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Correlation Between Serum Levels of Cardiac Troponin-T and the Severity of the Chronic Cardiomyopathy Induced by Doxorubicin

From the Division of Applied Pharmacology Research (HFD-910), Center for Drug Evaluation and Research, Food and Drug Administration, Laurel, MD; Division of Pediatric Cardiology, University of Rochester Medical Center, Rochester, NY; Department of Laboratory Medicine, Boston Childrens Hospital and Harvard Medical School, Boston, MA; and Pathology Section, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD.

Address reprint requests to Eugene H. Herman, PhD, Division of Applied Pharmacology Research (HFD-910), Center for Drug Evaluation and Research, Food and Drug Administration, 8301 Muirkirk Rd, Laurel, MD 20708; email hermaneu{at}cder.fda.gov

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To investigate, over a wide range of cumulative doxorubicin doses, the feasibility of using serum concentrations of cardiac troponin-T (cTnT) as a biomarker for doxorubicin-induced myocardial damage.

MATERIALS AND METHODS: Groups of spontaneously hypertensive rats (SHR) were given 1 mg/kg doxorubicin weekly for 2 to 12 weeks. Cardiomyopathy scores were assessed according to the method of Billingham and serum levels of cTnT were quantified by a noncompetitive immunoassay. Myocardial localization of cTnT was studied by immunohistochemical staining and confocal microscopy.

RESULTS: Increases in serum levels of cTnT (0.03 to 0.05 ng/mL) and myocardial lesions (cardiomyopathy scores of 1 or 1.5) were found in one out of five and two out of five SHR given 2 and 4 mg/kg doxorubicin, respectively. All animals given 6 mg/kg or more of doxorubicin had increases in serum cTnT and myocardial lesions. The average cTnT levels and the cardiomyopathy scores correlated with the cumulative dose of doxorubicin (0.13 v 0.4 ng/mL cTnT and scores of 1.4 v 3.0 in SHR given 6 and 12 mg/kg doxorubicin, respectively). Decreased staining for cTnT was observed in cardiac tissue from SHR receiving cumulative doses that caused only minimal histologic alterations (scores of 1 to 1.5). Staining for cTnT decreased simultaneously with increases in the severity of the cardiomyopathy scores.

CONCLUSION: cTnT is released from doxorubicin-damaged myocytes. Measurements of serum levels of this protein seem to provide a sensitive means for assessing the early cardiotoxicity of doxorubicin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NONINVASIVE TECHNIQUES FOR THE identification of patients who are at high risk of developing anthracycline cardiomyopathy are critically important for the prevention and management of this complication. Both echocardiography and radionuclide ventriculography¹ lack the sensitivity required to detect the early stages of the cardiomyopathy, in which deterioration of cardiac function has not yet developed. As a result, there is increasing interest in additional noninvasive methods for the detection of early myocardial alterations induced by anthracyclines.

A different approach for the detection of cardiac injury involves measurements of the serum concentrations of cytoplasmic enzymes (MB isoenzyme, isoenzyme of creatine kinase, lactate dehydrogenase-1) or other substances that are released from damaged myocytes.² Measurements of serum levels of cardiac troponin-T (cTnT), a component of the troponin complex of muscle cells, have begun to be used for the diagnosis of myocardial damage in various conditions,^2-5 including acute myocardial infarction, acute myocarditis, unstable angina, and isoproterenol-induced myocardial necrosis.⁶ The usefulness of this method has been aided by the development of a specific and sensitive immunoassay^7,8 that distinguishes the cardiac from the skeletal muscle isoforms of troponin-T. Troponin-T is thought to be responsible for positioning the troponin complex on the thin filaments of the sarcomeres by binding this complex to tropomyosin.⁹ Both cTnT and cardiac troponin-I are expressed in three different isoforms, slow- and fast-twitch skeletal muscle and cardiac muscle.^10,11 cTnT and cardiac troponin-I differ from their skeletal muscle isoforms by number of amino acid residues, six to 11 and 31, respectively. The first-generation assay for cTnT showed some degree of cross-reactivity between the cardiac and skeletal muscle isoforms, particularly in patients with chronic renal disease or skeletal myopathies.² However, the second-generation cTnT assay used in the present study has been found to be 10 times more cardiospecific, 100 times more reactive with cardiac than with skeletal muscle troponin-T,¹² and considerably less likely to give false-positive results in noncardiac conditions.¹³ In addition, the assay is able to detect cTnT whether the protein is released in the free or complexed form.¹⁴

