FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Minotti, G.
Right arrow Articles by Cairo, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Minotti, G.
Right arrow Articles by Cairo, G.
(The FASEB Journal. 1998;12:541-552.)
© 1998 FASEB

RESEARCH COMMUNICATION

The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium

Giorgio Minottia,1, Stefania Recalcatic, Alvaro Mordenteb, Giovanni Liberie, Antonio Maria Calafioree, Cesare Mancusoa, Paolo Preziosia, and Gaetano Cairod

a Department of Pharmacology, Catholic University School of Medicine, Rome, Italy
b Department of Biochemistry, Catholic University School of Medicine, Rome, Italy
c Department of Gastroenterology, University of Milan School of Medicine-IRCCS Ospedale Maggiore, Italy
d CNR Center for Cell Pathology, Milan, Italy
e Department of Cardiac Surgery, G. D'Annunzio University School of Medicine, Chieti, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anticancer therapy with doxorubicin (DOX) is limited by severe cardiotoxicity, presumably reflecting the intramyocardial formation of drug metabolites that alter cell constituents and functions. In a previous study, we showed that NADPH-supplemented cytosolic fractions from human myocardial samples can enzymatically reduce a carbonyl group in the side chain of DOX, yielding a secondary alcohol metabolite called doxorubicinol (DOXol). Here we demonstrate that DOXol delocalizes low molecular weight Fe(II) from the [4Fe-4S] cluster of cytoplasmic aconitase. Iron delocalization proceeds through the reoxidation of DOXol to DOX and liberates DOX-Fe(II) complexes as ultimate by-products. Under physiologic conditions, cluster disassembly abolishes aconitase activity and forms an apoprotein that binds to mRNAs, coordinately increasing the synthesis of transferrin receptor but decreasing that of ferritin. Aconitase is thus converted into an iron regulatory protein-1 (IRP-1) that causes iron uptake to prevail over sequestration, forming a pool of free iron that is used for metabolic functions. Conversely, cluster reassembly converts IRP-1 back to aconitase, providing a regulatory mechanism to decrease free iron when it exceeds metabolic requirements. In contrast to these physiologic mechanisms, DOXol-dependent iron release and cluster disassembly not only abolish aconitase activity, but also affect irreversibly the ability of the apoprotein to function as IRP-1 or to reincorporate iron within new Fe-S motifs. This damage is mediated by DOX-Fe(II) complexes and reflects oxidative modifications of -SH residues having the dual role to coordinate cluster assembly and facilitate interactions of IRP-1 with mRNAs. Collectively, these findings describe a novel mechanism of cardiotoxicity, suggesting that intramyocardial formation of DOXol may perturb the homeostatic processes associated with cluster assembly or disassembly and the reversible switch between aconitase and IRP-1. These results may also provide a guideline to design new drugs that mitigate the cardiotoxicity of DOX.—Minotti, G., Recalcati, S., Mordente, A., Liberi, G., Calafiore, A. M., Mancuso, C., Preziosi, P., Cairo, G. The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium. FASEB J. 12, 541–552 (1998)


Key Words: IRP-1 • cardiotoxicity • doxorubicin • alcohol metabolite • DOXol cluster disassembly • iron-responsive elements


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DOXORUBICIN (DOX)2 is an anthracycline antibiotic with broad spectrum antitumor activity; however, DOX can also induce cardiotoxicity and congestive heart failure, especially when the cumulative dose exceeds ~ 550 mg/m2 (1, 2). According to the prevailing hypotheses, the cardiotoxicity of DOX is mediated by metabolites that are formed within cardiomyocytes and alter critical cell constituents and functions (3, 4). Ethical and practical constraints preclude the identification and quantification of these metabolites in fine needle myocardial biopsies from DOX-treated patients. On the other hand, studies with laboratory animals do not always anticipate the yield and reactivity of a given metabolite in humans, presumably because the enzymes of biotransformation and the ultimate target (or targets) of toxicity may exhibit species-related differences (5). Reconstitution of DOX with myocardial fragments disposed during aorto-coronary bypass grafting provides a convenient and ethically acceptable strategy to obviate these problems and characterize the metabolism of this drug in the human heart. Using this approach, we have previously demonstrated that cytosolic fractions from human myocardium possess NADPH-dependent aldo-keto reductases that add two electrons to the C-13 carbonyl group in the side chain of DOX, forming a secondary alcohol metabolite called doxorubicinol (DOXol) (cf ref 6 and see Scheme I ). These studies have also shown that DOXol releases low molecular weight (low mol wt) Fe(II) from a protein (or proteins) other than ferritin, which per se would account for a major site of iron storage in the cytosolic milieu of human cardiomyocytes (6, 7). We have therefore hypothesized that DOXol might become cardiotoxic by removing iron from alternative sources such as iron-requiring enzymes (6).

This study was aimed at establishing whether DOXol removes iron from the [4Fe-4S] cluster of cytoplasmic aconitase, a protein displaying marked homology with the mitochondrial enzyme that converts citrate to isocitrate via the intermediate cis-aconitate in the Krebs cycle (8). The fourth ferrous iron of the cluster, referred to as Fea, is directly involved in enzymatic catalysis; limited removal of Fea would therefore abolish aconitase activity (8). Perhaps more important, dynamic processes of cluster disassembly and reassembly enable cytoplasmic aconitase to participate in iron metabolism and cell homeostasis. Cluster disassembly occurs when the process of iron removal extends beyond Fea and involves the three other centers, referred to as Feb1–3. This process liberates an apoprotein that binds to iron-responsive elements (IREs) in mRNAs for transferrin receptor or ferritin, increasing stability or decreasing translation, respectively (9–11). In so doing, the apoprotein functions as an iron regulatory protein-1 (IRP-1) that coordinately up-regulates the synthesis of transferrin receptor but down-regulates that of ferritin, causing iron uptake to prevail over sequestration and forming a pool of low mol wt Fe that is available for metabolic use. Under physiologic conditions, the balance between aconitase and IRP-1 is influenced by cell iron status. When iron is scarce, both cluster disassembly and de novo synthesis of the apoprotein shift the balance in favor of IRP-1, promoting the formation of low mol wt Fe (11). When iron is plentiful, new clusters are formed and the balance returns in favor of aconitase, decreasing low mol wt Fe. Reversible assembly or disassembly of [4Fe-4S] clusters can therefore be viewed as homeostatic mechanisms that constantly adapt the concentration of low mol wt Fe to the metabolic needs of the cell. Perturbing these mechanisms with DOXol or any other agent that inappropriately induces cluster iron delocalization and disassembly would predictably affect cell functions that require iron as a cofactor.

