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(Circulation. 1995;91:10-15.)
© 1995 American Heart Association, Inc.

Articles

Probucol Protects Against Adriamycin Cardiomyopathy Without Interfering With Its Antitumor Effect

N. Siveski-Iliskovic, MD; M. Hill, MSc; D.A. Chow, PhD; P.K. Singal, PhD

From the Division of Cardiovascular Sciences (N.S.-I., M.H., P.K.S.), Department of Physiology, and St Boniface General Hospital Research Center; and the Department of Immunology (D.A.C.), Faculty of Medicine, University of Manitoba, Winnipeg, Canada.

Correspondence to Dr P.K. Singal, St Boniface General Hospital Research Center, Rm R3022, 351 Tache Ave, Winnipeg, Manitoba, Canada R2H 2A6.

	Abstract

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Background The usefulness of adriamycin (ADR), a potent antitumor antibiotic, is limited by the development of life-threatening cardiomyopathy and congestive heart failure. Subcellular changes leading to heart failure are suggested to be mediated by a drug-induced increase in free radicals and lipid peroxidation. In an earlier study, concurrent treatment with probucol (PROB), a lipid-lowering drug with strong antioxidant properties, was shown to offer only partial protection against ADR cardiomyopathy. The present study had two aims: to determine whether this protective effect can be improved further by extended treatment with PROB, and to determine whether PROB affects the antitumor properties of ADR.

Methods and Results ADR (cumulative dose, 15 mg/kg body wt) was administered in rats in six equal injections (IP) over a period of 2 weeks. Three weeks after the end of treatment, cardiomyopathy and congestive heart failure were characterized by ascites, congested liver, depressed cardiac function, elevated left ventricular end-diastolic pressure, and myocardial cell damage. Myocardial glutathione peroxidase (GSHPx) activity was decreased and lipid peroxidation was increased. Administration of PROB (cumulative dose, 120 mg/kg body wt) in 12 equal injections (IP), before and concurrent with ADR, completely prevented these cardiomyopathic changes, normalized left ventricular function, lowered mortality, and eliminated ascites. Treatment with PROB was also accompanied by an increase in myocardial GSHPx and superoxide dismutase activities with a concomitant decrease in lipid peroxidation. Tumor regression in syngeneic DBA/2 mice inoculated with L5178Y-F9 lymphoma cells in the ADR+PROB group was significant and comparable to the ADR group.

Conclusions These data show for the first time that PROB can provide complete protection against ADR cardiomyopathy without interfering with antitumor properties of the drug. This protective effect of PROB may be related to the maintenance of the antioxidant status of the heart.

Key Words: antioxidants • heart failure

	Introduction

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Adriamycin, an anthracycline antibiotic, is a very good drug for the treatment of a variety of tumors. However, the development of cardiomyopathy and congestive heart failure has greatly limited the use of this highly effective antitumor drug. The risk of developing cardiomyopathy becomes unacceptably high beyond the cumulative dose of 550 mg/m² body surface area.¹ Although careful monitoring of patients as well as keeping the cumulative dose under the recommended limit of 550 mg/m² has significantly reduced the incidence of adriamycin-induced cardiomyopathy, smaller doses can also compromise function that is manifested when cardiac workload is increased.^{2 3} Furthermore, the incidence of heart disease can be enhanced by other treatments or drugs used in combination such as thoracic irradiation, cyclophosphamide, bleomycin, methotrexate, and cisplatin.^{4 5} Thus, cardiomyopathic changes may occur at a dose lower than the "safe upper limit" of 550 mg/m². Myocardial dysfunction, years after the therapy, has also been recognized.^{6 7} Clearly, myocardial protection during adriamycin treatment should remain the goal to enhance the beneficial effects of the drug as well as to remove the risk of short- or long-term cardiac problems.