Experience with cTnT as a biomarker of doxorubicin cardiotoxicity has been limited. Fink et al¹⁵ reported that serum concentrations of cTnT (measured using an early method) did not increase in children who had received three to five doses of anthracycline chemotherapy. In contrast, Ottlinger et al¹⁶ found that serum levels of cTnT increased from nonmeasurable to low in children receiving doxorubicin. Using the second-generation assay method, Lipshultz et al¹⁷ observed that low-level increases in serum concentrations of cTnT after the initial dose of doxorubicin were predictive of subsequent risk for left ventricular abnormalities, including dilatation and wall thinning in children. Seino et al¹⁸ detected elevated serum cTnT levels in spontaneously hypertensive rats (SHR) given eight weekly doses of 1.5 mg/kg doxorubicin. In a preliminary report, Herman et al¹⁹ found that the serum concentration of cTnT increased in SHR given cumulative doses of 7 to 12 mg/kg doxorubicin. In addition, they described a method for the immunohistochemical localization of cTnT. Using this technique, they demonstrated the loss of cTnT from the myocardium of doxorubicin-treated SHR. The present study was initiated to explore in more detail, over a range of cumulative doses of 2 to 12 mg/kg, the feasibility of using serum concentrations of cTnT as a specific biomarker for doxorubicin-induced myocardial damage. The SHR is an appropriate model for this type of study. Extensive studies have shown that the chronic administration of doxorubicin to these animals causes dose-related myocardial lesions that are consistent, reproducible, and comparable to those that develop in patients receiving this agent.^20,21 Detailed comparisons of the features of anthracycline cardiomyopathy in humans, the SHR model, and other animal models have been presented previously.^22,23 The results of this study show that in the SHR model, there are excellent correlations between the total cumulative dose of doxorubicin, the severity of the resulting cardiomyopathy, and the level of serum troponin-T.

	MATERIALS AND METHODS

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Adult male SHR (12 weeks old) were purchased from the Charles River Breeding Laboratory (Wilmington, MA). Each animal was housed individually and given rodent chow and water ad libitum. The experiment began after a 2-week acclimation period. All experimental procedures in the study were approved by the Center for Drug Evaluation and Research Institutional Animal Care and Use Committee and were in compliance with the guidelines for the Use and Care of Laboratory Animals (NIH publication 85-23).

Thirty-seven SHR were divided into six groups of five animals each and one group of seven. A control blood sample was obtained from the orbital sinus before administration of doxorubicin or saline. Groups 1 through 6 received intravenous injections of 1 mg/kg doxorubicin (via a tail vein) at weekly intervals for 2 weeks (group 1), 4 weeks (group 2), 6 weeks (group 3), 8 weeks (group 4), 10 weeks (group 5), or 12 weeks (group 6). Group 7 (saline control) received six (two animals), 10 (two animals), or 12 (three animals) weekly injections of comparable volumes of saline. Doxorubicin was obtained from Pharmacia (Columbus, OH) and was dissolved in physiologic saline to a concentration of 1 mg/mL immediately before use. The animals were observed daily and weighed weekly throughout the duration of the study.

Pathologic Evaluation of the Heart and Other Tissues
One week after the designated number of doses had been given, the animals were euthanized with an overdose of pentobarbital. Blood samples were collected for determination of serum levels of cTnT and the animals underwent complete necropsies.

Portions of the heart, kidneys, liver, lungs, and small intestine were fixed in phosphate-buffered 10% formalin, embedded in paraffin, and sectioned at a thickness of 5µm. These sections were stained with hematoxylin and eosin. Other portions of the heart were fixed as described above, embedded in glycol methacrylate resin, sectioned at a thickness of 1µm, and stained with alkaline toluidine blue. The frequency and severity of myocardial lesions induced by doxorubicin were assessed semiquantitatively by light microscopic examination.^20,21 The changes were graded on the basis of the number of myocytes showing myofibrillar loss and cytoplasmic vacuolization (score of 0 to 3 according to Billingham²⁴) in the toluidine blue–stained sections. Animals that died spontaneously during the study also underwent necropsy, but they were not included in the analyses of the data.