Our results demonstrate that DOXol delocalizes iron from [4Fe-4S] clusters and causes irreversible modifications of both aconitase and IRP-1 activities in human myocardium. These findings may help clarify how DOX becomes cardiotoxic, setting the stage for mechanism-oriented interventions that protect cardiomyocytes during anticancer therapy with this drug.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Lactose-free anthracyclines were obtained through the courtesy of Dr. Antonino Suarato (Pharmacia-Upjohn, Milan, Italy). Ammonium sulfate (ultrapure grade, FeIII<0.5 ppm) was purchased from Schwarz/Mann (Cleveland, Ohio). Cysteine, EDTA, and ferrous ammonium sulfate (FAS) were from Merck (Darmstadt, Germany). d,l-Fluorocitrate (barium salt) was a product of Pfaltz and Bauer and was obtained through the courtesy of Dr. Steven D. Aust (Biotechnology Center, Utah State University, Logan). cis-Aconitate, ferrozine, Sepharose 6B, bovine erythrocyte CuZn superoxide dismutase (SOD; EC 1.15.1.1), thymol-free bovine liver catalase (EC 1.11.1.6), and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.). Unless otherwise indicated, the experiments were carried out in 0.3 M NaCl, carefully adjusted to pH 7.0 just before use. This was done to avoid ligand-catalyzed interactions of most common buffers with iron (12). Although unbuffered, the pH of the reaction mixtures did not vary throughout the period of the experiment. All solutions were prepared with double-distilled water that had been passed through a Milli-Q Water System (Millipore; Marlborough, Mass.). Trace metals were eventually removed by ion-exchange chromatography on Chelex 100 (Bio-Rad; Richmond, Calif.).

Preparation of cytosols from myocardial biopsies
Small atrial samples (~0.1–0.2 g) were taken from 76 patients (45 M, 31 F, aged 61±4 years) undergoing aortocoronary bypass grafting. All biopsies were collected before cardiopulmonary bypass, using a previously described technique (6). This protocol had been approved by Institutional Review Boards, and informed consent was obtained from all patients. After storage at -80°C, pools of 15–20 myocardial biopsies were processed for cytosol preparation by sequential homogenization, ultracentrifugation, and overnight stirring of 105,000 g supernatants with 65% ammonium sulfate, as described previously (6). Calcium hydroxyapatite chromatography (6) was omitted in order to minimize cluster oxidation and other possible modifications of aconitase/IRP-1. After 20 min precipitation at 10,000 g, cytosolic proteins were suspended in a minimum volume of 0.3 M NaCl and dialyzed first against two 1-liter changes of 0.3 M NaCl–1 mM EDTA and then against two 1-liter changes of 0.3 M NaCl (to remove EDTA and EDTA–iron complexes). Previous studies have shown that EDTA would remove adventitious iron (6, 7) but not iron ions coordinated within the aconitase cluster (13). Cytosols prepared by this standard procedure were referred to as unmodified. Where indicated, cytosols (3–5 mg protein/ml) were incubated with cysteine and FAS at a final ratio of 1100 or 50 nmol/mg protein, respectively. This treatment was intended to reconstitute the [4Fe-4S] cluster of aconitase, a process requiring iron and thiols (13, 14). After 15 min at 4°C (13), unreacted FAS and cysteine were removed by gel filtration on a (1.5x12 cm) Sepharose 6B column equilibrated and eluted with 0.3 M NaCl, pH 7.0; protein-containing fractions were then incubated with 65% ammonium sulfate, which also contained 0.25 mM cis-aconitate, to protect the cluster against oxidative inactivation (13). After 4 h stirring, 10,000 g precipitates were dissolved in a minimum volume of 0.3 M NaCl and dialyzed sequentially against 1-liter changes of 0.3 M NaCl-0.1 mM cis-aconitate, EDTA-NaCl, and NaCl. These cytosols were referred to as iron-loaded. In other experiments, cytosols were dialyzed against two 1-liter changes of 100 mM Tris HCl, pH 8.9/40 mM KCl, diluted to 3 mg protein/ml with the same buffer, and incubated with 100 mM dithiothreitol (DTT) to remove iron from clusters (15). After 15 min at room temperature, these cytosols were subjected to Sepharose 6B chromatography, ammonium sulfate precipitation, and dialysis as described for the preparation of iron-loaded samples, with the exception that cis-aconitate was omitted throughout. Cytosols treated with DTT/pH 8.9 were referred to as iron-depleted.

In vitro RNA transcription and RNA–protein gel retardation assay for IRP-1
The pSPT-fer plasmid containing the IRE of human ferritin H chain (16) was linearized with BAM HI and transcribed in vitro with T7 RNA polymerase in the presence of 100 µCi of [{alpha}32-P]UTP (800 µCi/mmol; Amersham Co., Arlington Heights, Ill.). For IRP-1 assay, 2 µg protein samples were incubated with a molar excess of IRE probe, digested with Rnase T1, and treated with heparin as described previously (17). After separation on 6% nondenaturing polyacrylamide gels, IRP-1/IRE complexes were visualized by autoradiography and quantified by computer-assisted densitometry (Imaging Densitometer, G5670; Bio-Rad). Binding specificity has been demonstrated by competition studies (17). In selected experiments, iron-loaded cytosols (1.5–3 mg protein/ml) were assayed for IRP-1 after 1 h incubation at 37°C with cis-aconitate plus or minus DOX(ol) (100 and 4 nmol/mg protein, respectively). Preliminary experiments showed that DOX(ol) per se did not interfere with IRP-1 detection when added to cytosols just prior to the gel shift assay at a ratio of 0.5 to 5 nmol/mg protein; by contrast, >= 50 nmol cis-aconitate/mg protein decreased the formation and/or detection of IRE/IRP-1 complexes, in keeping with previous reports demonstrating similar effects of aconitase substrates (18). cis-Aconitate-treated cytosols were therefore subjected to Sepharose 6B chromatography, ammonium sulfate precipitation, and dialysis to ensure substrate removal. Where indicated, cytosols were treated with 2-mercaptoethanol (2-ME) just before the gel-shift assay to reduce disulfide bridges and regenerate -SH groups that mediate RNA binding (19, 20). Based on titration experiments, the concentration of 2-ME was optimized to 0.6% (v:v).

Assay for aconitase
Aconitase activity was determined spectrophotometrically by monitoring the disappearance of cis-aconitate ({epsilon}240=3.6 mM-1 cm-1) (21). The incubations (1 ml final volume) contained cytosols (80–300 µg protein) and 0.1 mM cis-aconitate in 0.3 M NaCl, pH 7.0, 37°C; one mU was defined as the amount of enzyme that consumed 1 nmol cis-aconitate/min (21). Where indicated, aconitase was measured in cytosols that had been treated with cis-aconitate plus or minus DOX(ol) and subsequently made free of substrate as described for IRP-1 assay.

Assays for aldo-ketoreductase activity and DOX metabolites
The anthracycline aldo-ketoreductase activity of human myocardium was measured as DOXol formation in 0.5 ml incubations containing cytosols (150 µg protein), NADPH, and DOX (both 150 µM) in 0.3 M NaCl, pH 7.0, 37°C. After 4 h, the reaction mixtures were extracted and analyzed by 2-dimensional thin layer chromatography (6). Mobile phases, standard preparations, metabolite assignement, and fluorimetric quantification have been described in detail (6, 22). Control experiments showed that variable amounts of DOXol hydrolyzed nonenzymatically and formed a polar metabolite (e.g., 4-demethyl-deoxy aglycones). Aldo-keto reductase activities were therefore expressed as nmol [DOXol]/mg protein/4 h, where [DOXol] indicates the metabolite per se and secondary products of nonenzymatic hydrolysis.

Assays for iron delocalization
Doxorubicinol-dependent delocalization of low mol wt Fe(II) was studied spectrophotometrically in 1 ml incubations containing cytosols (1 mg protein) in 0.3 M NaCl, pH 7.0, 37°C. Reactions were started by adding DOXol prepared by NaBH4 reduction of DOX and purified by 2-dimensional thin layer chromatography (6, 22). Delocalized Fe(II) was detected by chelation with 0.25 mM ferrozine, which per se would not remove iron from clusters (13). The formation of ferrozine-Fe(II) complexes [{epsilon}564 nm=27.9 mM-1 cm-1 (23)] was monitored against reference samples lacking the chromophore by using a Hewlett Packard 8453 diode array spectrophotometer with computer-assisted corrections for scatter and turbidity.

