Attenuation of Doxorubicin Chronic Toxicity in Metallothionein-overexpressing Transgenic Mouse Heart¹ Free

DOX³ is an anthracycline antibiotic and one of the most important anticancer agents. It is a valuable component of various chemotherapeutic regimens of breast carcinoma and small-cell lung carcinoma. In metastatic thyroid carcinoma, DOX is probably the best available agent. DOX is also an important ingredient for the successful treatment of Hodgkin’s disease and non-Hodgkin’s lymphomas. A clear dose-response relation for DOX in several curative regimens has been shown. 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 been described. First, acute myocardial injury, most often in the form of arrhythmia, occurs immediately after a single dose of DOX and is clinically manageable (2). Second, chronic cardiotoxicity resulting in cardiomyopathy represents a more common and clinically most important form of damage (3, 4, 5). Third, late-onset ventricular dysfunction and arrhythmia resulting from cardiomyopathy manifesting years to decades after DOX treatment has been increasingly recognized (6, 7, 8). The chronic and late-onset cardiotoxicity is dose-related and produces significant morbidity and mortality (9), and the incidence dramatically increases (in >20% of patients) at cumulative doses in excess of 550 mg/m² of body surface (10).

The proposed mechanism for the cytotoxic effect of DOX is the production of reactive oxygen species during its intracellular metabolism (11). In this context, many efforts have been made to increase myocardial antioxidant capacity as an approach to decrease the cardiotoxicity of DOX. MT is a highly conserved, low-M_r, thiol-rich protein. The mammalian MT has 61 amino acids, including 20 cysteine residues, but no aromatic amino acids or histidine or leucine (12). MT is highly inducible in biological systems under stresses such as the presence of heavy metals, starvation, heat, inflammation, or a diversity of pathological conditions (13, 14). That MT functions as a potent antioxidant has been demonstrated in both in vitro (15, 16, 17) and in vivo (18, 19, 20) studies. Zinc-MT has been shown to scavenge hydroxyl radicals in a cell-free system and to be more effective than GSH in preventing hydroxyl radical-induced DNA degradation (21). A study using HL-60 cells has demonstrated a direct reaction of hydrogen peroxide with the sulfhydryl groups of MT (22). The thiolate groups in the MT are the preferential attacking targets of hydrogen peroxide compared with the other sulfhydryl residues from GSH and protein fractions (22).

Several studies have been undertaken to explore whether MT can provide protection against DOX cardiotoxicity. Preinduction of MT by bismuth subnitrate in mice has been shown to decrease DOX-induced lipid peroxidation in the heart (23). Zinc, cadmium, cobalt, or mercury also induced MT expression in the heart and decreased DOX-related myocardial lipid peroxidation (23). The decreased drug toxicity parallels the level of cardiac MT (24), and the DOX-induced production of conjugated diene and malondialdehyde in the heart is negatively correlated with the concentration of MT in the tissue (25).

More convincing is the direct evidence that shows that DOX toxicity was greatly suppressed in the heart of MT-overexpressing transgenic mice. In these transgenic mice, MT was elevated only in the heart, not in the liver, kidneys, lungs, or skeletal muscles. Other antioxidant components including GSH, GSH peroxidase, GSH reductase, catalase, and superoxide dismutase were not altered in the MT-overexpressing heart. We have demonstrated that MT provides protection from DOX acute cardiotoxicity, including suppression of DOX-induced cardiac morphological changes, reduction in the level of serum creatine phosphokinase released from the heart, and inhibition of DOX-induced functional alteration in the isolated atrium (18). Furthermore, MT prevents DOX-induced myocardial apoptosis through inhibition of DOX-activated p38 mitogen-activated protein kinase (26) and of DOX-induced mitochondrial cytochrome c release and caspase-3 activation (27). These cardiac protective effects of MT correlate with its inhibition of DOX-generated reactive oxygen species and lipid peroxidation (17, 20).