Immunohistochemical Study
Paraffin sections of formalin-fixed heart tissue were used to demonstrate the immunohistochemical localization of cTnT. The sections were dehydrated in graded concentrations of ethanol and washed in phosphate-buffered saline containing 0.1% bovine serum albumin. Then the sections were pretreated with Glyca antigen retrieval solution (BioGenex, San Ramon, CA). The sections and the solution were placed in a pressure cooker, which was microwaved for two cycles of 15 minutes each, first at 100% power (800 watts) and then at 30% of full power. After cooling at room temperature for 20 minutes, nonspecific binding of immunoglobulins was suppressed by incubation with 5% normal horse serum (Vector Laboratories, Burlingame, CA) for 30 minutes. A mouse monoclonal antibody (clone T1/61; Serotec, Ltd, Oxford, England) directed against cTnT was used as the primary antibody; the sections were incubated overnight at 4°C with this antibody, diluted 1/10. After washing, the sections were treated with a horse antimouse immunoglobulin G conjugated with fluorescein isothiocyanate (Vector Laboratories) and diluted 1/50 for 1 hour at room temperature. Some of the sections were counterstained with propidium iodide for the demonstration of nuclear morphology. The sections were examined using a confocal microscope. For immunohistochemical negative control procedures, the primary antibody was either omitted or replaced by equivalent amounts of normal mouse immunoglobulin G. All microscopic studies were performed without knowledge of the drug treatment.

Serum Levels of cTnT
To monitor serum levels of cTnT, blood samples were collected before dosing (control) and 1 week after administration of two, four, six, eight, 10, or 12 weekly doses of doxorubicin. Blood samples were centrifuged and the sera were frozen at -40°C until assayed. Serum concentrations of cTnT were determined by immunoassay (Elecsys Stat; Roche Diagnostics, Indianapolis, IN).

Statistical Analysis
The Mann-Whitney test for nonparametric data was used to determine the significance of differences in the cardiomyopathy scores. The Tukey-Kramer multiple comparisons test was used to assess the significance of differences among the groups in cTnT concentrations in rat serum. P .05 was taken as the level of significance. Spearman's rank correlation coefficient was used to access the relationship between: (1) the dose of doxorubicin and the serum cTnT concentration; (2) the serum cTnT concentrations and the cardiomyopathy scores; and (3) the dose of doxorubicin and the cardiomyopathy scores.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
General Toxicity and Gross Anatomic Changes
Four of the SHR treated with doxorubicin died during the 13-week experimental period (one after eight doses, two after 10 doses, and one after 12 doses). No terminal blood samples for the cTnT analyses were available from these animals, and thus they were excluded from the study. At necropsy, excessive amounts of pericardial and peritoneal fluids were observed in one of five animals that received 6 mg/kg doxorubicin, in three of four SHR given 8 mg/kg, and in all animals given 10 or 12 mg/kg of the drug; excess fluid was also noted in all four SHR that died spontaneously. Accumulation of fluid was not found in SHR given saline. The kidneys and livers of animals having fluid accumulation appeared pale in comparison with those from the saline-treated control animals.

Myocardial Pathology
Doxorubicin caused myocardial alterations that were visible by light microscopy (cytoplasmic vacuolization and loss of myofibrils) at all dose levels. These lesions appeared similar to those induced by chronic administration of doxorubicin in earlier studies in SHR^20,21 and human patients.^23,25 Both types of changes were frequently seen in the same cell, and the numbers of affected cells became progressively more numerous as the lesions became more severe. Data on the incidence and severity of these lesions at the various cumulative doses of doxorubicin are summarized in Table 1.