Other assays
Proteins were determined by the bicinchoninic acid acid method (24). The cytosolic content of Fea was determined in 1 ml incubations containing cytosols (1–3 mg protein) and ammonium persulfate (1 mM), which served to induce slow cluster disassembly (13). Delocalized Fea was detected with ferrozine (0.25 mM), and readings were corrected for reference samples that contained 0.25 mM cis-aconitate to protect [4Fe-4S] clusters from oxidative decay (13). The difference between [ferrozine-Fe(II) (-cis-aconitate)] and [ferrozine-Fe(II) (+cis-aconitate)] gave a net measurement of cluster-delocalized Fe(II). Reactions were allowed to proceed until ammonium persulfate decreased aconitase activity to ~10% of initial values, indicative of near to complete removal of Fea (13).

Unless otherwise indicated, all data are expressed as the arithmetic mean ± SE. Statistical analyses were performed by paired and unpaired Student's t tests, and differences were considered significant when P < 0.05. Other conditions are indicated in the legends to the figures and tables.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Aconitase and IRP-1 in human myocardium cytosols
Both aconitase and IRP-1 were detected in unmodified cytosols from human myocardial biopsies, showing that these samples contained [4Fe-4S] holoproteins with enzymatic function as well as cluster-free apoproteins with RNA binding activity ( Fig. 1). Treatment with cysteine and FAS yielded iron-loaded cytosols bearing more [4Fe-4S] clusters; therefore, aconitase increased and IRP-1 decreased. By contrast, treatment with DTT/pH 8.9 yielded iron-depleted cytosol bearing fewer [4Fe-4S] clusters; hence, aconitase decreased whereas IRP-1 increased (see also Fig. 1). These findings showed that iron incorporation or removal and cluster assembly or disassembly made aconitase and IRP-1 activities mutually exclusive.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 1. Aconitase and IRP-1 activities in unmodified, iron-loaded, and iron-depleted cytosols. Cytosols were prepared and assayed for aconitase and IRP-1 as described in Materials and Methods. IRP-1/IRE complexes were quantified densitometrically and expressed as percent vs. unmodified cytosols. Values given as means ± SE of three to five determinations in duplicate. *, **P < 0.05 or P < 0.01 vs. unmodified cytosols, respectively. The bottom panel shows two representative gel shift assays of unmodified vs. iron-loaded or iron-depleted cytosols.

In addition to IRP-1, cells may contain variable amounts of an iron regulatory protein-2, which binds to IREs but lacks the ability to form the [4Fe-4S] cluster required for aconitase activity (25). Currently available techniques do not permit gel shift separation and detection of IRP-2 vs. IRP-1 in human tissues. Nonetheless, the observation that iron loading or unloading procedures were accompanied by coordinate but divergent changes of aconitase activity vs. IRE binding suggests that IRP-1 accounted for a major part of the biochemical features of our samples.

Doxorubicinol-dependent cluster iron delocalization: requirement for cis-aconitate
NADPH-supplemented cytosols were found to reduce DOX to DOXol, in keeping with the presence of aldo-keto reductases in human myocardium (6). Specific activities averaged 3.4 ± 0.4, 3.5 ± 0.7, and 3.9 ± 0.4 nmol DOXol/mg protein/4 h in unmodified, iron-loaded, and iron-depleted cytosols, respectively (n=3; P>0.05). Therefore, subsequent experiments to assess the metabolic fate of DOXol and its ability to release iron from aconitase were performed by reconstituting the purified metabolite with cytosols at a final ratio of 4 nmol/mg protein, reproducing the aldo-keto reductase activity of these samples. These experiments were performed in the presence or absence of cis-aconitate as a substrate intermediate of aconitase. This was done because substrates are known to modulate the response of cytoplasmic aconitase to redox-active agents that target its [4Fe-4S] cluster (20, 26). cis-Aconitate is most suitable for these studies (26) and readily isomerizes to both citrate and isocitrate within [4Fe-4S] clusters (8), reproducing physiologic equilibration of these three isomers during the aconitase reaction. As shown in Fig. 2A, incubation of DOXol with iron-loaded cytosols accompanied the recovery of DOX, indicative of a two-equivalent reoxidation of the secondary alcohol moiety at C-13. Formation of DOX was enhanced by cis-aconitate in a concentration-dependent manner, suggesting that DOXol oxidation was mediated by reactions with aconitase. In the experiments with 100 µM cis-aconitate, ~2 nmol DOXol converted to DOX; the remaining was recovered as 4-demethyl-deoxy DOXol aglycone, reflecting nonenzymatic hydrolysis and removal of the aminosugar bound to the anthracycline (cf Scheme I). However, the formation of this aglycone did not involve reactions with aconitase inasmuch as the relative yield was the same in the absence as in the presence of 100 µM cis-aconitate (1.8±0.2 vs. 1.9±0.3 nmol/mg protein; n=3).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. cis-Aconitate- and DOXol-dependent Fe(II) mobilization and DOX formation in different cytosol samples. Incubations (1 ml final volume) contained cytosols (1 mg protein), purified DOXol (4 µM), and increasing concentrations of cis-aconitate in 0.3 M NaCl, pH 7.0, 37°C. A) Incubations were extracted and assayed for DOX by 2-dimensional thin layer chromatography. B) The mobilization of Fe(II) was monitored by chelation with 0.25 mM ferrozine. Values are those determined at 1 h and are means ± SE of three to five experiments.

Having determined that DOXol oxidized with aconitase, we performed experiments to evaluate whether it also delocalized iron from the [4Fe-4S] cluster of this enzyme. As shown in Fig. 2B, reconstitution of DOXol with iron-loaded cytosols did accompany the mobilization of low mol wt Fe(II). This process was enhanced by cis-aconitate in a concentration-dependent manner, similar to the oxidation of DOXol to DOX, pointing to the release of iron ions associated with aconitase. It is important that the release of Fe(II) and the formation of DOX were both abolished by replacing iron-loaded cytosols with unmodified or iron-depleted samples having less or no [4Fe-4S] clusters, irrespective of the presence or absence of increasing concentrations of cis-aconitate ( Fig. 2A–B). Collectively, these experiments suggested that DOX and low mol wt Fe(II) were formed upon reaction of DOXol with the [4Fe-4S] cluster of aconitase. Moreover, the requirement for cis-aconitate gave preliminary evidence that DOXol–aconitase interactions were favored by the presence of physiologic substrates/intermediates within [4Fe-4S] clusters. To test this hypothesis, iron-loaded cytosols were coincubated with cis-aconitate and increasing concentrations of fluorocitrate, which competes for the cluster and inhibits aconitase (27). As shown in Fig. 3, fluorocitrate concentration-dependently suppressed aconitase activity and similarly decreased DOX formation and Fe(II) mobilization when cytosols were exposed to DOXol. These experiments gave more direct evidence that: 1) cytoplasmic aconitase was the source of DOXol-releasable iron; and 2) both DOXol oxidation and iron delocalization occurred when aconitase was involved in catalysis with physiologic substrate intermediates. Figure 3also shows that fluorocitrate did not modify the yield of 4-demethyl-deoxy DOXol aglycone, confirming that this metabolite was formed by aconitase-independent hydrolysis of DOXol.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Effects of fluorocitrate on aconitase activity and DOX formation or Fe(II) mobilization. Iron-loaded cytosols were assayed for aconitase as described in Materials and Methods, using 100 µM cis-aconitate plus or minus 10–200 µM fluorocitrate. Cytosols were also incubated with DOXol and assayed for DOX, 4-demethyl-deoxy DOXol aglycone, and low mol wt Fe(II) as described in Materials and Methods and the legend to Fig. 2, with the exception that cis-aconitate was held constant at 100 µM and added in conjunction with 10–200 µM fluorocitrate. Values are taken from a representative experiment and are expressed as percent to permit direct comparisons. In the absence of fluorocitrate, aconitase activity was 2.4 mU/mg protein, whereas 4-demethyl deoxy-DOXol aglycone, DOX, and low mol wt Fe(II) were 1.6, 1.8, and 3.4 nmol/mg protein, respectively. DOXol ag, 4-demethyl-deoxy DOXol aglycone.

