attenuation of doxorubicin chronic toxicity in metallothionein … · the chronic cardiomyopathy,...

[CANCER RESEARCH 61, 3382–3387, April 15, 2001]

Attenuation of Doxorubicin Chronic Toxicity in Metallothionein-overexpressingTransgenic Mouse Heart1

Xiuhua Sun, Zhanxiang Zhou, and Y. James Kang2

Departments of Medicine, and Pharmacology and Toxicology, University of Louisville, School of Medicine, [X. S., Z. Z., Y. J. K.], and Jewish Hospital Heart andLung Institute [Y. J. K.], Louisville, Kentucky 40202

ABSTRACT

Previous studies have shown that cardiac-specific metallothionein(MT)-overexpressing transgenic mice are highly resistant to acute cardio-toxicity induced by doxorubicin (DOX), a most effective anticancer agent.However, cumulative dose-dependent chronic cardiotoxicity attributableto long-term administration of DOX is a significant clinical problem.Because MT is a potent antioxidant and oxidative stress is criticallyinvolved in DOX-induced heart injury, the present study was undertakento test the hypothesis that MT also provides protection against DOXchronic cardiotoxicity. Transgenic mice containing high levels of cardiacMT and nontransgenic controls were treated with a cumulative dose of 40mg/kg of DOX in 10 equal i.v. injections over a period of 7 weeks. Threeweeks after the last injection, the mice were killed for an analysis ofcardiotoxicity. As compared with nontransgenic controls, DOX-inducedcardiac hypertrophy was significantly inhibited in the transgenic mice.Light microscopic examination revealed that DOX-induced myocardialmorphological changes were markedly suppressed or almost eliminated inthe transgenic mice. Under electron microscopy, extensive sarcoplasmicvacuolization and severe disruption of mitochondrial fine structure wereobserved in nontransgenic cardiomyocytes, but almost no sarcoplasmicvacuolization was observed, and the mitochondrial structural changeswere almost completely prevented in the transgenic cardiomyocytes. Theresults thus indicate that MT elevation is a highly effective approach toprevent chronic cardiomyopathy attributable to DOX. This study alsosuggests that oxidative stress is critically involved in the DOX-inducedchronic cardiotoxicity.

INTRODUCTION

DOX3 is an anthracycline antibiotic and one of the most importantanticancer agents. It is a valuable component of various chemother-apeutic regimens of breast carcinoma and small-cell lung carcinoma.In metastatic thyroid carcinoma, DOX is probably the best availableagent. DOX is also an important ingredient for the successful treat-ment of Hodgkin’s disease and non-Hodgkin’s lymphomas. A cleardose-response relation for DOX in several curative regimens has beenshown. Decreased doses result in inferior survival rates (1). However,increase in dose is very limited because of the severe cardiotoxicity,a major problem in the clinical application of DOX.

Three distinct types of DOX-induced cardiotoxicity have beendescribed. First, acute myocardial injury, most often in the form ofarrhythmia, occurs immediately after a single dose of DOX and isclinically manageable (2). Second, chronic cardiotoxicity resulting incardiomyopathy represents a more common and clinically most im-portant form of damage (3–5). Third, late-onset ventricular dysfunc-

tion and arrhythmia resulting from cardiomyopathy manifesting yearsto decades after DOX treatment has been increasingly recognized(6–8). The chronic and late-onset cardiotoxicity is dose-related andproduces significant morbidity and mortality (9), and the incidencedramatically increases (in.20% of patients) at cumulative doses inexcess of 550 mg/m2 of body surface (10).

