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Reviews |
Departments of
a Pharmacology and Pharmacognosy, G. D'Annunzio University School of Pharmacy, Chieti;
b General Pathology, University of Milan School of Medicine, Milan; and
c Structural and Functional Biology, University of Insubria School of Sciences, Varese, Italy
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ABSTRACT |
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 TOP ABSTRACT BACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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Key Words: mechanisms • cardioprotection • metallothionein • DOX
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BACKGROUND |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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Accordingto the prevailing hypothesis, the cardiotoxicity of anthracyclines is mediated by mechanisms that are distinct from those underlying the antitumor effects of these drugs, i.e., DNA intercalation and interference with the catalytic cycle of DNA topoisomerase II (2) . A major role in the development of cardiotoxicity has been assigned to iron, presumably because this metal can catalyze free radical reactions that overrule the antioxidant defenses of cardiomyocytes. Elegant reviews and editorials in this 3-5) and other journals (2 , 6-8 ) have contributed to the popularity of such hypothesis; however, there have also been reports showing that the `iron and free radical hypothesis' of cardiotoxicity does not withstand scrutiny according to some biochemical or pharmacological criteria (4 , 9 , 10 ). Hence, some investigators have proposed mechanisms of cardiotoxicity that are independent altogether of both iron and free radicals (4) . In an attempt to bridge the two extremes of this field, other studies have maintained a role for iron but not for free radicals, suggesting that anthracycline cardiotoxicity reflects disturbances in iron homeostasis within cardiomyocytes rather than the outcome of iron-catalyzed free radical injury (11 , 12 ). The aim of this review is to weigh biochemical and pharmacological arguments for and against the aforementioned hypotheses.
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DOES IRON MEDIATE ANTHRACYCLINE CARDIOTOXICITY? STUDIES WITH CHELATORS SEEM TO SAY YES |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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On the whole, one-electron redox cycling of anthracyclines increases
the intracellular levels of both low mol wt Fe(II) and
H2O2, setting the stage for the formation of
more damaging species. On the one hand, low mol wt Fe(II) can decompose
H2O2 into highly reactive hydroxyl radicals
( · OH) [Fe(II) + H2O2 
Fe(III) +
OH- + · OH]; on the other hand, Fe(II) itself can
be converted by H2O2 into ferryl
ions (FeIV=O) having the same reactivity as · OH
[Fe(II) + H2O2 FeIV=O +
H2O] (16)
. Both · OH and
FeIV=O can eventually cause cardiac dysfunction by
oxidizing lipids, proteins, and DNA, perhaps in concert with the redox
activity of DOX–iron complexes (Fig. 1
).
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In most cells, the chances for Fe(II) to form · OH or FeIV=O are kept to a minimum by the presence of detoxifying enzymes that concur in reducing O2·- and H2O2 to water (SOD, catalase, and glutathione peroxidase). Consistent with the `iron and free radical hypothesis', cardiomyocytes are ill equipped with these enzymes and thus may provide the ideal scenario for the formation of · OH or FeIV=O (5 , 6 ). Moreover, the earliest ultrastructural changes induced by DOX involve mitochondria and sarcoplasmic reticulum, as would be expected based on the presence in these organelles of NADH dehydrogenase or NADPH-cytochrome P450 reductase, respectively, which reduce DOX to semiquinone free radical (4 , 6 ). Dexrazoxane can protect against oxidative damage by entering cardiomyocytes and by undergoing stepwise hydrolysis of the two piperazine rings. This process releases one- and two- open ring by-products that chelate free iron or divert it from potentially toxic interactions with DOX (19 , 20 ). Initial studies in laboratory animals showed that dexrazoxane had unique pharmacokinetic, pharmacodynamic, and toxicologic properties. First, it did not interfere with anthracycline distribution, metabolism, or excretion (21) , nor did it appear to mitigate the antitumor potency of anthracyclines (22) . Second, it imposed relatively limited damage on mitotically active tissues such as bone marrow, testes, and gastrointestinal epithelia (23) . Finally, it protected against both acute and chronic cardiotoxicity in all the animal models tested, ranging from mice to dogs or swine (8) . These findings were largely confirmed in clinical trials showing that dexrazoxane could decrease the incidence or severity of anthracycline cardiotoxicity without affecting objective tumor response (24) . At longer follow-up, dexrazoxane also allowed patients to receive significantly greater cumulative doses of DOX without increasing its cardiotoxicity, the only drawback being a moderate to severe exacerbation of myelosuppression (25) . However, more recent studies have raised the possibility that dexrazoxane decreases tumor response in breast cancer patients treated with DOX (26) . These clinical findings appear to validate in vitro studies showing that dexrazoxane antagonizes the apoptotic and antiproliferative effects of DOX (27 , 28 ), presumably by interfering with anthracycline–topoisomerase II interactions (29) . Such interference is mediated by intact dexrazoxane but not by its hydrolysis products, indicating that a mechanism(s) other than iron chelation and prevention of free radical events may be involved (30) .