Although adriamycin-induced injury appears to be multifactorial,^{8 9 10 11 12 13 14 15 16 17 18} a common denominator to most of the proposed mechanisms is the mediation of oxygen radicals.^{2 19 20} Because of the presence of semiquinone in the tetracyclic aglycone molecule of adriamycin, the drug is reported to increase the oxygen radical activity^{17 18} as well as peroxidation of polyunsaturated fatty acids within the membrane phase.²⁰ This may also explain adriamycin-induced defects in membrane function due to use of this drug.^{13 19 21} In a recent study using a model of adriamycin-induced congestive heart failure in rats, we reported that concurrent treatment with probucol, a lipid-lowering drug with strong antioxidant property, offered partial protection against adriamycin-induced myocardial cell damage.²²

The present study was undertaken to examine whether this protection by probucol against adriamycin-induced cardiomyopathy can be improved by extending the drug exposure. The other important goal of the study was to test whether probucol has an effect on the antitumor properties of adriamycin.

	Methods

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Animal Model
Male Sprague-Dawley rats (body weight, 250±10 g) were maintained on normal rat chow. Rats were divided into four groups: CONT (control), ADR (adriamycin treated), PROB (probucol treated), and PROB+ADR (probucol plus adriamycin treated). Adriamycin (doxorubicin hydrochloride) was administered intraperitoneally in six equal injections (each containing 2.5 mg/kg ADR) to animals in ADR and PROB+ADR groups over a period of 2 weeks for a total cumulative dose of 15 mg/kg body wt as described previously.^{11 22} Probucol (cumulative dose, 120 mg/kg body wt) was also administered intraperitoneally to PROB and PROB+ADR groups in 12 equal injections (each treatment containing 10 mg/kg) over a period of 4 weeks, 2 weeks before adriamycin administration and 2 weeks alternating with adriamycin injections. CONT animals were injected with the vehicle alone (lactose, 75 mg/kg in saline) on the same regimen as ADR. Treated as well as control animals were observed for as long as 3 weeks after the last injection for their general appearance, behavior, and mortality. At the end of the 3-week posttreatment period, animals were assessed hemodynamically. Their hearts were used to study myocardial antioxidants, lipid peroxidation, and ultrastructure.

Hemodynamic Studies
Animals were anesthetized with sodium pentobarbital (50 mg/kg IP). A miniature pressure transducer (Millar Micro-Tip) was inserted into the left ventricle via the right carotid artery. Left ventricle systolic (LVSP), left ventricle end-diastolic (LVEDP), aortic systolic (ASP), and aortic diastolic (ADP) pressures were recorded on a Beckman Dynograph.

Bioassays
Catalase Assay
Ventricles were homogenized in 9 vol of 0.05 mol/L potassium phosphate buffer (pH 7.4) and centrifuged at 40 000g for 30 minutes. Supernatant (50 µL) was added to the cuvette containing 2.95 mL of 19 mmol/L H₂O₂ solution prepared in potassium phosphate buffer.²³ The color was read at 240 nm on a Zeiss spectrophotometer every minute for 5 minutes. Commercially available catalase was used as a standard. Specific activity of the enzyme was expressed as units per milligram of tissue protein.

Glutathione Peroxidase Assay
Glutathione peroxidase (GSHPx) activity was expressed as nanomoles of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidized to nicotinamide adenine dinucleotide phosphate (NADP) per minute per milligram of protein, with a molar extinction coefficient for NADPH at 340 nm of 6.22x10⁶.²⁴ Cytosolic GSHPx was assayed in a 3-mL cuvette containing 2.0 mL of 75 mmol/L phosphate buffer (pH 7.0). The following solutions were then added: 50 µL of 60 mmol/L glutathione, 100 µL glutathione reductase solution (30 U/mL), 50 µL of 0.12 mol/L NaN₃, 0.10 µL of 15 mmol/L Na₂EDTA, 100 µL of 3.0 mmol/L NADPH, and 100 µL of cytosolic fraction obtained after centrifugation at 20 000g for 25 minutes. Water was added to make a total volume of 2.9 mL. The reaction was started by the addition of 100 µL of 7.5 mmol/L H₂O₂, and the conversion of NADPH to NADP was monitored by a continuous recording of the change of absorbance at 340 nm at 1-minute intervals for 5 minutes. Enzyme activity of GSHPx was expressed in terms of milligrams of protein.