The serum cTnT levels (Table 2) and the total cumulative doses of doxorubicin showed a high degree of correlation (Table 3). Myocardial lesions were detected in one SHR treated with 2 mg/kg (cardiomyopathy score of 1) and two of five animals given 4 mg/kg doxorubicin (cardiomyopathy score of 1 in one animal and 1.5 in another). All SHR treated with 6 mg/kg or more of doxorubicin developed myocardial alterations (Table 1). Mild lesions (cardiomyopathy score of 1.5) were seen in five of six SHR given 6 mg/kg doxorubicin. The lesions in this group were significantly more severe than those in the groups receiving either 2 or 4 mg/kg doxorubicin (P < .05). One animal given 8 mg/kg doxorubicin had a mild lesion (cardiomyopathy score of 1.5), whereas three others had moderate lesions (cardiomyopathy score of 2). These lesions were significantly more severe than those in the groups that received 2, 4, or 6 mg/kg doxorubicin (P < .05). Cardiomyopathy scores of 2.5 were noted in three of the four SHR given 10 mg/kg doxorubicin. These lesions were more severe than those found in the groups given 8 mg/kg or less of doxorubicin (P < .05). The most severe lesions (cardiomyopathy scores of 3) were found in the hearts of all three SHR that tolerated 12 mg/kg doxorubicin. The hearts of all seven SHR given saline were normal (cardiomyopathy score of 0).

Immunohistochemical Observations
In preparations stained for the demonstration of cTnT, a green fluorescence was observed in the I bands of the myocytes of saline-treated SHR. In longitudinal sections of the myocytes, labeled I bands and unlabeled A bands of the myofibrils formed well-organized arrays (Fig 1A and 1B). In these normal myocytes, the intensity of the labeling of the I bands was strong and evenly distributed throughout the cytoplasm, in striking contrast to the unlabeled nuclei, the intercalated discs, and the interstitium (Fig 1A). In the cardiac myocytes of SHR treated with all doses of doxorubicin, the immunofluorescent staining resulted in a decreased reaction in the I bands, and this reduction varied directly with the total cumulative doses of the drug. At the low doses (2 to 4 mg/kg) of doxorubicin, the myocytes showed a mild, uneven reduction in the intensity of the staining, and the labeling of the I bands became less clearly discernible. Small, unstained vacuoles were scattered throughout the cytoplasm of some myocytes (Fig 1C and 1D). At the intermediate dose levels (6 to 8 mg/kg) of doxorubicin, the myocytes showed more diffusely reduced staining, the I bands were difficult to recognize, and the cytoplasmic vacuoles appeared larger (Fig 1E). These changes in labeling were associated with focal myofibrillar loss, as demonstrated by study with Nomarski optics (Fig 1F). At the high dose levels (10 to 12 mg/kg) of doxorubicin, the reaction for troponin-T was remarkably reduced in a large number of myocytes and the I bands were hardly discernible. Unlabeled, large-sized cytoplasmic vacuoles, profiles of different-sized myocytes indicative of hypertrophy and atrophy, and extensive myofibrillar loss and disarray were prominent (Fig 1G and 1H).

View larger version (127K): [in this window] [in a new window]   Fig 1. Matched pairs of confocal microscopic and Nomarski differential interference contrast images showing the localization of troponin-T (green fluorescence) and its correlation with histopathologic changes. Each, x 400. (A and B) Saline-treated SHR: Troponin-T is regularly distributed in the I bands of the myocytes (A). Nomarski image (B) demonstrates patterns of cross-striations in myocytes. (C and D) SHR treated with 4 mg/kg doxorubicin: Reduction in the intensity of the fluorescence of the I bands (C) and focal disruption of the cross-striations (D, arrow) are evident. (E and F) SHR treated with 8 mg/kg doxorubicin: Small cytoplasmic vacuoles (arrowhead in F) are discernible. Focal disruption, loss of I bands (arrow), and cytoplasmic vacuolization are shown. (G and H) SHR treated with 10 mg/kg doxorubicin: Extensive reduction of the green fluorescence of the I bands and many unstained cytoplasmic vacuoles are observed in the myocytes. Severe myofibrillar disruption is associated with cytoplasmic vacuolization.