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DOES IRON MEDIATE ANTHRACYCLINE CARDIOTOXICITY BY CATALYZING FREE RADICAL REACTIONS? STUDIES WITH ANTIOXIDANTS YIELD INCONCLUSIVE EVIDENCE |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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One additional strategy to probe and possibly counteract the role of
free radicals in small animal models of anthracycline cardiotoxicity
has relied on the use of low mol wt spin trapping agents such as
N-tert-butyl-
-phenylnitrone (PBN),
-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and
5,5-dimethyl-1-pyrroline N-oxide (DMPO). The rationale for using these
compounds has been twofold. The ability of spin traps to form stable
adducts with free radicals in intact tissues or cell-free systems, thus
allowing their detection by electron paramagnetic spectroscopy,
strongly suggested that they might prevent anthracycline cardiotoxicity
if free radicals were involved in the process. On the other hand, spin
traps can intercept both oxygen- and carbon-centered radicals, and thus
may prove to be more versatile and effective protectants than other
antioxidants. As expected from these biochemical premises, spin traps
were found to protect atria and whole heart preparations from
DOX-induced mechanical dysfunction (33)
. The degree of
protection achieved has been found to correlate with the lipophilicity
of these compounds, being highest with PBN, followed by POBN and DMPO
(CHCl3/H2O partition coefficients at 24°C:
199.0, 1.19, and 0.13, respectively) (34)
. Comparative
studies of DMPO vs. PBN have also shown that the former distributes
almost exclusively within the cytosolic compartment, whereas PBN can
also be found in mitochondria. The higher efficacy of PBN, subsequently
confirmed by in vivo studies (35)
, could thus
be explained by its ability to achieve substantial levels at the very
sites where DOX bioactivation initiates a cascade of free radical
reactions.
Research groups have recently readdressed the role of free radicals by evaluating the in vivo cardiotoxicity of DOX in transgenic mice overexpressing antioxidant defense systems. Such studies focused mainly on myocardial overexpression of catalase (36) and mitochondrial MnSOD (37) or the thiol-rich nonenzymatic protein metallothionein (MT), which has been shown to effectively scavenge · OH and to act as an iron chelator in vitro (38, and references therein). The transgenic mouse model was designed to elude the shortcomings due to poor cell penetration of exogenously administered proteins and to guarantee high catalase, MnSOD, and MT activity inside the cardiomyocytes. All three approaches proved successful in reducing acute myocardial damage by DOX, as assessed by ultrastructural lesions, intracellular enzyme release, and functional impairment. Although the transgenic approach may be impractical for clinical purposes, it clearly indicates that stable increases in intramyocardial levels of antioxidant proteins may effectively protect the heart from DOX, at least in small animal models of anthracycline cardiotoxicity.
Whereas the effects of antioxidants in rats and mice or in in vitro models of cardiotoxicity have lent credibility to the free radical hypothesis, similar studies in large animals have produced contrasting notions, especially when chronic anthracycline regimens were adopted. Although not all of the cardioprotective strategies outlined above have been tested in large animal models of anthracycline cardiotoxicity, carefully designed experiments with N-acetylcysteine or vitamin E have shown that neither compound would prevent or significantly reduce cardiac lesions induced by chronic treatment of dogs with DOX (23 , 39 ). Similar negative results have been obtained in clinical trials in which patients were given vitamin E (40) or N-acetylcysteine (41) prior to and/or concomitant with DOX. In none of these trials could antioxidants prevent cardiac dysfunction associated with multiple doses of the anthracycline. In the case of vitamin E, an intensified regimen was started as early as a week prior to DOX treatment, increasing the serum levels of this antioxidant by six- to eightfold. Such vigorous and pharmacokinetically optimized treatment was nonetheless insufficient to prevent heart failure and ultrastructural changes at endomyocardial biopsy (40) . To better appraise these negative findings, one should keep in mind that lesser increases in vitamin E plasma levels are sufficient to prevent or decrease the incidence of other cardiac events that presumably involve oxidative stress, such as nonfatal myocardial infarction in patients with coronary atherosclerosis (42) , arrhythmias and reinfarction after aortocoronary bypass (43) , and fatal infarction in smokers (44) .