Superoxide Dismutase Assay
Supernatant (20 000g for 20 minutes) was assayed for superoxide dismutase (SOD) activity by following the inhibition of pyrogallol autooxidation.²⁵ Pyrogallol (24 mmol/L) was prepared in 10 mmol/L HCl and kept at 4°C before use. Catalase (30 µmol/L stock solution) was prepared in an alkaline buffer (pH 9.0). Aliquots of supernatant (150 µg protein) were added to Tris · HCl buffer containing 25 µL pyrogallol and 10 µL catalase. The final volume of 3 mL was made up of the same buffer. Changes in absorbance at 420 nm were recorded at 1-minute intervals for 5 minutes. SOD activity was determined from a standard curve of percentage inhibition of pyrogallol autoxidation with a known SOD activity. This assay was highly reproducible, and the standard curve was linear up to 250 µg protein with a correlation coefficient of .998. Data are expressed as SOD units per milligram protein compared with the standard.

Malondialdehyde Assay
Measurement of lipid peroxidation by determining myocardial thiobarbituric acid reactive substance (TBARS) content was performed using a modified thiobarbituric acid (TBA) method.²⁶ Hearts were quickly excised and washed in buffered 0.9% KCl (pH 7.4). After the atria, extraneous fat, and connective tissue were removed, the ventricles were homogenized in the same buffer (10% w/v). The homogenate was incubated for 1 hour at 37°C in a water bath. A 2-mL aliquot was withdrawn from the incubation mixture and pipetted into an 8-mL Pyrex tube. One milliliter of 40% trichloroacetic acid (TCA) and 1 mL of 0.2% TBA were promptly added. To minimize peroxidation during the subsequent assay procedure, 2% butylated hydroxytoluene was added to the TBA reagent mixture.²⁷ Tube contents were vortexed briefly, boiled for 15 minutes, and cooled in a bucket of ice for 5 minutes. Two milliliters of 70% TCA was then added to all tubes, and the contents were again vortexed briefly. The tubes were allowed to stand for 20 minutes. This was followed by centrifugation of the tubes for 20 min at 3500 rpm. The color was read at 532 nm on a Zeiss spectrophotometer and compared with a known TBARS standard.

Ultrastructural Studies
For ultrastructural studies, three to five hearts in each group were processed as described.^{2 11} Hearts were washed in cold 0.1 mol/L sodium phosphate buffer (pH 7.4). Tissue samples, 4 to 6 mm in size, were taken from four different areas of the subendocardium as well as the subepicardium of the free left ventricle wall between the midregion and apex of the heart. The tissue pieces were immersed for 15 minutes in 0.1 mol/L phosphate buffer (pH 7.4) containing 3% glutaraldehyde. This briefly fixed tissue was further cut into pieces smaller than 1-mm cubes. Aldehyde fixation was continued for a total duration of 2 hours. The tissues were washed for 1 hour in the above phosphate buffer containing 0.05 mol/L sucrose. Postfixation was done in 2% OsO₄ for 1.5 hours, after which the tissue pieces were dehydrated in graded alcohol series. Tissue embedding was done in epon. Ultrathin sections were placed on Formvar-coated grids and stained with uranyl acetate and lead citrate. Electron micrographs of the subendocardial and subepicardial regions from the four groups were compared to establish ultrastructural differences.

Studies of Effect of Probucol on Antitumor Properties of Adriamycin
Male inbred DBA/2 mice (total number, 44) were inoculated with 10⁶ L5178Y-F9 cells.²⁸ A subcutaneous injection in 100-µL aliquot was made into the middle of a shaved area on the back of each syngeneic DBA/2 mouse. Tumor size was assessed as surface area by multiplying the larger tumor dimension by that at a 90-degree angle from it measured with a vernier caliper on the days on which adriamycin or probucol was administered. Probucol and adriamycin administrations were initiated 10 days after tumor inoculation. In ADR (n=10) and PROB+ADR (n=12) groups, each animal received a total cumulative dose of 15 mg/kg of adriamycin in six equal IP injections (ie, six treatments) over 2 weeks. In PROB (n=12) and PROB+ADR groups, each animal received a total cumulative dose of 60 mg/kg of probucol in six equal IP injections (ie, six treatments) over 2 weeks. The CONT group (n=10) received coconut oil (medium in which probucol was dissolved) in six IP injections for a total cumulative dose of 6 mL/kg.

Proteins and Statistical Analysis
Proteins were determined by the method of Lowry and associates.²⁹ Data were expressed as mean±SEM. For a statistical analysis of the data, group means were compared by one-way ANOVA, and Bonferroni's test was used to identify differences between groups. Statistical significance was acceptable to a level of P<.05.