Serum Troponin-T Levels
Data on the serum troponin-T concentrations found in SHR treated with the various doses of doxorubicin are summarized in Table 2. The mean concentration of troponin-T in the serum of all animals before dosing with doxorubicin was 0.02 ± 0.01 ng/mL. This value was not significantly different from the mean (0.01 ± 0.01 ng/mL) in the group given 2 mg/kg of doxorubicin; however, the only animal in this group that had myocardial lesions (cardiomyopathy score of 1) showed an elevation in serum level of cTnT to 0.03 ng/mL (Fig 2). Analysis of the data revealed a positive correlation between the serum cTnT concentrations and both the dose of doxorubicin and the cardiomyopathy scores (Table 3). After 4 mg/kg doxorubicin, the mean serum concentration of cTnT was 0.02 ± 0.02 ng/mL. However, two animals in this group had elevations to 0.05 ng/mL (Fig 2). Both of these SHR had myocardial lesions, with cardiomyopathy scores of 1 and 1.5. The cTnT concentrations in serum became considerably higher (P < .01) in the group receiving 6 mg/kg doxorubicin, in which they had a mean value of 0.13 ± 0.06 ng/mL. All six animals in this group had myocardial lesions (cardiomyopathy score of 1 in one animal and 1.5 in five animals). A further increase in serum cTnT concentrations (P < .05 in comparison to that in the 6-mg/kg group) and in the severity of the lesions was found after the administration of 8 mg/kg doxorubicin. The mean serum cTnT concentration in this group was 0.21 ± 0.05 ng/mL and the cardiomyopathy scores were 1.5 in one animal and 2.0 in three animals. The mean cTnT level of 0.24 ± 0.08 ng/mL found in the 10-mg/kg-doxorubicin group was not significantly different from that in the group given 8 mg/kg, but was significantly higher than that in the 6-mg/kg group (P < .05). Nevertheless, the cardiomyopathy scores found in these SHR (1.5 in one animal and 2.5 in three animals) were significantly more severe than those in the 8-mg/kg group (P < .05). All three animals that were given 12 mg/kg doxorubicin had increases in serum cTnT concentrations, which ranged from 0.19 to 0.66 ng/mL. Because of the small number of animals and the variability in cTnT concentrations, the mean value of 0.40 ± 0.24 ng/mL in this group was not significantly different from that in the 10- and 8-mg/kg groups. All three animals had a cardiomyopathy score of 3, which was significantly more severe than those found at lower dose levels (P < .01). At the end of the study, the mean serum troponin-T concentration in the saline-treated control SHR was 0.02 ± 0.01 ng/mL, and none of these animals had myocardial lesions.

View larger version (14K): [in this window] [in a new window]   Fig 2. Scatter diagram shows the relationship between the cumulative dose of doxorubicin (DXR) and the serum cTnT levels in individual SHR.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study demonstrates the usefulness of serum levels of cTnT for monitoring the extent of doxorubicin-induced cardiac toxicity in SHR. The data in the present study indicate that the magnitude of the increase in serumlevels of cTnT is dependent on the total cumulative dose of doxorubicin (Fig 2). Furthermore, this increase shows a positive correlation with the cardiomyopathy scores derived from microscopic examination of cardiac tissues. Serum levels of cTnT have been shown to increase in conditions, such as myocardial infarction, unstable angina, myocarditis, and heart contusions, in which irreversible cellular injury is a prominent feature.^2,3,5,26-28 In contrast, doxorubicin-induced cardiomyopathy causes diffuse degeneration of myocytes. As shown in the present study, the release of cTnT can also be used to assess this type of nonnecrotic heart damage.

The cTnT released from necrotic cells can be derived from two separate troponin pools in the myocyte. After a myocardial infarction,² cTnT levels begin to increase within 3 to 6 hours and remain elevated for several days. The early increase in cTnT is thought to come from a soluble cytosolic pool (approximately 5%), which can be released rapidly after myocyte damage.² The largest pool of cTnT in myocytes is bound to the contractile apparatus, and the breakdown of these organelles seems to account for the subsequent prolonged elevation of serum levels of cTnT after a myocardial infarction.² It seems likely that both the cytosolic and the myofibrillar forms of cTnT contributed to the elevated serum levels of cTnT observed in the doxorubicin-treated SHR. Some of the cytosolic pool of cTnT could leak from the myocyte as a result of doxorubicin-induced oxidative damage to the sarcolemma.^29,30 Myofibril-bound cTnT could also be released because myofibrillar loss occurs as a consequence of exposure to doxorubicin. Evidence for release of cTnT from the myofibrils was detected in the present study, in which the intensity of the immunohistochemical staining for cTnT was found to be decreased in the myocytes of SHR showing increased serum levels of this protein. The time course of the release of cTnT after each of the individual doses of doxorubicin remains to be evaluated. In each treatment group, determinations of serum levels of cTnT were made 1 week after the final doxorubicin dosing. Because the half-life of cTnT in blood is approximately 4 hours,^3,4,31 it seems likely that this protein is released continuously after doxorubicin-induced myocyte damage has begun to occur.

