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.
G Minotti, … , R Zamparelli, G Possati
J Clin Invest.
1995;
95(4)
:1595-1605.
https://doi.org/10.1172/JCI117833
.
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
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
Giorgio Minotti,*Anna Franca
Cavaliere,*
AlvaroMordente,5 MarcoRossi,11
RoccoSchiavello,11
RobertoZamparelli,11
andGianFederico
PossatiI
Departmentsof*Pharmacology,
'Obstetrics
andGynecology, §BiologicalChemistry,IlAnesthesiology
and Intensive Care, and 1Cardiac Surgery, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome,ItalyAbstract
Introduction
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
Fe(ll)
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
(2.1±0.1,
1.2±0.2, and0.6±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
radicals.
cardiotoxicity
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;
02-,
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
Methods
0
R= #C-CH0
DOX
H OH
R= sC-CH2OH
DOXol
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 1.1.1.21] (15) or carbonyl reductases [EC
1.1.1.184]
(16). Doxorubicinol is the primary circulatingme-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
otheranthracyclinesinmyocardialsamplesfrompatients
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
patients.
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 1.15.1.1), 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
CH3COOC2H5/
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
polar
metabolites.Enzymatic
(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
by
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
1.18.1.2) (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
adding
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
10'
C.)
0i)
4
1-- 0-
1--E
5.
Complete
- 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
Ag
protein -30nmoliron) and ceruloplasmin-recon-stituted rLF (166 ,ug protein -90 nmol of iron). The column waswashed 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
MyocardialSamples*
Sample Unmodified cytosol Iron-depleted cytosol
nmolDOXol/mgproteinper 4h
Postmortem 0.7±0.2 3.9±0.4t
Exvivo
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
nmol
Fe(L1l)/mg
protein (n = 8). The two preparations from ex vivoMaze'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.
Results
(a) NADPH- andDOX-dependent
Fe(HI)
mobilization inun-modified
oriron-depleted cytosols. NADPH and DOX-supple-mentedcytosolicfractionsfrom humanmyocardiumwerefoundtorelease
Fe(11).
Asshown inFig. 2, comparableresultswereobtained withcytosols fromselected postmortemsamples and
cytosols from ex vivo pre- orpost-CPB samples. NADPH or
DOX persemobilized very littleFe(II);moreover, the mobili-zation of
Fe(H)
was notobservedwhen thecytosols
hadpre-viously been depleted of nonheme Fe(III) (see also
Fig. 2).
These resultsshowedthat themobilizationof
Fe(11)
was medi-atedbyDOX metabolites which reduced nonhemeFe(III).(b) NADPH-dependentDOX metabolism in unmodifiedor
iron-depleted cytosols.
NADPH-supplemented
unmodifiedcy-tosols from postmortem orex vivo
samples
werefoundto re-duce DOXto DOXol, however,theyield
of DOXol increaseddramatically when the cytosols had
previously
beendepleted
of nonheme
Fe(I1)
(Table I).Omission of NADPHorcytosols
abolished DOXolformation, indicatingthatDOX reductionwas mediatedbyanenzymaticmechanism. Doxorubicin-metaboliz-ing enzymesarealso knowntocleave theglycosidic
bondbe-tween daunosamine and the tetracycline ring
(26), therefore,
DOX- andDOXolaglyconeswere measured inunmodifiedor
iron-depleted cytosols.As shown in TableII,
NADPH-supple-mented unmodified
cytosols
from postmortemsamples
werecapableofformingDOXaglyconeand
7-deoxy
DOXaglycone,
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
detect-able.
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
Fe(ll)
wasparalleledbyatenacious bindingofFe(IH)tocytosolic proteins (Fig. 3 B). These results showed that cytosols could incorporate Fe(lI) by oxidizingFe(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
Fe(lI).
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 DOXolwas inversely related to the cytosolic content ofFe(IH) and mobilization of
Fe(ll)
(compare with Fig. 2 and Tables I andII). 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
E
IV
450- 400
350
0 30 60 90 120 150 130
min
30 =
01 20
_ ,{ *
~~~~~~~Fe(Hl)
oxidation10 Fe(IM
incorporation
0
0 30 60 90 120 150 180
min
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.
4
3
U
X 2
-61
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.
5
04
U o3
1
a O
A
0 5 10 15 20 25
nmol
Fe(IU)
/mgproteinFe(I)
10 B
04
_
E /:
0
0 5 10 15 20 25
nmol
Fe(III)
/mgprotein
c
0
NADPH+H!f3' 2Fe(H) N+H
NADP l 2Fe(III)
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
Fe(ll)
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,
coupled
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
keeping
with theoxidation ofoneDOXolTable 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
stoichiometry
ofDOXoloxidation 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
DOXol:Fe(IH)
ratio exceeded 0.5, i.e., when the electron donor (DOXol) was in excess of the electron acceptor(Fe")
(see also TableHI). Similar studies with chemical DOXol gave
a .90% experimental agreement (not shown). At this time,
evidence for a 0.5
stoichiometry
of DOXol oxidation vsFe(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
Fe(LI)
reduction of 0.54±0.04. Similar calculations with ex vivo pre-orpost-CPB samples gave stoichiometries of 0.54or0.53,respectively.