Mechanisms and stoichiometry of cluster iron delocalization
cis-Aconitate and DOXol-dependent iron release was further characterized from mechanistic and stoichiometric viewpoints. A series of control experiments showed that neither cis-aconitate nor DOXol per se could release iron; a combination of cis-aconitate with DOX was similarly ineffective ( Table 1). Thus, iron release was mediated by a concerted action of cis-aconitate with the secondary alcohol moiety of DOXol. In a subsequent set of experiments, we characterized whether the simultaneous formation of DOX and low mol wt Fe(II) reflected a redox coupling of DOXol with oxygen, yielding superoxide anion (O2·-) and hydrogen peroxide (H2O2) as the ultimate mediators of iron delocalization. Duplicate determinations with 80–92% experimental agreement showed that cis-aconitate and DOXol-dependent release of 3.9 nmol Fe(II) was not appreciably affected by SOD (100 U/ml), catalase (400 U/ml) or a combination of the two enzymes, yielding 3.6, 3.8, and 4.1 nmol Fe(II)/mg protein, respectively. Redox coupling of DOXol with oxygen was therefore irrelevant to the mechanisms of iron release, which instead reflected direct reactions of DOXol with clusters. Not surprisingly, the data in Fig. 2A–B had shown that replacing iron-loaded cytosols with unmodified or iron-depleted samples was sufficient to abolish the formation of DOX, implying that DOXol oxidized with cluster iron rather than with oxygen.


View this table:
[in this window]
[in a new window]
  		Table 1. cis-Aconitate- and/or DOX(ol)-dependent mobilization of low mol wt Fe(II) in iron-loaded cytosolsa

From a stoichiometric viewpoint, we thought it was important to establish whether iron delocalization was limited to Fea or extended to Feb1–3. For this purpose, cytosols were assayed for Fea by limited cluster disassembly with ammonium persulfate (13). Using this method, we could calculate that unmodified cytosols contained 0.4 ± 0.1 nmol Fea/mg protein, which increased to 1.1 ± 0.3 in iron-loaded cytosols and decreased down to <0.2 in iron-depleted samples (n=3). Changes in Fea were therefore consistent with the procedures that had been used to promote cluster assembly or disassembly. With particular reference to iron-loaded samples, a value of ~1 nmol Fea/mg protein was opposed to the ability of cis-aconitate and DOXol to release up to ~4 nmol Fe(II) (cf Fig. 2B). This comparison strongly suggested that cis-aconitate and DOXol-dependent iron delocalization exceeded limited removal of Fea and involved Feb1–3, setting the premises for cluster disassembly and conversion of aconitase to the corresponding apoprotein/IRP-1. After this characterization, it also became evident that the oxidation of two DOXol was sufficient to mobilize the four iron centers of aconitase, as attested by the ability of 100 µM cis-aconitate to couple the formation of ~2 nmol DOX with the mobilization of ~4 nmol Fe(II) (cf Fig. 2A–B). This 1:2 stoichiometry was in keeping with an earlier suggestion that the two reducing equivalents of a secondary alcohol moiety can be returned to two independent iron ions (6).

Effects of DOXol on aconitase and IRP-1 activities
Having demonstrated that cis-aconitate coupled DOXol oxidation with cluster iron delocalization, we performed experiments to evaluate functional consequences on the enzymatic or RNA binding activities of this protein. As shown in Fig. 4A, treatment of iron-loaded cytosols with DOXol and cis-aconitate almost abolished aconitase activity, consistent with the removal of cluster iron that occurred under these conditions. Doxorubicinol and cis-aconitate per se did not decrease aconitase, nor was the enzyme activity affected by cis-aconitate plus DOX (see also Fig. 4A). This confirmed that cluster iron delocalization and consequent loss of enzyme activity reflected a concerted action of cis-aconitate with the secondary alcohol moiety of DOXol. On the other hand, cis-aconitate and DOXol could abolish aconitase activity irrespective of the presence or absence of SOD and/or catalase, as one would expect if O2·- and H2O2 were not involved in cluster disassembly ( Fig. 4B).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 4. Effects of DOXol on aconitase activity. Iron-loaded cytosols were assayed for aconitase before and after 1 h incubation with cis-aconitate and/or DOX(ol), as described in Materials and Methods. Where indicated, the incubations contained SOD (100 U/ml) and/or catalase (400 U/ml). Values are means ± SE of three determinations. cis-acon, cis-aconitate; CAT, catalase.

After these characterizations, cytosols were assessed for their ability to undergo reversible disassembly and reassembly of [4Fe-4S] clusters. For this purpose, iron loaded cytosols were treated with DTT/pH 8.9 or cis-aconitate plus DOXol to remove cluster iron and abolish aconitase activity; both samples were then reconstituted with cysteine and FAS to assist the formation of new Fe-S motifs. As shown in Fig. 5, cytosols that had been treated with DTT/pH 8.9 responded to FAS and cysteine by forming new [4Fe-4S] clusters, as evidenced by excellent recovery of aconitase activity. In contrast, cytosols treated with cis-aconitate and DOXol failed to recover enzymatic function. These comparative experiments suggested that cis-aconitate- and DOXol-dependent iron removal was accompanied by apoprotein modifications that precluded iron reincorporation and cluster reassembly.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Recovery of aconitase activity after cluster iron removal with DTT/pH 8.9 or cis-aconitate plus DOXol. Iron-loaded cytosols were assayed for aconitase activity before and after cluster iron removal by DTT/pH 8.9 (A) or cis-aconitate plus DOXol (B), as described in Materials and Methods. After these treatments, cytosols were exposed to cysteine and FAS and assayed for recovery of aconitase activity. Values are means ± SE of three determinations. cys, cysteine; FAS, ferrous ammonium sulfate; cis-acon, cis-aconitate.