The proposed mechanism for the cytotoxic effect of DOX is theproduction of reactive oxygen species during its intracellular metab-olism (11). In this context, many efforts have been made to increasemyocardial antioxidant capacity as an approach to decrease the car-diotoxicity of DOX. MT is a highly conserved, low-Mr, thiol-richprotein. The mammalian MT has 61 amino acids, including 20 cys-teine residues, but no aromatic amino acids or histidine or leucine(12). MT is highly inducible in biological systems under stresses suchas the presence of heavy metals, starvation, heat, inflammation, or adiversity of pathological conditions (13, 14). That MT functions as apotent antioxidant has been demonstrated in bothin vitro (15–17) andin vivo (18–20) studies. Zinc-MT has been shown to scavenge hy-droxyl radicals in a cell-free system and to be more effective thanGSH in preventing hydroxyl radical-induced DNA degradation (21).A study using HL-60 cells has demonstrated a direct reaction ofhydrogen peroxide with the sulfhydryl groups of MT (22). The thio-late groups in the MT are the preferential attacking targets of hydro-gen peroxide compared with the other sulfhydryl residues from GSHand protein fractions (22).

Several studies have been undertaken to explore whether MT canprovide protection against DOX cardiotoxicity. Preinduction of MTby bismuth subnitrate in mice has been shown to decrease DOX-induced lipid peroxidation in the heart (23). Zinc, cadmium, cobalt, ormercury also induced MT expression in the heart and decreasedDOX-related myocardial lipid peroxidation (23). The decreased drugtoxicity parallels the level of cardiac MT (24), and the DOX-inducedproduction of conjugated diene and malondialdehyde in the heart isnegatively correlated with the concentration of MT in the tissue (25).

More convincing is the direct evidence that shows that DOXtoxicity was greatly suppressed in the heart of MT-overexpressingtransgenic mice. In these transgenic mice, MT was elevated only inthe heart, not in the liver, kidneys, lungs, or skeletal muscles. Otherantioxidant components including GSH, GSH peroxidase, GSH re-ductase, catalase, and superoxide dismutase were not altered in theMT-overexpressing heart. We have demonstrated that MT providesprotection from DOX acute cardiotoxicity, including suppression ofDOX-induced cardiac morphological changes, reduction in the levelof serum creatine phosphokinase released from the heart, and inhibi-tion of DOX-induced functional alteration in the isolated atrium (18).Furthermore, MT prevents DOX-induced myocardial apoptosisthrough inhibition of DOX-activated p38 mitogen-activated proteinkinase (26) and of DOX-induced mitochondrial cytochromec releaseand caspase-3 activation (27). These cardiac protective effects of MTcorrelate with its inhibition of DOX-generated reactive oxygen spe-cies and lipid peroxidation (17, 20).

These studies, however, addressed only the role of MT in protectionagainst DOX acute cardiotoxicity. In cancer chemotherapy, chronicrather than acute drug toxicity is a complex and significant problem.

Received 10/17/00; accepted 2/16/01.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants CA68125 and HL59225, an Established InvestigatorAward (9640091N) from the American Heart Association National Center, and a researchgrant from the Jewish Hospital Foundation, Louisville, KY (to Y. J. K.). Y. J. K. is aUniversity scholar of the University of Louisville.

2 To whom requests for reprints should be addressed, at Department of Medicine,University of Louisville School of Medicine, 511 South Floyd Street, MDR 530, Louis-ville, KY 40202. E-mail: [email protected].

3 The abbreviations used are: DOX, doxorubicin; GSH, glutathione; MT, metallothio-nein.

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The chronic cardiomyopathy, resulting in congestive heart failure, isthe major drawback of DOX in the clinical application. Therefore,more comprehensive experimental approaches should be developed tounderstand the role of MT in cardiac protection against chronictoxicity induced by DOX. The present study was thus undertaken toexamine whether MT suppresses DOX-induced chronic cardiotoxicity.

MATERIALS AND METHODS

Animals. FVB mice obtained from the University of Louisville ResearchCenter were housed at the University animal quarters that were maintained at22°C with a 12-h light/dark cycle. They had free access to standard rodentchow and tap water. Cardiac-specific, MT-overexpressing, transgenic micewere produced from the FVB strain. Detailed descriptions for the developmentand characterization of these transgenic mouse lines were reported previously(18). The transgenic founders were bred with the nontransgenic FVB mice tomaintain genetic stability. Transgenic mice were identified from the heterozy-gous littermates by a PCR procedure. Several transgenic lines have beenproduced and all of them have been tested in their responses to DOX toxicity.There were no any other phenotypes observed in these transgenic mice excepttheir resistance to DOX cardiotoxicity. All of the animal procedures wereapproved by the Institutional Animal Care and Use Committee, which iscertified by the American Association of Accreditation of Laboratory AnimalCare.