In summary, chelating iron with dexrazoxane mitigates both acute and chronic cardiotoxicity, and such protection can be seen in patients as well as in all the animal models tested. In contrast, antioxidant interventions afford protection in small animals, but not in patients or large animals at risk for chronic cardiomyopathy. This discrepancy raises a potentially critical issue that needs to be addressed from mechanistic and pathophysiologic viewpoints.
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DO ANTHRACYCLINES GENERATE TOO MANY RADICALS FOR THEM TO MEDIATE CARDIOTOXICITY? |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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H2O + O2·- + H+]
(47)
. Substantial amounts of · OH could thus be
diverted from reacting with lipids or other targets. One last
possibility is that oxidative damage proceeds through reactive species
that are different from · OH and cannot be formed when
anthracyclines generate a stoichiometric excess of
H2O2 over Fe(II). In particular, early studies
by Aust and co-workers (48
, 49
), and later
observations by several other investigators (reviewed in ref 16
), have
suggested that oxidative damage could be mediated by oxygen-bridged
Fe(II)-Fe(III) complexes or equally reactive perferryl ions
[Fe(II)O2/Fe(III)O2·-]. Both these
species are formed when some Fe(II) oxidizes with oxygen or
less than stoichiometric amounts of H2O2,
yieldingFe(II):Fe(III) ratios that impart unusual reactivity to
these iron–oxygen complexes (16)
. Neither complex would
therefore be formed when semiquinone free radicals generate
H2O2 in excess of Fe(II), causing the oxidation
of too much Fe(II) and shifting the Fe(II)/Fe(III) ratio
toward the unreactive Fe(III) (16)
. Studies in microsomal
systems (50)
or cell cultures (51)
exposed to
DOX suggest that this may be the case. In these systems, lipid
peroxidation and other indices of damage were significantly enhanced by
addition of catalase or by genetic manipulations leading to catalase
overexpression, respectively, as would be expected if excess
H2O2 were negating moderate oxidation of Fe(II)
with oxygen and consequent formation of optimal Fe(II)/Fe(III) ratios.
Keeping these mechanisms in mind, one might conclude that antioxidants
cannot protect against cardiotoxicity for the very reason that
anthracyclines possibly prevent, rather than induce, oxidative damage.
Studies in isolated heart models or in whole animals (reviewed in ref 4
) appear to validate these conclusions, showing that cardiotoxicity
may sometimes develop in the absence of lipid peroxidation or other
indices of oxidative damage. Such a possibility is more difficult to
prove in patients, as both ethical and practical constraints preclude
biochemical measurements of oxidative stress in fine needle myocardial
biopsies. To overcome these problems, cardiac lipid peroxidation has
been assessed by collecting blood samples from the coronary sinus and
by measuring the plasma levels of lipid conjugated dienes and
hydroperoxides before and after DOX infusions. These studies have shown
that DOX does not increase but actually decreases the myocardial
release of conjugated dienes and hydroperoxides, apparently confirming
that a free radical-generating drug can paradoxically abolish oxidative
damage (10)
. These new findings call for a critical reassessment of previous studies supporting the oxidative nature of anthracycline cardiotoxicity, especially regarding the transgenic mice models. For instance, one would expect overexpression of MnSOD to increase the dismutation of O2·- without significantly affecting iron delocalization by semiquinone free radicals, thus increasing the availability of H2O2 for reaction with Fe(II) and formation of · OH or FeIV=O. Nevertheless, cardiac damage is halted by MnSOD overexpression (37) , suggesting that 1) neither · OH nor FeIV=O contribute to toxicity, and 2) tissue damage probably is mediated by iron–oxygen complexes, which become redox-inactive when excess dismutation of O2·- to H2O2 shifts Fe(II)/Fe(III) ratios toward unreactive Fe(III). Similar considerations can be extended to the catalase studies. In fact, cardiac lipid peroxidation was suppressed at catalase expression levels approaching values 100-fold over the wild-type; however, higher levels of catalase expression did not prevent, but actually enhanced, lipid peroxidation (36) . While indicating that catalase overexpression provides a very narrow window of protection, these studies indirectly confirm that free radical injury is promoted when Fe(II) is `protected' from excessive oxidation by H2O2, which presumably occurs at very high levels of enzyme expression. On balance, the results obtained with SOD- or catalase-overexpressing mice are open to debate and do not provide unequivocal evidence that anthracycline cardiotoxicity is indeed mediated by oxidative processes.