	Results

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General Observations and Hemodynamics
Within 1 week after the completion of treatment with adriamycin, animals in the ADR-only group had enlarged abdomen, developed ascites, and appeared weaker and lethargic. At death, all ADR group animals had a significant amount of peritoneal fluid (Table 1). In the PROB+ADR animals, the amount of peritoneal fluid was insignificant. Of 14 animals examined for ascites in the PROB+ADR group, 12 animals had no ascites, 1 animal had 6 mL of ascites, and 1 animal had 13 mL of ascites. During the posttreatment period, the mortality rate was approximately 32% in the ADR group. There were no deaths in the CONT, PROB, and PROB+ADR groups (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effects of Probucol Pretreatment and Concurrent Treatment on Adriamycin-Induced Changes

ASP and LVSP were significantly depressed, whereas LVEDP was significantly elevated in the ADR group alone. In the PROB+ADR group, these parameters were no different from those of the CONT and PROB groups (Table 1).

Ultrastructure
Morphological changes in the ADR group were typical for adriamycin-induced cardiomyopathy and included swelling of mitochondria, vacuolization of the cytoplasm, formation of lysosomal bodies, and dilation of the sarcotubular system (Fig 1, top). Ultrastructure of hearts from the PROB+ADR group was indistinguishable from that of the CONT group and had regular myofibrillar arrangement, maintained sarcotubular system, and preserved mitochondria (Fig 1, bottom).

View larger version (157K): [in this window] [in a new window]   Figure 1. Photomicrographs of adriamycin-induced changes included loss of myofibrils, swelling of mitochondria (M), and sarcoplasmic reticulum (arrow). Vacuolization (*) and dense bodies (double arrow) are also apparent. Bottom, Hearts from the PROB+ADR (probucol and adriamycin) group did not show any of these changes. Mitochondria (M), myofibrils (MF), sarcoplasmic reticulum (arrow), and other cellular details are normal. Magnification line in both figures is 1 µm. For ultrastructural studies, three to five hearts in each group were processed as described.

Antioxidants
In addition to the study of different antioxidant enzyme activities, the amount of lipid peroxidation was determined by evaluating myocardial TBARS content (Table 2). GSHPx activity was reduced and TBARS were increased significantly in the ADR group (Table 2). In the PROB+ADR group, GSHPx activity as well as TBARS were near control levels. Total SOD activity in the PROB and PROB+ADR groups was significantly higher, whereas catalase activity did not show change in any group.

View this table: [in this window] [in a new window]   Table 2. Effects of Probucol Pretreatment and Concurrent Treatment on Adriamycin-Induced Changes in Antioxidant Enzyme Activities and Lipid Peroxidation

Antitumor Effect
To assess the effects of probucol on the antitumor efficacy of adriamycin, subcutaneous tumor growth was studied in mice (Fig 2). The L5178Y-F9 lymphoma model in mice was chosen because it was cloned directly from the L5178Y,³⁰ one of the standard experimental tumors used to examine chemotherapeutic efficacy of different anticancer drugs, including adriamycin and its derivatives.³¹ A significant reduction in the tumor size was seen in the ADR group as well as the PROB+ADR group compared with the CONT and PROB groups. There was no significant difference in the tumor size between ADR and PROB+ADR groups.

View larger version (26K): [in this window] [in a new window]   Figure 2. Plot of effects of ADR (adriamycin), PROB (probucol), and PROB+ADR (probucol plus adriamycin) on the regression in tumor size in lymphoma-bearing DBA/2 mice. Tumor size in the ADR group and PROB+ADR group was significantly less compared with the CONT and PROB groups. Data are mean±SEM of 10 to 12 animals. *P<.05 using ANOVA and P<.05 using ANOVA and Bonferroni's post-hoc test indicate significant differences from the corresponding points in the CONT and PROB groups.