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
anthracycine-supplemented
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
DOXol:Fe(IH)
ratio exceeded an optimum value of - 0.5 (compare with TableIll).
This meansthat 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 [\
IL)
.iX. 1R ;.i1tX -.;,,..
[>N't
Figure6.C-13
metabo-lite formation and redox couplingwithiron: com-parisonsofDOX with
DNR,4'-epiDOX,and
4-demethoxyDNR. In
(A)NADPH- and an- thracycline-supple-mentediron-depleted cytosolsfrom
postmor-temsampleswere
as-sayedfor C-13secondary alcohol formation. Val-° DOXol ues aretnose
ueterrrneu
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
4-de-methoxyDNRolwere
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
was(P<0.05)vs
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
nmolFe(III)
Cytosol
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
IleoSt,
90
-60
-
0--
I--3 30
I2
0
v
0
ironloaded cyto9o01i
/ unmodified
ct1osol
1I
H-rHF- rH.F
30
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
ceruloplasmin-reconstituted
rHF(corresponding
protein, respectively).Under theseconditions,ne
Fe(II)couldbeassayed, suggestingthatDOXo]
couplewith rHF-boundFe(III). Negativeresultf
tainedbyreplacingrHFwithequimolariron bou
or
ceruloplasmin-reconstituted
rLF(correspondi
Iug
protein,
respectively); (b) commercially
Etransferrin (2 atoms
Fe(HI)/molecule,
corresl nmolFe(llI)/0.4
mgprotein);or(c) partially ( transferrin (0.76atomsFe(llI)/molecule,
corre: nmolFe(HI)/1.1
mgprotein).The latter had beremoving iron from holotransferrin under redu(
andbypresenting
apotransferrin
with anitrilot complex,according
toestablishedprocedures (3'
the formation of 4.8 or4.9nmol DOX/h,
andtia b c I of 9.5 or 9.8 nmol
Fe(H)/h,
were observed by incubating 594000 nmol of DOXol with 10 nmol of
Sepharose
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
ae
20.100
availability
of alternativesources of iron.14,400 Discussion
In this study we have demonstrated, for the first time, that
human cardiomyocytes possess the NADPH-dependent
enzy--_____
matic machinery which reduces DOX to DOXol. Evidence for45 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
iromatographed
framework to discuss the potential role of DOXol in the clinicaled,concentrated settings of DOX toxicity.
The insetshows
Previous studies
onthe molecular mechanisms of DOXol
5
Mg
of thefrac- toxicity have emphasized that it inhibits the ATPases ofsarco-with 10
tg
of lemma, sarcoplasmic reticulum, or mitochondrial membranes loadedwith the fromdog
orrabbit heart(36, 37).
This inhibitionwas shownmine
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
mitochon-dvolume
(deter-
drialF1
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 membranecolumn 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
species
thatwasliabletoatenceof strong reductive mobilization by DOXol.Inlightof theseresults,we
e calcium sites propose that thecardiotoxicityof DOX
might
be the reflectionpredominate
in ofaredoxcouplingbetween DOXol andtightlyboundcytosolic
wondered how iron.
itins rich ofH Enzymatic experimentswith NADPHandDOX,aswellas
andFe(II)mo- nonenzymatic experiments with
purified
DOXol, indicatethatnol
of purified 1 mol of this metabolite releases 2molofFe(ll),
i.e.,thetwoand to self- or electrons needed forreducingtheC- 13
carbonyl moiety
of DOXto58or50 fig are returned to two independent Fe(III)
ions, according
to a zither DOXnor - 0.5stoichiometry ofDOXol oxidation vsFe(I1)
reduction. I didnotredox These resultspointto avery efficient and novellinkage
betweenswerealsoob- DOX bioactivation and iron
reduction,
inasmuchas the DOX ndto:(a) self- semiquinonefreeradical/O2'
-couple
would reduce< 1Fe(mI)
ngto 150or18 (12). Moreover, we have shown that the redox
coupling
ofavailable holo- DOXol with iron doesnotinvolve
O2--
(compare
withResults,
ponding to 10 section C), suggesting that SODmight
not be the first-line 38%)saturated defenseagainstDOXtoxicity.
This could very wellaccountforsponding to 10 thefindingthat
copper-deficient
diets decrease the tissue levelsenprepared by of SOD activity, but fail to exacerbate the
development
of acing conditions cardiomyopathy in DOX-treated animals
(41).
riacetic-Fe(RI)
Anintriguing
feature of theanthracycline
antibiotics is that 5). By contrast, minor chemical modificationsusually
result in rathermajor
he mobilization changes ofcardiotoxicity.
Forexample,
DNR is in all similarto 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
Fe(ll)
because theyare 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
enzymatic
formationofDOXol(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
car-diotoxicity.
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 largeexcess 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.
Acknowledgments
This workwassupportedin partbyaNorth AtlanticTreatyOrganization
Collaborative Research Grant (901102)toG. Minotti.
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