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IS ANTHRACYCLINE CARDIOTOXICITY INDEPENDENT OF IRON AND FREE RADICALS? |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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Alternative mechanisms of cardiotoxicity have implicated DOX metabolites other than semiquinone free radicals. This is the case for doxorubicinol (DOXol), a hydroxy metabolite formed upon two-electron reduction of the C-13 carbonyl group in the side chain of DOX:
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DOXol is formed by cytosolic NADPH-dependent enzymes sharing similarities with the multigene family of carbonyl and aldo-keto reductases (58) . From a pharmacokinetic standpoint, DOXol accumulates in the heart of rats receiving multiple DOX injections; such accumulation reflects intramyocardial DOX metabolism rather than metabolite uptake from the bloodstream (4) . This has suggested that the delayed cardiotoxicity induced by chronic treatment with DOX in laboratory animals may reflect a unique tendency of cardiomyocytes to form and retain DOXol. This also seems to occur in DOX-treated patients, as limited studies in postmortem tissues have demonstrated that DOXol can be detected within cardiomyocytes well after completion of anthracycline treatments (59) . Experiments in ex vivo samples, obtained from patients undergoing aortocoronary bypass grafting, have confirmed that human cardiomyocytes can reduce DOX to DOXol by virtue of cytosolic aldo-keto reductases (11) . From a pharmacodynamic viewpoint, the C-13 secondary alcohol moiety of DOXol is redox-inactive toward oxygen and thus is unable to initiate a free radical cascade. Nonetheless, DOXol can potently and directly inhibit the Ca2+-Mg2+ ATPase of sarcoplasmic reticulum, the f0-f1 proton pump of mitochondria, and the Na+-K+ ATPase and Na+-Ca2+ exchanger of sarcolemma (60 , 61 ). DOXol can therefore affect myocardial energy metabolism, ionic gradients, and Ca2+ movements, ultimately impairing cardiac contraction and relaxation.
By rationalizing anthracycline cardiotoxicity as a free radical-independent disease, the two mechanisms described above may explain why antioxidants cannot protect patients or large animals. At the same time, these mechanisms are difficult to reconcile with the protective efficacy of dexrazoxane (8) or with the ability of iron to potentiate anthracycline toxicity in cultured cardiomyocytes (62) , unless we assume that iron mediates DOX disruption of gene expression or DOXol inhibition of key enzymes. Inasmuch as these two possibilities remain unverified at the present time, one can only conclude that anthracycline cardiotoxicity is a multifactorial process involving both iron-independent and iron-mediated processes. It would also appear that the role of iron cannot be confined to the promotion of free radical events, as not only dexrazoxane, but also antioxidants would protect patients or large animals if this were the case. To better appraise the role of iron, one should probably address the following questions. Can anthracyclines perturb the mechanisms whereby iron is handled as an essential cofactor rather than as a free radical catalyst? Further, can dexrazoxane antagonize the effect of DOX on iron homeostasis and related cell functions?
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THE IRP-IRE MACHINERY: A KEY MECHANISM OF IRON HOMEOSTASIS AND A NOVEL TARGET OF ANTHRACYCLINE CARDIOTOXICITY |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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Definition of the structure of IRP-1 provided a clue to understanding how this protein can sense iron levels. IRP-1 is the cytosolic counterpart of mitochondrial aconitase, the enzyme that isomerizes citrate into isocitrate in the Krebs cycle by virtue of a catalytic [4Fe-4S] cluster (70) . When cells are iron depleted, the [4Fe-4S] cluster is disassembled and the cytosolic aconitase switches to IRP-1; when cells are iron repleted, the cluster is reconstituted and IRP-1 switches back to aconitase 66-68) (Fig. 2 ).The -SH groups of several cysteine residues have been implicated in RNA binding or cluster iron coordination, but only Cys437 appears to participate in both processes (71) . Anthracyclines have the potential to disrupt this simple but highly efficient mechanism of iron homeostasis. In fact, recent studies from our laboratories have shown that reconstitution of DOXol with cytosolic fractions from human myocardial samples causes delocalization of low mol weight Fe(II) from the [4Fe-4S] cluster of aconitase (12) . This reaction proceeds via redox mechanisms, which regenerate DOX and form DOX-Fe(II) complexes as ultimate by-products; the latter can, in turn, inhibit IRP-1 and prevent its physiologic switch to aconitase (12) . Sulfydryl reductants like ß-mercaptoethanol cannot rescue either IRP-1 or aconitase activities, and this implies that DOX–Fe(II) complexes can irreversibly modify Cys437 to sulfinic/sulfonic derivatives that cannot be converted back to -SH (12) . Although in vitro (68 , 72 ) and in vivo (73) studies have implicated O2·- and H2O2 as pathophysiologic modulators of aconitase/IRP-1, neither SOD nor catalase appear to protect this protein from the action of DOXol (12) . These findings confirm that C-13 metabolites can cause cardiac damage by virtue of oxyradical-independent mechanisms.