Under the same conditions as described in Fig. 1, iron-loaded cytosols consistently exhibited less IRP-1 activity than did unmodified cytosols, in agreement with the inverse relationship between cluster iron saturation and RNA binding ( Fig. 6, lanes a, b). However, subsequent treatment of iron-loaded cytosols with DOXol and cis-aconitate, while releasing iron from the cluster, did not increase but actually suppressed the formation of IRE/IRP-1 complexes ( Fig. 6A, lane c). To better characterize these findings, cytosols were treated with 2-ME just prior to the gel shift assay in order to reduce disulfide bridges and regenerate -SH groups that facilitate RNA binding (19, 20). As also shown in Fig. 6A (lanes d, e), 2-ME slightly increased IRP-1 activity in both unmodified or iron-loaded cytosols; therefore, IRP-1 remained ~ 50% lower in iron-loaded cytosols. 2-Mercaptoethanol had a more visible effect on iron-loaded cytosols that had been treated with cis-aconitate and DOXol; nonetheless, 2-ME could not restore IRP-1 activity to the same levels as observed in cytosols that had not been treated with these compounds ( Fig. 6A, lane f). Collectively, these results suggested that: 1) the different RNA binding activity of unmodified and iron-loaded cytosols reflected the degree of cluster iron saturation more than the availability of -SH groups; and 2) treatment of iron-loaded cytosols with cis-aconitate and DOXol converted sulphydryl groups to disulfides, but also to more complex species that could not be reduced by 2-ME, causing the irreversible inactivation of IRP-1. Two lines of evidence confirmed that IRP-1 was inactivated by a concerted action of cis-aconitate and the alcohol moiety of DOXol. First, increasing concentrations of DOXol per se had no effect on RNA binding ( Fig. 6B). Second, neither cis-aconitate nor cis-aconitate plus DOX could modify IRP-1 activity ( Fig. 6C).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Effects of cis-aconitate and/or DOX(ol) on IRP-1 activity. A) IRP-1 was assayed in unmodified cytosols (lanes a, d), iron-loaded cytosols (lanes b, e), and cis-aconitate/DOXol-treated, iron-loaded cytosols (lanes c, f). Where indicated, samples were mixed with 0.6 % 2-ME just prior to the assay. cis-Aconitate/DOXol treatments were performed as described in Materials and Methods and the gel is representative of three similar experiments. B) Iron-loaded cytosols (1 mg protein/ml) were incubated with 0.5–5 µM purified DOXol in 0.3 M NaCl, pH 7.0, 37°C. After 1 h, aliquots were taken and assayed for IRP-1. C) Unmodified cytosols (lane a) were made iron loaded with cysteine/FAS (lane b) and subsequently treated with cis-aconitate (lane c) or cis-aconitate plus DOX (lane d). 2-ME, 2-mercapto~ethanol.

Experiments were performed to establish how cis-aconitate and DOXol failed to increase but actually suppressed IRP-1 activity, although they effectively removed iron from the cluster of aconitase. In a first set, neither SOD nor catalase or a combination of the two enzymes was found to prevent the decline of IRP-1 in iron-loaded cytosols exposed to cis-aconitate and DOXol ( Fig. 7A). This showed that O2·- and H2O2 were not involved as causative agents of IRP-1 dysfunction, in keeping with previous findings that they were similarly unable to delocalize cluster iron. In a second set, we attempted to establish whether IRP-1 was affected by the two specific products of cis-aconitate- and DOXol-dependent cluster disassembly, i.e., ~2 nmol DOX and ~4 nmol low mol wt Fe(II) (cf Fig. 2A–B). For this purpose, we prepared iron-depleted cytosols having high levels of IRP-1; these samples were then exposed to DOX, Fe(II) or a mixture of DOX with Fe(II). As reported in Fig. 7B, neither DOX nor Fe(II) affected IRP-1 activity; however, a significant decrease was observed when DOX and Fe(II) were both included. A series of parallel determinations showed that a mixture of 2 nmol DOX with 4 nmol Fe(II) changed the spectral characteristics of the drug, causing a decrease in absorbance at 477 nm and an increase at 600 nm (not shown). These spectral changes reflected the formation of DOX-Fe(II) complexes that converted to DOX-Fe(III) by oxidizing with oxygen (28). Comparative analyses against known amounts of DOX-Fe(III) also allowed us to calculate that ligand interactions of 4 nmol Fe(II) with 2 nmol DOX approached completion in ~5 min and yielded 0.9 ± 0.1 nmol Fe(III) (n=3). Within the same experimental period, unchelated Fe(II) did not appreciably oxidize with oxygen, as judged by a complete recovery after chelation with ferrozine (3.9±0.05 nmol; n=3). Taking these findings into account, we concluded that IRP-1 was targeted by DOX-Fe(II) complexes in which iron had become redox unstable. Superoxide dismutase and catalase were nonetheless unable to prevent the inactivation of IRP-1 by DOX-Fe(II), showing that drug–iron complexes acted by redox intermediates other than O2·- and H2O2 (see also Fig. 7B).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 7. Mechanisms of cis-aconitate- and DOXol-dependent IRP-1 inactivation. A) Iron-loaded cytosols were assayed for IRP-1 after 1 h incubation with cis-aconitate plus DOXol, as described in Materials and Methods. Where indicated, incubations were carried out in the presence of SOD (100 U/ml) and/or catalase (400 U/ml). B) Iron-depleted cytosols (1 mg protein/ml) were incubated with DOX (2 µM) and/or FeSO4 (4 µM), plus or minus SOD and catalase, in 0.3 M NaCl, pH 7.0, 37°C. After 1 h, aliquots were taken and assayed for IRP-1. Values are taken from a representative experiment and are expressed as percent to permit comparisons. cis-acon, cis-aconitate; CAT, catalase.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Iron is an important cofactor of several enzymes and cellular functions, varying from respiration to DNA synthesis (11). To maintain homeostasis, cells have evolved cytoplasmic aconitase/IRP-1, a bifunctional protein that modulates iron uptake vs. sequestration and regulates the concentration of metabolically available low mol wt Fe. In this study, we have shown that an anticancer drug like DOX may become cardiotoxic through the action of DOXol, a secondary alcohol metabolite that irreversibly inactivates aconitase/IRP1. Inasmuch as these processes cannot be studied in cardiac biopsies from cancer patients, we have used cytosolic fractions from myocardial samples obtained during surgery. Moreover, the experiments have been performed with concentrations of DOXol that reproduce those formed by the aldo-keto reductases of human myocardium. Results obtained under these conditions may therefore be relevant to pathologic conditions in vivo.

The aconitase reaction relies on [4Fe-4S] clusters in which Fea and Feb1 have distinct ferrous properties and Feb2–3 tends to stabilize as Fe(II)-Fe(III) dimers (29). Previous evidence for a possible reaction of DOXol with iron was obtained in cytosols that had been chromatographed on calcium hydroxyapatite or subjected to iron loading in the absence of reducing agents (6). Both of these manipulations may have caused cluster iron oxidation, forming protein–iron complexes that contained predominantly Fe(III) (6). Under those conditions, DOXol was found to reduce Fe(III) and mobilize Fe(II) by virtue of reaction mechanisms that did not require additional cofactors (6). In the present study, hydroxyapatite chromatography has been omitted and iron loading has been performed in the presence of cysteine (see Materials and Methods). These modifications have allowed us to stabilize some iron in a ferrous form attributable to the Fea of properly assembled [4Fe-4S] clusters. Under these newly defined conditions, DOXol inactivates cytoplasmic aconitase by delocalizing both Fea and Feb1–3 in a low mol wt Fe(II) form. Iron mobilization proceeds through the reoxidation of DOXol to DOX and is accompanied by irreversible modifications that preclude cluster reassembly and recovery of aconitase activity (cf Fig. 4and Fig. 5).