Drug Treatment. Male MT-transgenic mice and nontransgenic controls[8-week old; body weight, 20–30 g] were treated with DOX (Sigma ChemicalCo., St. Louis, MO) according to a procedure described previously (28). Inbrief, DOX was dissolved in saline and injected i.v. at 4 mg/kg (10 ml/kg ofbody weight) twice a week (Monday and Thursday) for a total of 10 injections.Control animals were injected with the same volume of saline. Animals werenot treated for 2 weeks between the first four injections and the last sixinjections to allow the recovery of bone marrow depression. The animals werekilled 3 weeks after the last injection. This treatment protocol was developedand standardized based on clinical application of DOX and the clinicallyrelevant cardiomyopathy that was developed from this treatment in the mousemodel (28).

Cardiac MT Measurement. MT concentrations in the heart were deter-mined by a cadmium-hemoglobin affinity assay (29). Briefly, heart tissueswere homogenized in four volumes of 10 mM Tris-HCl buffer (pH 7.4) at 4°C.After centrifugation of the homogenate at 10,0003 g for 15 min, 200ml ofsupernatant were transferred to microtubes for MT analysis as describedpreviously (18). The MT concentrations in the heart from nontransgenic andtransgenic mice, treated with or without DOX, are expressed as microgramsper gram of heart tissue.

Light and Electron Microscopy. After being anesthetized with sodiumpentobarbital (60 mg/kg body weight), the hearts of all of the experimentalanimals were fixedin situ by vascular perfusion with saline for 10 minfollowed by a Karnovsky’s fixative [2% paraformaldehyde and 2.5% glutar-aldehyde in cacodylate buffer (pH 7.4)] for 15 min. Thein situ fixativeperfusion procedure was described previously (17). The fixed mouse heartswere removed and weighed. The tissue samples taken from the left ventricleswere cut into 1-mm3 blocks, and kept in the same fixative overnight at 4°C.After rinsing with the same buffer, the blocks were postfixed in 1% osmiumtetroxide, dehydrated in a graded ethanol series with 100% propylene oxide asa transitional solvent, and embedded in LX-112 resin (LADD Research Indus-

tries Co.). Both semithin and ultrathin sections were obtained with a LKBultramicrotome. The semithin sections were stained with 1% azure II in 1%borax, and observed with light microscopy. The ultrathin sections were stainedwith uranyl acetate and lead citrate and observed with a Philip transmissionelectron microscope.

For the scoring procedure to estimate the extent of myocardial damageunder light microscopy, we used the guidelines as outlined in Table 1 (28).

Statistical Analysis. Data are expressed as mean6 SD values. The histo-logical parameters were evaluated using Kruskal-Wallis nonparametricANOVA analysis. Scheffe’sF test was used for further determination of thesignificance of differences. Differences between MT-overexpressing trans-genic mice and nontransgenic controls were considered significant atP , 0.05.

RESULTS

Effects of DOX on MT Concentrations in the Heart. MT con-centrations in transgenic and nontransgenic mouse hearts were meas-ured after DOX treatment. As shown in Table 2, MT concentrations inthe transgenic mouse heart were about 60-fold higher than in thenontransgenic mouse heart in the saline-treated controls. After DOXtreatment for a total of 40 mg/kg, MT concentrations were slightly,statistically insignificantly, elevated in nontransgenic mouse hearts.Interestingly, MT concentrations in transgenic mouse hearts weresignificantly (P , 0.05) decreased after the treatment with DOX; a15% reduction was observed.