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Besides IRP-1, cells may contain an IRP-2 with similar binding affinity and specificity to consensus IREs (68 , 74 ). However, IRP-2 cannot form [4Fe-4S] clusters and hence cannot sense iron levels by switching between aconitase and IRE binding activities, as does IRP-1. The mechanism whereby IRP-2 contributes to iron homeostasis is regulated by its ubiquitination and proteasome-mediated degradation (75) . When iron is abundant, IRP-2 is degraded; when iron is scarce, IRP-2 is protected from degradation and can bind to IREs, leading to up-regulation or down-regulation of TfR or ferritin mRNA translation, respectively. A unique 73 amino acid sequence serves as the degradation domain of IRP-2 (75) . Currently available techniques do not always allow the separation of IRP-1 from IRP-2 in human samples; hence, we do not know whether DOXol inactivation of aconitase/IRP-1 is accompanied by a simultaneous loss of IRP-2. However, binding of IRP-2 to mRNAs is mediated by cysteine residues that are particularly susceptible to chemical modifications, similar to the cysteines of IRP-1 (76) . It is therefore conceivable that the DOX–Fe(II) complexes that inactivate IRP-1 can do the same with IRP-2.
What are the pathologic consequences of disrupting the IRP/IRE
machinery? Broadly speaking, one can expect that cardiomyocytes become
unable to sense iron levels and to coordinate iron trafficking between
transferrin receptor, ferritin, and the different sites of iron
metabolic use. Disruption of the IRP/IRE machinery might also affect
posttranscriptional processes and metabolic events not immediately
related to the TfR–ferritin pathway. This is suggested by the presence
of IRE motifs in the 5' regions of mRNAs for mitochondrial aconitase,
Drosophila succinate dehydrogenase, and erythroid
-aminolevulinate synthase (68)
, as well as in the 3'
region of mRNA for mammalian proton-coupled metal ion transporters
(e.g., DCT1/Nramp2) (77)
. The effect of IRP-1
modifications on cellular iron homeostasis has indeed been demonstrated
by several studies. Cells overexpressing iron-insensitive IRP-1 mutants
cannot withstand iron supplementation (78)
, and
inappropriately high IRP activity has been been found in duodenal
(79)
or reticuloendothelial cells (80)
from
iron overload-prone hemochromatosis patients. In contrast, low IRP
activity has been found in monocytes from patients with inflammation,
providing a mechanistic link between enhanced iron sequestration in the
reticuloendothelial system, reduced iron availability for
hematopoiesis, and persistent anemia in chronic diseases
(81)
.
By disrupting cardiac iron homeostasis, the decline in IRP activity would also lead to inadequate reconstitution of several enzymes and oxygen binding proteins that function by virtue of this metal (cytochromes, catalase, lipoxygenases, myoglobin, etc.). Additional consequences of DOXol/IRP interactions might be expected after misplacement of iron ions at cellular sites critical to the contraction–relaxation cycle. This is best exemplified by recent studies showing that chronic anthracycline treatment desensitizes sarcoplasmic reticulum to Ca2+-induced Ca2+ release, indicating a dysfunction of the Ca2+-gated/ryanodine receptor-2 Ca2+ release channel (82) . Whereas anthracycline treatment definitely decreases the levels of channel mRNA (55) and immunoreactive proteins (83) , a corollary mechanism for channel dysfunction has been identified in its sterical occupancy by iron ions. In this context, Fe(II) is remarkably more inhibitory than Fe(III), presumably because it has greater affinity for the cysteine residues lining the channel (84) . Collectively, these studies suggest that Ca2+ channels and IRPs share similar mechanisms of inactivation by Fe(II) and foretell that changes in the IRP/IRE machinery may lead to anomalous iron movements and sterical association with cell constituents other than storage proteins or iron-dependent enzymes.