Although cis-aconitate is often shown to protect Fe-S motifs from chemical agents (13, 26), our studies demonstrate that it represents an absolute requirement and cofactor for DOXol oxidation and iron release to occur (cf Fig. 2A–B and Table 1). This finding is pathophysiologically relevant, as [4Fe-4S] clusters would almost certainly contain some substrate in vivo (20). To achieve some insight into the role of cis-aconitate, one must remember that the aconitase reaction accompanies sterical and redox transients, reflecting different modes of Fea ligation by citrate or isocitrate and reversible intensification of the ferrous character of Fea as opposed to the unchanged Fe(II)/Fe(III) behavior of Feb1–3 (29). cis-Aconitate is an obligatory intermediate of these processes, inasmuch as it isomerizes to both citrate and isocitrate and can bind to Fea either mode by rotating °180 (29). Sterical and redox transients at or near Fea might thus enable DOXol to introduce reducing equivalents into the cluster, explaining how four iron atoms with relatively different characteristics can eventually be delocalized in a ferrozine-chelatable form having net Fe(II) properties. In keeping with this possible mechanism, the effects of cis-aconitate on DOXol oxidation and Fe(II) release decrease remarkably when aconitase is exposed to fluorocitrate, forming by-products that bind tightly to Fea and displace the physiologic substrate intermediates otherwise involved in sterical and redox transients (cf Fig. 3and ref 27). Under the same experimental conditions, fluorocitrate would not affect drug metabolites that are formed by aconitase-independent mechanisms, e.g., 4-demethyl-deoxy DOXol aglycone (cf Fig. 3).

Experiments with unmodified vs. iron-loaded or iron-depleted cytosols indicate that cluster assembly or disassembly generates an inverse relationship between aconitase and IRP-1 in human myocardium (cf Fig. 1). This regulatory mechanism is lost when cis-aconitate and DOXol remove cluster iron, forming an apoprotein that lacks aconitase activity but also fails to function as IRP-1 (cf Fig. 6A). Under these particular conditions, the physiologic switch of aconitase to IRP-1 is precluded by oxidative modifications of cysteine residues—most notably Cys437—that mediate interactions of the apoprotein with IREs (19, 20, 26). In fact, cis-aconitate and DOXol-treated cytosols can visibly recover IRP-1 activity after exposure to 2-ME, a reducing agent that converts disulfides to -SH groups (see also Fig. 6A). Treatment with 2-ME is nonetheless insufficient to achieve a complete reactivation of IRP-1, suggesting that some oxidative modifications have proceeded beyond the formation of disulfides, yielding sulfenic, sulfinic, or sulfonic moieties that cannot be reverted to -SH by 2-ME (30). A series of reconstitution experiments indicates that IRP-1 is inactivated by the two specific products of DOXol-induced cluster disassembly, i.e., DOX and Fe(II), forming redox-active DOX–Fe(II) complexes (cf section on "Effects of DOXol on aconitase and IRP-1 activities" and Fig. 7B). Previous studies by other investigators have similarly suggested a possible role for DOX-iron complexes in protein thiol oxidation (31) and cardiotoxicity (32–34). Our present findings extend those reports, showing that cytoplasmic aconitase/IRP-1 is both a source of DOXol-releasable iron and a target of DOX–iron complexes that react in situ with critical residues of the apoprotein. Cys437 can also contribute along with Cys503 and Cys506 to the process of cluster iron coordination, conferring aconitase activity on the apoprotein (19, 20). The dual role of cysteines in RNA binding and cluster assembly would adequately explain how cis-aconitate and DOXol treatments preclude iron reincorporation and recovery of aconitase activity (cf Fig. 5).

The observation that DOXol can irreversibly inactivate IRP-1 anticipates several pathologic consequences, the most obvious being a disturbance in the regulation of transferrin iron uptake vs. ferritin iron sequestration. Cardiomyocytes would thus become unable to sense iron levels and coordinate iron movements between the different sites of deposition or metabolic use. In addition to these changes, there may be other and more subtle consequences of DOXol-cluster interactions. For example, the simultaneous loss of aconitase activity should increase the cytosolic levels of citrate, altering the putative role of this compound as a chelator and transporter of intracellular iron (35). Recent studies have also shown that IRP-1 can bind to IREs in mRNA for mitochondrial aconitase, decreasing the synthesis of this enzyme (36, 37). The biologic significance of this regulatory connection is unknown, but these findings reinforce the idea that a reversible switch between aconitase and IRP-1 modulates several homeostatic processes. Interrupting this switch with DOXol should predictably expose cardiomyocytes to a multifactorial damage, especially if one appreciates how sensitive these cells can be to anomalous changes in iron and energy metabolism (38). De novo synthesis of IRP-1 would probably fail to compensate for these disorders, inasmuch as the apoprotein is itself a target of DOX-iron complexes. Similar reasonings might perhaps be applied to IRP-2, which is also redox-regulatable at critical -SH residues (25, 26).

The mechanisms described above must be contrasted with other prevailing hypotheses of cardiotoxicity, mostly involving a quinone group placed in the tetracycline ring of DOX (cf Scheme I). Several studies have shown that this moiety is liable to one-electron addition by NAD(P)H oxidoreductases of intracellular organelles, yielding a semiquinone free radical that readily regenerates the parent quinone by reducing oxygen to O2·- and H2O2 (3, 4). During this redox cycling, O2·- can reductively release some iron from ferritin (39, 40). Inasmuch as cardiomyocytes are relatively deficient in oxyradical-detoxifying enzymes such as SOD, catalase, or glutathione peroxidase, one-electron redox cycling of DOX is often said to facilitate intracardiac accumulation and reaction of O2·- and H2O2 with delocalized Fe(II), forming hydroxyl radicals or iron–oxygen complexes that eventually promote lipid peroxidation (3, 4). Attempts to validate this biochemical hypothesis in human subjects have produced conflicting results. We have recently demonstrated that DOX infusions do not increase but actually suppress cardiac lipid peroxidation in cancer patients, as evidenced by a decrease of lipid-conjugated dienes and hydroperoxides in blood samples collected from the coronary sinus (7). These findings cast doubt on the importance of lipid peroxidation and explain other negative reports showing that antioxidants like {alpha}-tocopherol or N-acetylcysteine would not protect cancer patients from DOX toxicity (41, 42). A significant degree of protection has nonetheless been observed with dexrazoxane, a bis-keto-piperazinedione that hydrolyzes intracellularly and liberates a diacid diamide that chelates iron (1, 2, 33, 34). Keeping this in mind, one cannot escape the conclusion that dexrazoxane mitigates cardiotoxic events other than iron-dependent lipid damage. The experiments described in our present study may help reconcile these inconsistencies. For example, dexrazoxane might reequilibrate iron between ferritin, enzymes, or putative transporters like citrate, thereby relieving the disturbances otherwise associated with the inactivation of aconitase/IRP-1 and consequent changes in iron uptake vs. sequestration or metabolic use. Alternatively, dexrazoxane might compete with DOX for iron and prevent formation of those drug–iron complexes that interrupt the physiologic interconversion between aconitase and IRP-1. In either case, dexrazoxane would prevent homeostatic disorders rather than lipid peroxidation or equivalent free radical reactions, making it possible to rationalize the protective effects of chelation therapy as opposed to unsuccessful interventions with antioxidants.