These studies, however, addressed only the role of MT in protection against DOX acute cardiotoxicity. In cancer chemotherapy, chronic rather than acute drug toxicity is a complex and significant problem. The chronic cardiomyopathy, resulting in congestive heart failure, is the major drawback of DOX in the clinical application. Therefore, more comprehensive experimental approaches should be developed to understand the role of MT in cardiac protection against chronic toxicity induced by DOX. The present study was thus undertaken to examine whether MT suppresses DOX-induced chronic cardiotoxicity.

FVB mice obtained from the University of Louisville Research Center were housed at the University animal quarters that were maintained at 22°C with a 12-h light/dark cycle. They had free access to standard rodent chow and tap water. Cardiac-specific, MT-overexpressing, transgenic mice were produced from the FVB strain. Detailed descriptions for the development and characterization of these transgenic mouse lines were reported previously (18). The transgenic founders were bred with the nontransgenic FVB mice to maintain genetic stability. Transgenic mice were identified from the heterozygous littermates by a PCR procedure. Several transgenic lines have been produced and all of them have been tested in their responses to DOX toxicity. There were no any other phenotypes observed in these transgenic mice except their resistance to DOX cardiotoxicity. All of the animal procedures were approved by the Institutional Animal Care and Use Committee, which is certified by the American Association of Accreditation of Laboratory Animal Care.

Male MT-transgenic mice and nontransgenic controls [8-week old; body weight, 20–30 g] were treated with DOX (Sigma Chemical Co., St. Louis, MO) according to a procedure described previously (28). In brief, DOX was dissolved in saline and injected i.v. at 4 mg/kg (10 ml/kg of body weight) twice a week (Monday and Thursday) for a total of 10 injections. Control animals were injected with the same volume of saline. Animals were not treated for 2 weeks between the first four injections and the last six injections to allow the recovery of bone marrow depression. The animals were killed 3 weeks after the last injection. This treatment protocol was developed and standardized based on clinical application of DOX and the clinically relevant cardiomyopathy that was developed from this treatment in the mouse model (28).

MT concentrations in the heart were determined by a cadmium-hemoglobin affinity assay (29). Briefly, heart tissues were homogenized in four volumes of 10 mm Tris-HCl buffer (pH 7.4) at 4°C. After centrifugation of the homogenate at 10,000 × g for 15 min, 200 μl of supernatant were transferred to microtubes for MT analysis as described previously (18). The MT concentrations in the heart from nontransgenic and transgenic mice, treated with or without DOX, are expressed as micrograms per gram of heart tissue.

After being anesthetized with sodium pentobarbital (60 mg/kg body weight), the hearts of all of the experimental animals were fixed in situ by vascular perfusion with saline for 10 min followed by a Karnovsky’s fixative [2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer (pH 7.4)] for 15 min. The in situ fixative perfusion procedure was described previously (17). The fixed mouse hearts were removed and weighed. The tissue samples taken from the left ventricles were 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% osmium tetroxide, dehydrated in a graded ethanol series with 100% propylene oxide as a transitional solvent, and embedded in LX-112 resin (LADD Research Industries Co.). Both semithin and ultrathin sections were obtained with a LKB ultramicrotome. The semithin sections were stained with 1% azure II in 1% borax, and observed with light microscopy. The ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Philip transmission electron microscope.

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

Data are expressed as mean ± SD values. The histological parameters were evaluated using Kruskal-Wallis nonparametric ANOVA analysis. Scheffe’s F test was used for further determination of the significance of differences. Differences between MT-overexpressing transgenic mice and nontransgenic controls were considered significant at P < 0.05.

MT concentrations in transgenic and nontransgenic mouse hearts were measured after DOX treatment. As shown in Table 2, MT concentrations in the transgenic mouse heart were about 60-fold higher than in the nontransgenic mouse heart in the saline-treated controls. After DOX treatment 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 were significantly (P < 0.05) decreased after the treatment with DOX; a 15% reduction was observed.