The `IRP/IRE hypothesis' of cardiotoxicity has two additional convincing pros. First, the cardiotoxicity scores of structurally distinct anthracyclines correlate better with the intracardiac formation of C-13 metabolites than with systemic metabolism and disposition (11) . This suggests that the cardiotoxic potential of anthracyclines might be reliably predicted from their affinity for myocardial aldo-keto reductases, regardless of drug-to-drug variability in extracardiac pharmacokinetics. Second, the `IRE-IRP hypothesis' may help clarify the role of dexrazoxane from novel standpoints. Dexrazoxane might protect cardiomyocytes by competing with DOX for iron and preventing formation of those drug–iron complexes that inactivate aconitase/IRP-1 and, perhaps, IRP-2, as previously hinted. In so doing, dexrazoxane can mitigate disturbances in iron movements, metabolic use, and sterical association with proteins or enzymes. In either case, dexrazoxane would act on homeostatic processes rather than free radical reactions, possibly explaining why antioxidant interventions are less protective than chelation therapy against chronic cardiotoxicity. The IRE/IRP hypothesis of cardiotoxicity nonetheless has its drawbacks. For example, there have been sporadic reports of delayed cardiotoxic effects in laboratory animals after a single DOX administration, in spite of the fact that DOXol accumulates to a minimal extent under these conditions and eventually disappears within a week after drug treatment (85) . Again, studies in laboratory animals have shown that 5-iminodaunorubicin can form a C-13 hydroxy metabolite and yet is less cardiotoxic than other anthracyclines (86 , 87 ). Inasmuch as the C-13 derivatives from structurally distinct anthracyclines have been shown to retain comparable iron reactivity (11) , it is unlikely that the reduced cardiotoxicity of 5-iminodaunorubicin may be attributed to decreased interactions of its hydroxymetabolites with aconitase/IRPs or other targets. What is peculiar to 5-iminodaunorubicin is that its quinone moiety has been chemically modified so as to generate much less O2·- and H2O2 (86 , 87 ). This suggests that anthracyclines can be made less cardiotoxic by decreasing generation of oxyradicals rather than of C-13 metabolites, in the face of conceptual efforts to identify mechanisms alternative to oxidative stress! Although the clinical use of 5-iminodaunorubicin has been precluded by severe extracardiac toxicity, preclinical evidence accumulated with this drug clearly argues against the DOXol and IRE/IRP hypothesis.
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ROLE OF IRON IN ANTHRACYCLINE CARDIOTOXICITY: IS THERE ROOM FOR A UNIFYING HYPOTHESIS? |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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A key to a unifying hypothesis might be to separate the mechanism(s) of acute cardiotoxicity from those of chronic cardiomyopathy and to see whether the former can be preparatory to the latter. As for the acute cardiotoxicity, biochemical indices of oxidative damage and effective protection by both antioxidants and dexrazoxane indicate that, with the possible exception of few experimental conditions (4) , iron-catalyzed free radical injury is important in this setting. As mentioned, this is more difficult to prove in patients, who respond to acute DOX treatments with a paradoxical inhibition of cardiac lipid peroxidation rather than with stimulation (10) . This is a unique situation, perhaps because cancer patients present with spontaneously up-regulated prooxidant tone and react to DOX metabolism and H2O2 formation with unproductive processes that annihilate · OH or iron–oxygen complexes (cf. Section IV and ref 10 ).
As anthracyclines are given chronically, depletion of antioxidant defenses such as glutathione and related enzymes (4) and the consequent accumulation of H2O2 may likewise divert · OH from oxidative damage or inactivate iron–oxygen complexes, thereby terminating free radical injury. The transition from acute to chronic effects is therefore accompanied by a decline of the protective efficacy of antioxidants but not of dexrazoxane, perhaps because this is the time when iron moves from the free radical arena to the homeostatic disorders associated with DOXol formation and consequent inactivation of the IRE/IRP machinery (Fig. 3 ).Can this hypothesis incorporate a mechanistic link between acute and chronic cardiotoxicity? We propose that it can. In fact, recent studies in smooth muscle cells have shown that aldo-keto reductase induction may represent a common response in tissues exposed to H2O2 and certain by-products of lipid peroxidation (e.g., 4-hydroxy-nonenal) (88) . Whereas the induction of these enzymes enables cells to detoxify lipid aldehydes formed during the acute phase of cardiotoxicity, it also paves the road to time-delayed reduction of DOX to DOXol and consequent inactivation of IRPs. Thus, chronic cardiotoxicity might be `primed' by events occurring at earlier stages, such as the redox cycling of anthracyclines with oxygen (yielding H2O2) and the promotion of iron-catalyzed damage (causing lipid peroxidation) (cf. Fig. 3 ). Direct evidence for anthracycline induction of aldo-keto reductases is lacking, but the observation that intracardiac levels of DOXol increase with time and repeated dosing of DOX strongly indicates that this may be the case.