A role for O2·- and H2O2 might be maintained if these species mediate the deleterious effects of DOXol on aconitase/IRP-1. Our studies indicate that this is not the case. In fact, neither SOD nor catalase or a combination of the two enzymes can prevent DOXol-dependent cluster disassembly, in keeping with the earlier suggestion that DOXol interacts with iron by direct electron transfer mechanisms (see section on "Mechanisms and stoichiometry of cluster iron delocalization" and ref 6). Likewise, SOD and catalase cannot prevent the bifunctional inactivation of IRP-1 and aconitase, perhaps because the oxidation of DOX-Fe(II) with oxygen generates Fe(II)/Fe(III) complexes that modify biologic targets by O2·- and H2O2-independent mechanisms (cf refs 28, 39 and Fig. 4B, Fig. 7B). Critical appraisal of the literature also suggests that O2·- and H2O2 have multiple and often diverging effects on Fe-S clusters and aconitase/IRP-1; however, neither species would impose the same bifunctional and irreversible inactivation as imposed by DOXol (43–48).

In conclusion, our studies demonstrate that the interaction of DOXol with cytoplasmic aconitase/IRP-1 encircles various aspects of DOX toxicity and provides a framework to optimize cardioprotection with dexrazoxane or other newly designed chelators. The unusual reactivity of DOXol with aconitase/IRP-1 vis a vis its marginal contribution to the tumoricidal effects of DOX (49) also implies there may be additional means to decrease cardiotoxicity without affecting anticancer activity. Potential strategies include the development of drugs that inhibit aldo-keto reductases or the identification of DOX analogs that form fewer alcohol metabolites.



View larger version (14K):
[in this window]
[in a new window]
  		Figure . Structures of DOX and DOXol.


   ACKNOWLEDGMENTS
 
This work was supported by grants from MURST (to G.M., Special Project ex 40% "New assessment approaches in toxicology," 1996) and from Consiglio Nazionale delle Ricerche.


   FOOTNOTES
 
1 Correspondence: Department of Pharmacology, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome, Italy. E-mail: ibifa{at}rm.unicatt.it

2 Abbreviations: DOX, doxorubicin: DOXol, doxorubicinol; IRP-1, iron regulatory protein-1; IREs, iron-responsive elements; low mol wt, low molecular weight; FAS, ferrous ammonium sulfate; DTT, dithiothreitol; 2-ME, 2-mercaptoethanol; O2·-, superoxide anion; H2O2, hydrogen peroxide; SOD, superoxide dismutase.