The hearts of DOX-treated nontransgenic mice were dilated and both the atrium and the ventricle were enlarged and hypertrophic (Fig. 1). The heart weight and the ratio of heart weight:body weight were significantly (P < 0.05) increased by DOX (Table 3). The cardiac hypertrophy and the increases in both the heart weight and the ratio of heart weight:body weight attributable to DOX treatment were almost completely prevented in the MT-overexpressing transgenic mice (Table 3; Fig. 1).

In comparison with the saline-treated controls (Fig. 2, A and B), the myocardium from DOX-treated nontransgenic mice was characterized by prominent and diffuse vacuolization (Fig. 2,C). In contrast, only microvacuolar morphological changes were observed in a few small areas in the sections from DOX-treated MT-transgenic mouse hearts (Fig. 2,D). The scores recorded for the myocardial lesions resulting from DOX administration are presented in Table 4. Nontransgenic mice treated with DOX had the most severe myocyte damage according to the recorded scores (all scored 6). However, cardiomyopathy scores ranged only from 1 to 2 in the MT-transgenic mice, significantly lower than that in the nontransgenic mice (P < 0.05).

Compared with the cardiomyocytes of both nontransgenic and MT-transgenic mice treated with saline (Fig. 3, A and B), altered structural integrity, vacuoles, and extensive loss of myofibrils took place in the heart of nontransgenic mice treated with DOX (Fig. 3,C). The heart from MT-transgenic mice treated with DOX, as shown in Fig. 3,D, appeared almost normal. Close examination at higher magnification revealed that the cytoplasmic vacuolization mainly resulted from dilation of sarcoplasmic reticulum in the cardiomyocytes of nontransgenic mice treated with DOX (Fig. 3,E). The structural disruption of mitochondrial organization with a decrease in the total number of cristae or a complete disappearance of cristae was also observed. The MT-transgenic mouse hearts did not display obvious cytoplasmic vacuolization and retained the fine ultrastructure of mitochondria even in the cardiomyocytes of myofilament disarray (Fig. 3 F).

The present study provides direct evidence that MT prevents DOX chronic cardiotoxicity. The chronic cardiotoxicity was examined specifically at three levels. First, the gross anatomical changes of the heart treated with DOX showed a typical chronic toxic response including ventricular dilation, cardiac hypertrophy, and overall enlargement of the heart as determined by the absolute heart weight and the ratio of the heart weight:body weight. Second, histological examination revealed that macrovacuolization predominates in the DOX-treated myocardium. Third, ultrastructural alterations including sarcoplasmic reticulum vacuolization, mitochondrial swelling, and other fine-structure disruption occurred widely in the DOX-treated cardiomyocytes. However, all of these changes are dramatically inhibited in the MT-overexpressing transgenic mouse heart. In particular, the gross anatomical changes were almost completely prevented.

DOX is an important anticancer agent. It is irreplaceable in the treatment of some cancers (30). Therefore, there have been tremendous efforts attempting to reduce the cardiotoxicity of DOX. These attempts include the following: (a) to decrease myocardial concentrations of DOX by methods including alternative dosing regimens, slowly infusing the drug to keep plasma concentrations low, and/or binding the drug to carrier molecules to decrease the availability of the drug to myocytes; (b) a considerable effort has been directed to the synthesis and development of new compounds that will retain significant anticancer activity while decreasing cardiac toxicity; and (c) finally, many substances have been tested experimentally as potential cardiac protective agents that can be concurrently administered with DOX. These attempts, however, have achieved limited success (30). Therefore, alternative experimental and clinical approaches are required to improve the therapeutic efficacy of this agent.