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Our hypothesis may also help address two additional questions. The first question is: Can iron-independent mechanisms exacerbate the iron-based ones? Again, we propose that it can. For example, free radicals generated during the acute phase of cardiotoxicity might damage nucleic acids without causing overt cardiac dysfunction until myocytes fail to repair or replace important enzymes and proteins with low turnover rates (35) . The ability of DOX to concomitantly disrupt cardiac gene expression in an iron-independent fashion might definitely exacerbate this pathway of cardiotoxicity. Likewise, the ability of DOXol to directly inhibit constitutive or de novo synthesized enzymes would amplify the consequences of free radical-mediated DNA damage and free radical-independent impairment of gene expression (Fig. 3) .
The second question to be answered is: Can our unifying hypothesis explain the decreased cardiotoxicity of 5-iminodaunorubicin and reconcile this with the DOXol and IRE/IRP story? We propose that positive answers can be given with respect to both acute and chronic cardiotoxicity mechanisms. Due to chemical modifications at the quinone moiety, 5-iminodaunorubicin cannot reduce oxygen nor can it delocalize iron from cellular stores; therefore, it clearly fails to promote iron- and free radical-dependent acute toxicity. Because of its lower redox activity, 5-iminodaunorubicin might similarly fail to link the acute toxicity to the free radical-independent phase of cardiac damage, as the latter must be `prepared' by H2O2 or lipid aldehyde induction of the aldo-keto reductases that form C-13 metabolites. The lower toxicity of 5-iminodaunorubicin can thus be reconciled with both free radical-independent and -dependent mechanisms.
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NOVEL HYPOTHESES CAN HELP DESIGN BETTER PROTECTIVE STRATEGIES |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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A `good protectant' has been identified in amifostine (WR2721), a phosphorylated aminothiol that exposes a hyperreactive sulfydryl moiety (WR1065) upon dephosphorylation by membrane-bound alkaline phosphatase (89) . Amifostine dephosphorylation occurs in normal tissues more effectively than in cancer foci, presumably because of 1) higher alkaline phosphatase expression in the capillary endothelium of well-perfused normal tissues, and 2) impaired alkaline phosphatase activity in malignant tissues, reflecting the low pH values found within hypoxic tumor masses (8) . Amifostine can thus afford selective protection by shielding cellular thiols from ionizing radiations (90) or alkylating antineoplastic agents (91) . Drug metabolism studies have indicated that WR1065 can be recovered from cells or whole organs in the form of symmetrical disulfides and both protein and nonprotein mixed disulfides, similar to other thiol-based antioxidants (92) . However, WR1065 can also be recovered in a form of sulfinic/sulfonic derivatives (92) , the same that probably form in IRP-1 when DOX-Fe(II) damages critical cysteines (12) . This indicates that WR1065 might have the potential to protect IRPs and perhaps other proteins from oxidation of critical —SH residues. In addition, amifostine is characterized by selective uptake and retention in the heart and has the ability to scavenge oxygen free radicals produced by DOX (93) , leaving the antitumor efficacy of the anthracycline virtually unchanged. Based on these premises and other encouraging results in experimental models (8) , amifostine can be expected to mitigate both acute and chronic cardiotoxicity, proving more versatile than N-acetylcysteine or other thiol-based protectants.
Another cardioprotective approach that would fit well into our
unifying hypothesis of cardiotoxicity is based on low mol wt nitroxyl
radicals (nitroxides, >N=O
). Due to their chemical stability toward
most diamagnetic molecules, nitroxides have long been used in the
experimental settings as biophysical probes, spin labels, or contrast
agents in nuclear magnetic resonance and EPR imaging (94)
.
More recently, nitroxides, particularly piperidine derivatives, have
been found to protect cells (95)
, organs
(96)
, and whole animals (97
, 98
)
in a number of experimental models of oxidative stress. Such protective
effects have been attributed to the ability of the nitroxyl moiety to
dismutate O2·-, detoxify semiquinone free radicals,
reduce FeIV=O, and terminate radical–chain reactions by
direct interaction with alkyl, alkoxyl, and alkylperoxyl radicals (100,
and references therein). Recent studies suggest that some of these
antioxidant effects might be mediated at least in part by the
hydroxylamine (>N-OH) resulting from one-electron reduction of
nitroxyl (101)
.