	Discussion

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Development of cardiomyopathy and congestive heart failure due to adriamycin in the present study was established by the myocardial cell damage, depressed systolic pressures, increase in LVEDP, ascites, and congestive changes in liver. In a previous study, we reported that simultaneous treatment with probucol mitigates adriamycin-induced cardiomyopathic changes as well as congestive heart failure.²² However, the protection achieved was not complete, and the conclusion reached was that concurrent treatment with probucol delayed or decreased development of cardiomyopathy. The present study demonstrates for the first time that an extended exposure to probucol before and during treatment completely prevents the development of adriamycin cardiomyopathy as indicated by zero mortality in the PROB+ADR group and maintenance of the hemodynamic function as well as myocardial ultrastructure in these animals. In the previous study that used only concurrent therapy with probucol, some ultrastructural injury due to adriamycin, as indicated by perimitochondrial edema, was still present at 3 weeks after treatment.²² In the present study, no such ultrastructural injury was apparent.

For any practical application of probucol in combination with adriamycin, however, it was important to examine whether probucol modified the antitumor properties of adriamycin. In this regard, another important finding in the present study is that in an established tumor model in syngeneic DBA/2 mice,²⁸ probucol had no effect on the antitumor activity of adriamycin. A comparable tumor regression seen in the ADR and the PROB+ADR groups further supports the potential usefulness of combination therapy. Because of the two phenolic groups in its molecular structure, probucol has been reported to be a strong antioxidant.^{32 33}

Beneficial effects of probucol may be independent of its cholesterol-lowering property and may involve antioxidant mechanisms.²² It is important to note that adriamycin has been shown to promote the production of free radicals,^{17 18} and these toxic species are known to cause myocardial dysfunction.^{34 35} Data on lipid peroxidation are also in concert with this suggestion, as probucol caused complete prevention of the adriamycin-induced increase in TBARS. Another important factor in the improvement of antioxidant status is the prevention of an adriamycin-induced decrease in GSHPx activity. Instead, in the PROB+ADR group, there was a significant increase in the GSHPx as well as SOD activities. Thus, probucol clearly improved "endogenous antioxidant reserve," and the latter has been suggested to improve myocardial structure and function.³⁴ Although mechanisms for probucol-induced increase in antioxidants (GSHPx and SOD) are not clear, the study clearly demonstrates that probucol may be providing protection by acting as an antioxidant as well as by promoting endogenous antioxidants. A reason for the partial protection achieved with parallel treatment with probucol and the complete protection achieved with pretreatment and concurrent treatment may be that pretreatment further strengthened antioxidant defenses and better prepared the heart for the oxidative stress due to adriamycin.

It should also be noted that some of the signs and symptoms of heart failure seen in this study could result from nephrotoxicity. In this regard, development of chronic glomerulonephritis with a nephritic syndrome due to anthracyclines has been reported.³⁷ Although physical findings (ascites, edema) are mutual for nephrotic syndrome and congestive heart failure, hemodynamic data, myocardial ultrastructural injury, and enzyme changes seen in our study clearly document myocardial dysfunction. The prevention of myocardial changes along with a lack of these signs and symptoms suggests an important significance for myocardial changes. Whether nephrotoxicity also occurred and was corrected will have to be determined in a separate study. At any rate, it does not diminish the suggested principle of the use of protection with an antioxidant.

Other approaches to prevent cardiotoxicity have met with limited success. For example, dose reduction with continuous infusion ^{38 39} and weekly low-dose schedule⁴⁰ reduced cardiotoxicity possibly by avoiding high peak concentrations. However, a progressive fall in resting left ventricular ejection fraction,⁴¹ as well as the occurrence of cardiomyopathy several years after the therapy, have also been reported.⁴² The ICRF 187 given with an anthracycline has also been reported to reduce cardiotoxicity.⁴³ However, leukopenia and thrombocytopenia were the major dose-limiting toxic side effects of this combination.⁴⁴ Thus, safer combination therapy for adriamycin is still needed.

Because probucol prevents adriamycin-induced cardiomyopathy but does not affect antitumor properties of the drug, a combination therapy for the treatment of patients with a variety of soft and solid malignancies holds great promise. Clinical trials are required to establish the beneficial effects of this approach in patients. Further studies are also needed to elucidate the mechanisms by which probucol influences endogenous antioxidant activities.

	Acknowledgments

 
This work was supported by grants from the Manitoba Heart and Stroke Foundation and Medical Research Council of Canada. Dr Siveski-Iliskovic was supported by a fellowship from the Faculty of Graduate Studies, University of Manitoba.

Received September 6, 1994; accepted October 24, 1994.

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