Gross Anatomical Changes of the DOX-treated Heart.Thehearts of DOX-treated nontransgenic mice were dilated and boththe atrium and the ventricle were enlarged and hypertrophic (Fig. 1).The heart weight and the ratio of heart weight:body weight weresignificantly (P , 0.05) increased by DOX (Table 3). The cardiachypertrophy and the increases in both the heart weight and the ratio ofheart weight:body weight attributable to DOX treatment were almostcompletely prevented in the MT-overexpressing transgenic mice (Ta-ble 3; Fig. 1).

Morphological Changes under Light Microscopy. In compari-son with the saline-treated controls (Fig. 2,A andB), the myocardiumfrom DOX-treated nontransgenic mice was characterized by promi-nent and diffuse vacuolization (Fig. 2C). In contrast, only microvacu-olar morphological changes were observed in a few small areas in thesections from DOX-treated MT-transgenic mouse hearts (Fig. 2D).The scores recorded for the myocardial lesions resulting from DOXadministration are presented in Table 4. Nontransgenic mice treatedwith DOX had the most severe myocyte damage according to the

Table 1 Criteria for morphological evaluation of cardiotoxicity

Degree Severity

1 Sarcoplasmic microvacuolizations and/or interstitial or cellular edema.2 Same as 1 plus sarcoplasmic macrovacuolizations or atrophia,

necrosis, fibrosis, endocardial lesions, and thrombi.Extension

0 No lesions.0.5 ,10 single altered myocytes in the whole heart section.1 Scattered single altered myocytes.2 Scattered small groups of altered myocytes.3 Widely spread small groups of altered myocytes.4 Confluent groups of altered myocytes.5 Most cells damaged.

Table 2 MT concentrations in the heart from nontransgenic and transgenic micetreated with saline and DOX

MT (mg/g tissue)

Saline DOX

Non-TGa 5.36 1.5 8.06 2.4MT-TG 323.46 13.7 273.86 6.0b

a TG, transgenic.b Significantly different from the saline-treated mouse heart (P, 0.05;n 5 5).

Table 3 Effect of MT on DOX-induced changes in heart weight and heart weight:bodyweight ratio

Treatment AnimalsHeart weight

(mg)Heart weight (mg):Body

weight (g) ratio

Saline Non-TG 1756 4 5.96 0.2MT-TG 1746 8 5.86 0.3

DOX Non-TGb 2106 4 8.96 0.7MT-TG 1806 6 7.36 0.3

a TG, transgenic.b Significantly different from the saline-treated nontransgenic mouse heart (P, 0.05;

n 5 5).

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MT INHIBITS DOX CARDIOTOXICITY



recorded scores (all scored 6). However, cardiomyopathy scoresranged only from 1 to 2 in the MT-transgenic mice, significantlylower than that in the nontransgenic mice (P , 0.05).

Ultrastructural Alterations. Compared with the cardiomyocytesof both nontransgenic and MT-transgenic mice treated with saline(Fig. 3,A andB), altered structural integrity, vacuoles, and extensiveloss of myofibrils took place in the heart of nontransgenic mice treatedwith DOX (Fig. 3C). The heart from MT-transgenic mice treated with

DOX, as shown in Fig. 3D, appeared almost normal. Close examina-tion at higher magnification revealed that the cytoplasmic vacuoliza-tion mainly resulted from dilation of sarcoplasmic reticulum in thecardiomyocytes of nontransgenic mice treated with DOX (Fig. 3E).The structural disruption of mitochondrial organization with a de-crease in the total number of cristae or a complete disappearance ofcristae was also observed. The MT-transgenic mouse hearts did notdisplay obvious cytoplasmic vacuolization and retained the fine ul-trastructure of mitochondria even in the cardiomyocytes of myofila-ment disarray (Fig. 3F).