The pathways by which DOX generates reactive oxygen species have been extensively studied. One is the formation of a DOX-iron complex (31). The DOX-iron complex spontaneously reacts to generate hydrogen peroxide and hydroxyl radical, leading to oxidative damage (31). Dexrazoxane (ICRF-187, ADR 529) reacts directly with the DOX-iron complex to promote the opening of the amide ring of dexrazoxane with a simultaneous transfer of the iron from DOX to the carboxylamine generated by the ring opening (32). This compound has been studied both experimentally and clinically for its potential as a cardioprotective agent (33). Protection against DOX cardiotoxicity with this agent has been observed, but the protection has never been satisfactory (34). This may be attributable, at least in part, to other important pathways of reactive oxygen species generation by DOX. In addition, clinical use of dexrazoxane has been seriously questioned because of the associated severe myelosuppression, which is actually potentiated by DOX (35). The possibility that dexrazoxane may interfere with cancer therapy has also been raised (36).

The flavin reductases, including cytochrome P-450 reductase, cytochrome 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 rapidly to reduce the oxygen to superoxide with regeneration of intact DOX. The superoxide is rapidly converted to hydrogen peroxide, which is in turn converted to hydroxyl radical. The DOX semiquinone also reacts with hydrogen peroxide to yield hydroxyl radical. Another pathway is through the binding of DOX to the endothelial isoform of nitric oxide synthase (eNOS) which subsequently undergoes eNOS-mediated reduction (38). This reduces DOX to the semiquinone radical. As a consequence, superoxide formation is enhanced and nitric oxide production is decreased. This may lead to the generation of peroxynitrite and hydrogen peroxide; both are further converted to hydroxyl radical. Neither of these two pathways of reactive oxygen species generation by DOX is sensitive to the action of iron chelators. The unique feature of MT thus allows cardiac protection from the toxicity of reactive oxygen species generated from DOX through the latter pathways. This scenario was clearly demonstrated in the present study.

It has been observed in our previous studies (39) that treatment with a single high dose of DOX induces MT expression in the heart. In the present study, we also observed that chronic treatment with low dose of DOX also increased MT concentrations in nontransgenic mouse hearts. This elevation, however, was apparently not high enough to provide protection against DOX cardiotoxicity. An interesting observation in the present study was that MT concentrations in the transgenic mouse heart were significantly decreased after the chronic treatment with DOX.

It has been demonstrated that the cluster structure of zinc-MT provides a chemical basis by which the cysteine ligands can induce oxidoreductive properties (40). This structure allows for thermodynamic stability of zinc in MT, while permitting zinc to retain kinetic lability. This is demonstrated by the fast zinc exchange between MT isoforms (41), between MT and the zinc cluster in the Gal4 transcription factor (41), and between MT and the apoforms of various zinc proteins (42). Importantly, mobilization of zinc from MT is triggered by oxidative stress (42). This either may constitute a general pathway by which zinc is distributed in the cell or may be restricted to conditions of stress in which zinc is needed in antioxidant defense systems. The oxidative stress condition is certainly applicable to the DOX-treated myocardium. Interaction between zinc-MT and oxidants, whose concentrations increase in the myocardium under DOX treatment, will cause zinc release from MT. Because metals protect MT from degradation (43), a decrease in total MT concentrations attributable to the loss of zinc would be observed. This may explain the observed reduction of MT concentrations in the DOX-treated transgenic mouse heart.

The results obtained here demonstrate that MT is a powerful cardioprotectant in preventing DOX chronic cardiotoxicity. The antioxidant action of MT (44) would be highly responsible for this cardioprotetion. MT is highly inducible under a wide diversity of stress conditions, including oxidative stress. The regulation of MT expression has been well studied, and several agents, such as bismuth subnitrate (25), isoproterenol (45), and tumor necrosis factor-α (46), have been identified to selectively elevate MT levels in the heart. Therefore, the basis for developing pharmaceutical agents to increase MT concentration in the heart already exists. Exploring the potential for MT to protect against DOX cardiotoxicity would likely result in novel approaches to this clinical problem and could positively influence clinical outcomes.

We thank Donald Mosley and Cathy Caple for technical assistance.