon metabolic needs, for the synthesis of

hemeproteins (60). Alternatively, these pools of iron might identify with enzymes which operate by virtue of essential non-heme Fe(III) moieties (61). In either case, DOXol-dependent

delocalization ofthese sources of iron would interrupt important cell functions, irrespective of any subsequent involvement of delocalized Fe(II) infree radical reactions and pharmacologic

interventionwith ironchelators. This scenario might very well

providean additionalexplanation for the incomplete

cardiopro-tection afforded bydexrazoxane.

Invitro studies withtumor cell lines indicate that

anthracy-cline C-13 metaboliteshave much lesstumoricidalactivity than

havethe parent compounds, with the possible exception of

4-demethoxy DNRol being equipotentas 4-demethoxy DNR (37,

62). On the other hand,ourstudies indicatethat an intramyocar-dial mobilization ofFe(II)dependsverycritically on the forma-tionofC-13metaboliteswhich reduceFe(III).This mechanistic

dissociation,aswell asthe clinicalneed for "better"

anthracy-cline regimens and cardioprotective interventions (6), would

possibly identify inhibitors of anthracyclineC-13 reductases as a newclass ofdrugsthatmitigate cardiotoxicitywithout affect-ing antitumor activity. Thepurificationand characterization of intracardiac C-13 reductases would helpto selectpotential

in-hibitors and accelerate their in vivotestingascardioprotectants.


This workwassupportedin partbyaNorth AtlanticTreatyOrganization

Collaborative Research Grant (901102)toG. Minotti.


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