DISCUSSION

The present study provides direct evidence that MT prevents DOXchronic cardiotoxicity. The chronic cardiotoxicity was examined spe-cifically at three levels. First, the gross anatomical changes of theheart treated with DOX showed a typical chronic toxic responseincluding ventricular dilation, cardiac hypertrophy, and overall en-largement of the heart as determined by the absolute heart weight andthe ratio of the heart weight:body weight. Second, histological exam-ination revealed that macrovacuolization predominates in the DOX-treated myocardium. Third, ultrastructural alterations including sar-coplasmic reticulum vacuolization, mitochondrial swelling, and otherfine-structure disruption occurred widely in the DOX-treated car-

Table 4 Cardiomyopathy scores recorded from nontransgenic and MT-transgenic micetreated with DOX

Treatment AnimalsNo. ofanimals

Cardiomyopathy score

0 1 2 4 6 8

Saline Non-TGa 6 6 0 0 0 0 0MT-TG 6 6 0 0 0 0 0

DOX Non-TGb 5 0 0 0 0 5 0MT-TGb,c 5 0 4 1 0 0 0

a TG, transgenic.b Significantly different from saline-treated controls (P, 0.05).c Significantly different from nontransgenic treated with DOX (P, 0.05).

Fig. 1. Gross anatomical changes of DOX-treated mouse heart and the effect of MT.Both nontransgenic (WT) and transgenic mice (TG) were treated with DOX at 4 mg/kg,i.v., twice a week for 10 injections over 7 weeks. The hearts were obtained 3 weeks afterthe last injection, and the DOX-treated heart was compared with the saline (SLN)-treatedcontrol.

Fig. 2. Light micrograph demonstrating the effect ofMT on DOX-induced myocardial injury.A, nontrans-genic saline-treated control;B, MT-transgenic saline-treated control;C, nontransgenic heart treated with DOX;D, MT-transgenic heart treated with DOX.Arrows, thevacuolization of the myocardium.3250.

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diomyocytes. However, all of these changes are dramatically inhibitedin the MT-overexpressing transgenic mouse heart. In particular, thegross anatomical changes were almost completely prevented.

DOX is an important anticancer agent. It is irreplaceable in thetreatment of some cancers (30). Therefore, there have been tremen-dous efforts attempting to reduce the cardiotoxicity of DOX. Theseattempts include the following: (a) to decrease myocardial concentra-tions of DOX by methods including alternative dosing regimens,slowly infusing the drug to keep plasma concentrations low, and/orbinding the drug to carrier molecules to decrease the availability of thedrug to myocytes; (b) a considerable effort has been directed to thesynthesis and development of new compounds that will retain signif-icant anticancer activity while decreasing cardiac toxicity; and (c)finally, many substances have been tested experimentally as potentialcardiac protective agents that can be concurrently administered withDOX. These attempts, however, have achieved limited success (30).Therefore, alternative experimental and clinical approaches are re-quired to improve the therapeutic efficacy of this agent.

The pathways by which DOX generates reactive oxygen specieshave been extensively studied. One is the formation of a DOX-ironcomplex (31). The DOX-iron complex spontaneously reacts to gen-erate hydrogen peroxide and hydroxyl radical, leading to oxidativedamage (31). Dexrazoxane (ICRF-187, ADR 529) reacts directly withthe DOX-iron complex to promote the opening of the amide ring ofdexrazoxane with a simultaneous transfer of the iron from DOX to the

carboxylamine generated by the ring opening (32). This compoundhas been studied both experimentally and clinically for its potential asa cardioprotective agent (33). Protection against DOX cardiotoxicitywith this agent has been observed, but the protection has never beensatisfactory (34). This may be attributable, at least in part, to otherimportant pathways of reactive oxygen species generation by DOX. Inaddition, clinical use of dexrazoxane has been seriously questionedbecause of the associated severe myelosuppression, which is actuallypotentiated by DOX (35). The possibility that dexrazoxane mayinterfere with cancer therapy has also been raised (36).