In all but one doxorubicin-treated animal, serum cTnT levels of 0.2 ng/mL or greater were indicative of myocardial cardiomyopathy scores of 2 or higher. The highest cTnT concentrations were detected in animals that had the most extensive myocyte degeneration (Tables 1 and 2). The lower limit of sensitivity for the cTnT assay used in the present study was 0.0123 ng/mL.¹² More than 30 control serum samples obtained from SHR before doxorubicin treatment or from saline-treated SHR were analyzed for cTnT, and except for two samples, they were found to be below the level of assay detection. The two samples with slight increases above the limit of detection were terminal samples from saline-treated SHR (two of seven) and had cTnT values of 0.013 and 0.02 ng/mL. Therefore, it seems that normal or baseline cTnT levels will not pose a problem in the analysis of doxorubicin cardiotoxicity. Increases in serum levels of cTnT were detected in all animals in which the hearts showed evidence of myocyte damage. An important observation was the clear increase in serum levels of cTnT detected after the administration of low cumulative doses of doxorubicin. Seven of 10 SHR treated with either 2 or 4 mg/kg doxorubicin had neither detectable morphologic alterations nor changes in cTnT concentrations. However, three other animals in these treatment groups had cardiomyopathy scores of 1 or 1.5 and slight increases in serum levels of cTnT (0.03 ng/mL in one animal and 0.05 ng/mL in two animals). Each of these levels was above the limit of sensitivity of the assay and the highest nontreatment control concentration (0.02 ng/mL). In the SHR model, the 6-mg/kg cumulative dose of doxorubicin seems to be the threshold dose that causes cardiac lesions with a score of 1.5 and elevated serum levels of cTnT (averaging 0.13 ng/mL) in all animals. These findings strongly indicate that the detection of cTnT provides information both as to the occurrence and the severity of doxorubicin-induced cardiotoxicity.

The amounts of cTnT released into the circulation seem to vary according to the type of myocyte injury. Under conditions of acute myocardial necrosis, cTnT concentrations of more than several ng/mL have been detected.² The highest cTnT concentration detected in the present study was 0.66 ng/mL in an animal that received 12 mg/kg doxorubicin and had a cardiomyopathy score of 3. All other doxorubicin-treated SHR with myocardial lesions had cTnT concentrations of 0.30 ng/mL or less. Ottlinger et al³² reported that serum levels of cTnT increased from a pretreatment value of 0.01 ng/mL to that of 0.03 to 0.09 ng/mL in children treated with doxorubicin. Experimental data show that the serum concentration of cTnT in dogs and rats subjected to ischemia (temporary coronary occlusion for 90 minutes) increased to approximately 13 ng/mL after 4.5 hours of reperfusion in dogs and 100 ng/mL after 130 minutes of reperfusion in rats.³³ The serum cTnT concentration in rats increased to 3.75 ng/mL within 6 hours after treatment with two doses of isoproterenol,⁶ which induces myocardial necrosis. Thus, considerably less cTnT is released when myocytes undergo doxorubicin-induced degeneration than when they develop necrosis.

In conclusion, the present study demonstrates the usefulness of measurements of serum levels of cTnT for the assessment of the severity of doxorubicin-induced chronic cardiomyopathy in the SHR model. This concept is supported by a highly positive correlation between the elevations in serum levels of cTnT, the cardiomyopathy scores, and the total cumulative doses of doxorubicin administered to the SHR. Additional evidence for these relationships was obtained by confocal microscopy studies demonstrating the loss of cTnT from doxorubicin-damaged cardiac myocytes.

Comparison with the results of radionuclide ventriculography and myocardial biopsies in a large number of patients receiving chemotherapy with anthracyclines would seem necessary to evaluate directly the usefulness of measurements of serum cTnT levels for the early, noninvasive detection of the cardiotoxicity induced by these agents.

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Submitted December 17, 1998; accepted February 26, 1999.

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