Received for publication November 17, 1997. Accepted for publication December 22, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Weiss, R. B. (1992) The anthracyclines: will we ever find a better doxorubicin? Sem. Oncol. 19, 670–686[Medline]
  2. Dorr, R. T. (1996) Cytoprotective agents for anthracyclines. Sem. Oncol. 23 (Suppl. 8), 23–34[Medline]
  3. Olson, R. D., and Mushlin, P. S. (1990) Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J. 4, 3076–3086[Abstract]
  4. Powis, G. (1989) Free radical formation by antitumor quinones. Free Rad. Biol. Med. 6, 63–101[Medline]
  5. Nies, A.S., and Spielberg, S.P. (1996) Principles of therapeutics. In Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th Ed (Hardman, J. G., Limbird, L. E., Molinoff, P. B., and Ruddon, R. W., eds) pp. 43–62, McGraw-Hill Co., New York
  6. Minotti, G., Cavaliere, A. F., Mordente, A., Rossi, M., Schiavello, R., Zamparelli, R., and Possati, G. F. (1995) Secondary alcohol metabolites mediate iron delocalization in cytosolic fractions of myocardial biopsies exposed to anticancer anthracyclines. J. Clin. Invest. 95, 1595–1605
  7. Minotti, G., Mancuso, C. Frustaci, A., Mordente, A., Santini, S. A., Calafiore, A. M., Liberi, G., and Gentiloni, N. (1996) Paradoxical inhibition of cardiac lipid peroxidation in cancer patients treated with doxorubicin. J. Clin. Invest. 98, 650–661[Medline]
  8. Beinert, H., and Kennedy, M. C. (1993) Aconitase, a two-faced protein: enzyme and iron regulatory factor. FASEB J. 7, 1442–1449[Abstract]
  9. Klausner, R. D., Rouault, T. A., and Harford, J. B. (1993) Regulating the fate of mRNA: the control of cellular iron. Cell 72, 19–28[Medline]
  10. Harrison, P. M., and Arosio, P. (1996) The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1275, 161–203[Medline]
  11. Hentze, M. W., and Kuhn, L. C. (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc. Natl. Acad. Sci. USA 93, 8175–8182[Abstract/Free Full Text]
  12. Miller, D. M., Buettner, G. R., and Aust, S. D. (1990) Transition metals as catalysts of 'autoxidation' reactions. Free Rad. Biol. Med. 8, 95–108[Medline]
  13. Kennedy, M. C., Emptage, M. H., Dreyer, J.-L., and Beinert, H. (1979) The role of iron in the activation-inactivation of aconitase. J. Biol. Chem. 258, 11098–11105[Abstract/Free Full Text]
  14. Constable, A., Quick, S., Gray, N. K., and Hentze, M. W. (1992) Modulation of the RNA-binding activity of a regulatory protein by iron in vitro: switching between enzymatic and genetic function? Proc. Natl. Acad. Sci. USA 89, 4554–4558[Abstract/Free Full Text]
  15. Haile, D. J., Rouault, T. A., Tang, C. K., Chin, J., Harford J., and Klausner, R. D. (1992) Reciprocal control of RNA-binding and aconitase activity in the regulation of the iron-responsive element binding protein: Role of the iron-sulfur cluster. Proc. Natl. Acad. Sci. USA 89, 7536–7540[Abstract/Free Full Text]
  16. Mullner, E. W., Neupert, B., and Kuhn, L. C. (1989) A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell 58, 373–382[Medline]
  17. Cairo, G., and Pietrangelo, A. (1994) Transferrin receptor gene expression during rat liver regeneration. Evidence for post-transcriptional regulation by iron regulatory factor B, a second iron-responsive element binding protein. J. Biol. Chem. 269, 6405–6409[Abstract/Free Full Text]
  18. Haile, D. J., Rouault, T. A., Harford, J. B., Kennedy, M. C., Blondin, G. A., Beinert, H., and Klausner, R. D. (1992) Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc. Natl. Acad. Sci. USA 89, 11735–11739[Abstract/Free Full Text]
  19. Philpott, C. C., Haile, D., Rouault, T. A., and Klausner, R. D. (1993) Modification of free Fe-S cluster cysteine residue in the active iron-responsive element-binding protein prevents RNA binding. J. Biol. Chem. 268, 17655–17658[Abstract/Free Full Text]
  20. Hirling, H., Henderson, B. R., and Kuhn, L. C. (1994) Mutational analysis of the [4Fe-4S] cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase. EMBO J. 13, 453–461[Medline]
  21. Drapier, J. C., and Hibbs, J. B., Jr. (1986) Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. J. Clin. Invest. 78, 790–797
  22. Takanashi, S., and Bachur, N. R. (1976) Adriamycin metabolism in man. Evidence for urinary metabolism. Drug. Metab. Dispos. Biol. Fate Chem. 4, 79–87[Abstract]
  23. Aust, S. D., Miller, D. M., and Samokyszin, V. M. (1990) Iron redox reactions in lipid peroxidation. Methods Enzymol. 186, 457–463[Medline]
  24. Stoscheck, C. M. (1990) Quantitation of protein. Methods Enzymol. 182, 50–68[Medline]
  25. Phillips, J. D., Guo, B., Yu, Y., Brown, F. M., and Leibold, E. A. (1996) Expression and biochemical characterization of iron regulatory proteins 1 and 2 in Saccharomices cerevisiae. Biochemistry 35, 15704–15714[Medline]
  26. Bouton, C., Hirling, H., and Drapier, J.-C. (1997) Redox modulation of iron regulatory proteins by peroxynitrite. J. Biol. Chem. 272, 19969–19975[Abstract/Free Full Text]
  27. Lauble, H., Kennedy, M. C., Emptage, M. H., Beinert, H., and Stout, D. C. (1996) The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proc. Natl. Acad. Sci. USA 93, 13699–13705[Abstract/Free Full Text]
  28. Minotti, G. (1990) NADPH and adriamycin dependent microsomal release of iron and lipid peroxidation. Arch. Biochem. Biophys. 268, 398–403
  29. Beinert, H., and Kennedy, M. C. (1989) Engineering of protein-bound iron sulfur clusters. Eur. J. Biochem. 189, 5–15
  30. Torchinsky, Y. M. (1981) Sulfur in Proteins, pp. 48–56, Pergamon Press, New York
  31. Vile, G., and Winterbourn, C. C. (1990) Thiol oxidation and inhibition of Ca-ATPase by adriamycin in rabbit heart microsomes. Biochem. Pharmacol. 39, 769–774[Medline]
  32. Gianni, L., Corden, B. J., and Myers, C. E. (1983) The biochemical basis of anthracycline toxicity and antitumor activity. In Reviews in Biochemical Toxicology (Hodgson, E., Bend, J., and Philpot, P. M., eds) pp. 1–82, Elsevier-North Holland, New York
  33. Doroshow, J. H. (1991) Doxorubicin-induced cardiotoxicity. New Engl. J. Med. 324, 843–845[Medline]
  34. Buss, J. L., and Hasinoff, B. B. (1993) The one-ring open hydrolysis product intermediates of the cardioprotective agent ICRF-187 (dexrazoxane) displace iron from iron-anthracyclines complexes. Agents Actions 40, 86–95[Medline]
  35. Paraskeva, E., and Hentze, M. W. (1996) Iron-sulfur clusters as genetic regulatory switches: The bifunctional iron regulatory protein-1. FEBS Lett. 389, 401–403
  36. Gray, N. K., Pantopoulos, K., Dandekar, T., Ackrell, B. A. C., and Hentze M. W. (1996) Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron responsive elements. Proc. Nal. Acad. Sci. USA 93, 4925–4930[Abstract/Free Full Text]
  37. Kim, H.-Y., La Vaute, T., Iwai, K., Klausner, R. D., and Rouault, T. A. (1996) Identification of a conserved and functional iron-responsive element in the 5'-untranslated region of mammalian mitochondrial aconitase. J. Biol. Chem. 271, 24226–24230[Abstract/Free Full Text]
  38. Schoen, F. J. (1994) The heart. In Robbins Pathologic Basis of Disease, 5th Ed (Cotran, R. S., Kumar, V., and Robbins S. L., eds) pp. 517–582, W. B. Saunders Co., Philadelphia
  39. Minotti, G. (1993) Sources and role of iron in lipid peroxidation. Chem. Res. Toxicol. 6, 134–146[Medline]
  40. Ryan, T. P., and Aust, S. D. (1992) The role of iron in oxygen-mediated toxicities. Crit. Rev. Toxicol. 22, 119–141[Medline]
  41. Weitzman, S. A., Lorell, B., Carey R. W., Kaufman, S., and Stossel, T. P. (1980) Prospective studies of tocopherol prophylaxis for anthracycline cardiac toxicity. Curr. Therap. Res. 28, 682–686
  42. Myers, C., Bonow, R., Palmieri, S., Jenkins, J., Corden, B., Locker, G., Doroshow, J., and Epstein, S. A. (1983) A randomized controlled trial assessing the prevention of doxorubicin cardiomyopathy by N-acetylcysteine. Sem. Oncol. 10 (Suppl.), 53–55[Medline]
  43. Gardner, P. R., Ranieri, I., Epstein, L. B., and White C. R. (1995) Superoxide radical and iron modulate aconitase activity in mammalian cells. J. Biol. Chem. 270, 13339–13405
  44. Pantopoulos, K., Mueller, S., Atzberger, A., Ansorge, W., Stremmel, W., and Hentze, M. W. (1997) Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intracellular oxidative stress. J. Biol. Chem. 272, 9802–9808[Abstract/Free Full Text]
  45. Rouault, T. A., and Klausner, R. D. (1996) Iron-sulfur clusters as biosensors of oxidants and iron. Trends Biochem. Sci. 21, 174–177[Medline]
  46. Hentze, M. W. (1996) Iron-sulfur clusters and oxidant stress responses. Trends Biochem. Sci. 21, 282–283[Medline]
  47. Pantopoulos, K., and Hentze, M. W. (1995) Rapid response to oxidative stress mediated by iron regulatory protein. EMBO J. 14, 2917–2924[Medline]
  48. Cairo, G., Castrusini, E., Minotti, G., and Bernelli-Zazzera, A. (1996) Superoxide and hydrogen peroxide-dependent inhibition of iron regulatory protein activity: a protective stratagem against oxidative injury. FASEB J. 10, 1326–1335[Abstract]
  49. Kuffel, M. J., Reid, J. M., and Ames, M. M. (1992) Anthracyclines and their C-13 alcohol metabolites: growth inhibition and DNA damage following incubation with tumor cell lines. Cancer Chemother. Pharmacol. 30, 51–57[Medline]



This article has been cited by other articles:


Home page
 Mol. Interv.Home page
Y. Chen, P. Jungsuwadee, M. Vore, D. A. Butterfield, and D. K. St. Clair
Collateral Damage in Cancer Chemotherapy: Oxidative Stress in Nontargeted Tissues
Mol. Interv., June 1, 2007; 7(3): 147 - 156.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Salvatorelli, S. Guarnieri, P. Menna, G. Liberi, A. M. Calafiore, M. A. Mariggio, A. Mordente, L. Gianni, and G. Minotti
Defective One- or Two-electron Reduction of the Anticancer Anthracycline Epirubicin in Human Heart: RELATIVE IMPORTANCE OF VESICULAR SEQUESTRATION AND IMPAIRED EFFICIENCY OF ELECTRON ADDITION
J. Biol. Chem., April 21, 2006; 281(16): 10990 - 11001.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
X. Xu, H. L. Persson, and D. R. Richardson
Molecular Pharmacology of the Interaction of Anthracyclines with Iron
Mol. Pharmacol., August 1, 2005; 68(2): 261 - 271.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, and L. Gianni
Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity
Pharmacol. Rev., June 1, 2004; 56(2): 185 - 229.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Corna, P. Santambrogio, G. Minotti, and G. Cairo
Doxorubicin Paradoxically Protects Cardiomyocytes against Iron-mediated Toxicity: ROLE OF REACTIVE OXYGEN SPECIES AND FERRITIN
J. Biol. Chem., April 2, 2004; 279(14): 13738 - 13745.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
S. FOGLI, P. NIERI, and M. C. BRESCHI
The role of nitric oxide in anthracycline toxi