The flavin reductases, including cytochrome P-450 reductase, cy-tochrome b5 reductase, NADH dehydrogenase, and xanthine oxidase,have the capacity to reduce DOX to DOX semiquinone free radical(37). In the presence of oxygen, the DOX semiquinone reacts rapidlyto reduce the oxygen to superoxide with regeneration of intact DOX.The superoxide is rapidly converted to hydrogen peroxide, which is inturn converted to hydroxyl radical. The DOX semiquinone also reactswith hydrogen peroxide to yield hydroxyl radical. Another pathway isthrough the binding of DOX to the endothelial isoform of nitric oxidesynthase (eNOS) which subsequently undergoes eNOS-mediated re-duction (38). This reduces DOX to the semiquinone radical. As aconsequence, superoxide formation is enhanced and nitric oxide pro-duction is decreased. This may lead to the generation of peroxynitriteand hydrogen peroxide; both are further converted to hydroxyl radical.Neither of these two pathways of reactive oxygen species generation

Fig. 3. Electron micrograph demonstrating the effect ofMT on DOX-induced cardiomyopathy.A, nontransgenic con-trol treated with saline;B, MT-transgenic control treated withsaline;C and E, nontransgenic hearts treated with DOX;DandF, MT-transgenic hearts treated with DOX.M, mitochon-dria;p, damaged mitochondria and swollen tubular system.A,B: 310,000;C, D: 31,500;E, F: 315,000.

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by DOX is sensitive to the action of iron chelators. The unique featureof MT thus allows cardiac protection from the toxicity of reactiveoxygen species generated from DOX through the latter pathways. Thisscenario was clearly demonstrated in the present study.

It has been observed in our previous studies (39) that treatment witha single high dose of DOX induces MT expression in the heart. In thepresent study, we also observed that chronic treatment with low doseof DOX also increased MT concentrations in nontransgenic mousehearts. This elevation, however, was apparently not high enough toprovide protection against DOX cardiotoxicity. An interesting obser-vation in the present study was that MT concentrations in the trans-genic mouse heart were significantly decreased after the chronictreatment with DOX.

It has been demonstrated that the cluster structure of zinc-MTprovides a chemical basis by which the cysteine ligands can induceoxidoreductive properties (40). This structure allows for thermody-namic stability of zinc in MT, while permitting zinc to retain kineticlability. This is demonstrated by the fast zinc exchange between MTisoforms (41), between MT and the zinc cluster in the Gal4 transcrip-tion factor (41), and between MT and the apoforms of various zincproteins (42). Importantly, mobilization of zinc from MT is triggeredby oxidative stress (42). This either may constitute a general pathwayby which zinc is distributed in the cell or may be restricted toconditions of stress in which zinc is needed in antioxidant defensesystems. The oxidative stress condition is certainly applicable to theDOX-treated myocardium. Interaction between zinc-MT and oxi-dants, whose concentrations increase in the myocardium under DOXtreatment, will cause zinc release from MT. Because metals protectMT from degradation (43), a decrease in total MT concentrationsattributable to the loss of zinc would be observed. This may explainthe observed reduction of MT concentrations in the DOX-treatedtransgenic mouse heart.

The results obtained here demonstrate that MT is a powerfulcardioprotectant in preventing DOX chronic cardiotoxicity. The anti-oxidant action of MT (44) would be highly responsible for thiscardioprotetion. MT is highly inducible under a wide diversity ofstress conditions, including oxidative stress. The regulation of MTexpression has been well studied, and several agents, such as bismuthsubnitrate (25), isoproterenol (45), and tumor necrosis factor-a (46),have been identified to selectively elevate MT levels in the heart.Therefore, the basis for developing pharmaceutical agents to increaseMT concentration in the heart already exists. Exploring the potentialfor MT to protect against DOX cardiotoxicity would likely result innovel approaches to this clinical problem and could positively influ-ence clinical outcomes.

ACKNOWLEDGMENTS

We thank Donald Mosley and Cathy Caple for technical assistance.

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2001;61:3382-3387. Cancer Res Xiuhua Sun, Zhanxiang Zhou and Y. James Kang Metallothionein-overexpressing Transgenic Mouse HeartAttenuation of Doxorubicin Chronic Toxicity in

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