The ability of nitroxides to intercept such a wide array of free radicals suggests that they might prove better protectants than N-acetylcysteine or vitamin E, as also mentioned for spin traps. Accordingly, the piperidine nitroxide TEMPOL has been reported to afford remarkable protection in an in vitro model of anthracycline-induced acute cardiotoxicity (102) . Two additional considerations may concur to explain this effect. First, substantial amounts of TEMPOL can be detected in subcellular compartments that are critical sites of DOX metabolism and oxidative damage (e.g., mitochondria and sarcoplasmic reticulum) (102) . Second, nitroxides can oxidize Fe(II) to Fe(III) (94 , 102 ), thereby decreasing the formation of · OH or equivalent iron complexes from reaction of Fe(II) with H2O2 or oxygen (cf. Section IV ). Besides hampering the oxidative damage underlying acute cardiotoxicity, nitroxide oxidation of Fe(II) might interfere with processes occurring during the chronic phase of cardiac damage, such as DOX-Fe(II) inactivation of IRPs and consequent misplacement of unregulated Fe(II) at the Ca2+ release channel of sarcoplasmic reticulum or other cellular sites (cf. Section VI ). Thus, the ability to oxidize Fe(II) might enable nitroxides to protect against both acute and chronic events of cardiotoxicity.
Recent studies with TEMPOL have shown that protective interventions with nitroxides may have two additional advantages. First, nitroxides are remarkably more toxic to neoplastic cells than to their normal counterparts; second, TEMPOL is toxic to both wild-type and multidrug resistant tumor cells (103) . Inasmuch as nitroxides per se cannot interfere with DOX-induced double strand breaks and cell survival (104) , these findings suggest that such compounds can improve the therapeutic index of anthracyclines not only by protecting cardiac tissues, but also by imposing their own toxicity on cancer cells. This unexpected observation in cancer cells has been tentatively explained by one-electron oxidation of the nitroxyl moiety of TEMPOL to an oxo-ammonium cation species (>N+-O) with the potential to oxidize critical cellular components (100) . The in vivo efficacy of nitroxides might be limited by their too rapid reduction to the corresponding hydroxylamines, which are probably less effective as antioxidants and almost completely devoid of cytotoxic effects on tumor cells. This problem might be solved by adoption of slow-release devices and/or by the recent development of a polynitroxylated form of human serum albumin that reoxidizes hydroxylamine, shifting the equilibrium in favor of nitroxides (105) .
One last approach to cardiac protection might be to decrease the formation of oxyradicals or C-13 metabolites, thus interfering with either or both steps in our tentative scheme (cf. Fig. 3 ). Lessons from 5-iminodaunorubicin indicate that chemical modifications of the quinone moiety decrease the formation of oxyradicals and attenuate cardiotoxicity; however, extracardiac toxicity can unpredictably occur after these manipulations. Attempts to modify the yield of C-13 metabolites have not been pursued so far, but should be given priority in both academic and industrial environments for at least two different reasons. First, C-13 metabolites do not contribute significantly to the antitumor activity of anthracyclines (106) . Drugs inhibiting aldo-keto reductases or anthracyclines forming fewer C-13 metabolites should therefore prove useful to prevent cardiac toxicity without affecting tumor response. Second, our scheme suggests that C-13 metabolites are the ultimate and most critical mediators of chronic cardiotoxicity. Decreasing the formation of these metabolites would generate a bottleneck in the process of cardiotoxicity, irrespective of prior contributions from oxyradicals and related oxidative events.
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CONCLUSIONS |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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Note added in proof: For a different picture of the same topic, the reader may want to consult two reviews that appeared while our article was in press [Myers, C. E. (1998) The role of iron in doxorubicin-induced cardiomyopathy, Semin. Oncol. 25 (Suppl. 10), 10–14; Singal, P. K., and Iliskovic, N. (1998) Doxorubicin-induced cardiomyopathy N. Engl. J. Med. 339, 900–905].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: DOX, doxorubicin; DOXol, doxorubicinol;
O2·-, superoxide anion;
H2O2, hydrogen peroxide; low mol wt, low
molecular weight; · OH, hydroxyl radical; FeIV=O,
ferryl ion; SOD, superoxide dismutase; PBN,
N-tert-butyl--phenylnitrone; POBN,
-(4-pyridyl-1-oxide)-N-tert-butylnitrone; DMPO,
5,5-dimethyl-1-pyrroline N-oxide; MT, metallothionein; TfR, transferrin
receptor; IRP, iron regulatory protein; IREs, iron-responsive elements;
TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl.
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REFERENCES |
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TOPABSTRACTBACKGROUNDDOES IRON MEDIATE ANTHRACYCLINE...DOES IRON MEDIATE ANTHRACYCLINE...DO ANTHRACYCLINES GENERATE TOO...IS ANTHRACYCLINE CARDIOTOXICITY...THE IRP-IRE MACHINERY: A...ROLE OF IRON IN...NOVEL HYPOTHESES CAN HELP...CONCLUSIONSREFERENCES |
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-tocopherol on adriamycin cardiotoxicity. Ann. N.Y. Acad. Sci. 1982;393:411-418.[Medline]
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