Cardioprotective effect of curcumin against doxorubicin-induced myocardial toxicity in albino rats.

OBJECTIVES: To study the preventive role of curcumin against doxorubicin (Dox)-induced myocardial toxicity in rats.
MATERIALS AND METHODS: Cardiotoxicity was produced by cumulative administration of Dox (15 mg/kg for two weeks). Curcumin (200 mg/kg, po) was administered as pretreatment for two weeks and then for two alternate weeks with Dox. The general observations, mortality, histopathology, biomarker enzymes like lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), biochemical parameters such as aspartate aminotransferase (AST) alanine aminotransferase (ALT) and alkaline phosphatase (ALP), antioxidant enzymes such as glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were monitored after three weeks of last dose.
RESULTS: The repeated administration of Dox induced cardiomyopathy associated with an antioxidant deficit and increased level biomarkers. Pretreatment with the curcumin significantly protected myocardium from the toxic effects of Dox by reducing the elevated level of biomarker enzymes like LDH and CPK and biochemical parameters such as AST, ALT and ALP back to normal. Curcumin increased the reduced level of GSH, SOD and CAT and decreased the elevated level of malondialdehyde (MDA) in cardiac tissue.
CONCLUSION: The biochemical and histopathology reports support the cardioprotective effect of curcumin which could be attributed to antioxidant.

Introduction

Doxorubicin/adriamycin (Dox) is a powerful, well-established and highly efficacious drug in many kinds of cancers like solid tumors, leukemia, soft tissue sarcoma, breast cancer, small cell carcinoma of the lung and esophageal carcinomas. But its clinical usefulness is still restricted due to its specific toxicities to cardiac tissues.[1] Congestive heart failure, cardiomyopathy, and electrocardiographic changes were demonstrated after cumulative Dox administration.[2] The possible mechanisms proposed for cardiotoxic effects of Dox include free radical induced myocardial injury, lipid peroxidation,[3] mitochondrial damage,[4] decreased activity of Na+–K+ adenosine triphosphate,[5] vasoactive amine release[6] and cellular toxicity.[7] Increased oxidative stress[8] and release of free radicals, including super oxide anion (O2-) and other reactive oxygen intermediates as well as endogenous antioxidant deficits have been suggested to play a major role in Dox-induced cardiomyopathy and heart failure.[9] Our earlier studies have demonstrated that polyherbal formulation/antioxidant compounds show protective effects in Dox-induced cardiotoxicity,[10] without reducing their therapeutic efficacy. Moreover, in recent years, it has been observed that there is a growing interest in the usage of natural antioxidants as a protective strategy against cardiovascular-related problems in experiments such as ischemia reperfusion[11] and Dox-induced cardiotoxicity.[12]

Curcumin (Cur), a yellow pigment obtainable from the rhizomes of the plant Curcuma longa, is a crystalline compound that has been traditionally used in medicine and culinary practices in India. Recently, curcumin was found to possess chemopreventive effect against skin cancer, forestomach cancer, colon cancer and oral cancer in mice. It possesses various pharmacological effects viz antioxidant, anti-inflammatory, anti-thromobotic, anti-apoptotic and hepatoprotective. Review of literature reveals that curcumin shows protective role against isoproterenol-induced myocardial infarction[13] and reperfusion-induced myocardial injuries in rats.[14] The non-toxic nature of curcumin[15] as well as its multiple beneficial effects has made it one of the most attractive compounds to explore for its protective role. Therefore, the present attempt has been made to investigate the role of curcumin on biochemical alterations and oxidative status against Dox-induced cardiotoxicity in rats.

Materials and Methods

Chemicals and drugs

Dox was a gift from Get Well Pharmaceuticals, India. Curcumin was purchased from Himedia Labs, India. Other chemicals were obtained from Sigma and analyzing kits were obtained from ERBA. All the chemicals were of the analytical grade.

Animals

Albinorats of either sex weighing 150-200 g procured from animal house of K.L.E.S's College of Pharmacy, Hubli, were used for the study after the clearance from Institutional Animal Ethical Committee (Ref. No. KLESCOPH/IAEC.Clear/06-07/08). Animals were acclimatized for one week to laboratory conditions before starting the experiment, they had free access to water and standard rat feed but 12 h prior to an experiment, the rats were deprived of food but not water.

Experimental protocol

After one week of acclimatization, the animals were randomly divided into 4 groups of 6 animals in each. Group 1 served as normal control and received normal saline 5 ml/kg body weight (i.p.). Group 2 animals were treated with Dox (2.5 mg/kg body weight, i.p.) in 6 equal injections alternatively for two weeks to make a total cumulative dose of 15 mg/kg body weight. Group 3 animals received curcumin (200 mg/kg body weight po) for two weeks and then alternatively with vehicle for next two weeks. Group 4 animals received curcumin (200 mg/kg body weight for two weeks) as a pretreatment followed by Dox administration as in group 2.

Enzyme assays

Thirty six hour after the last treatment, orbital blood samples were obtained under light ether anesthesia using heparinized microcapillaries for the estimation of biomarkers CPK[16] and LDH.[17] Normal as well as treated animals were observed for a period of three weeks after the last injection for the general appearance, behavior and mortality. At the end of three weeks post treatment period, animals were sacrificed under ether anesthesia and a midline abdominal incision was performed and heart tissue was quickly dissected out and washed in ice cold saline, dried on filter paper and weighed immediately. A portion of each heart was taken from all the groups and a 30% w/v homogenate was prepared in 0.9% buffered KCl (pH 7.4) for the estimation of glutathione (GSH),[18] malondialdehyde (MDA)[19] superoxide dismutase (SOD)[20] and catalase (CAT).[21] Orbital blood samples were collected before sacrificing the animals and used for estimation of AST, ALT[22] and ALP.[23] The remaining portion of the heart tissue was used for histopathological studies.

Histopathological studies

The heart tissue sections were fixed in 10% formalin. The specimens were processed by standard procedure and embedded in paraffin wax. The blocks were sectioned from the ventricular portion and stained according to the hematoxylin and eosin method and were examined by light microscopy.

Statistical analysis

The results are expressed as mean ± S.E.M. The results were analyzed using one-way ANOVA followed by Dunnett's multiple comparison test. Data was computed for statistical analysis by using Graph Pad Prism 5 Software.

Results

Effect of curcumin on Dox-induced cardiac toxicity was established by measuring cardiac biomarker enzymes and endogenous antioxidants and heart tissue histopathology.

General observation and mortality

The general appearance of all groups of animals was recorded throughout the study. In Dox-treated group, the animal fur became scruffy and developed a pink tinge. These rats also had red exudates around the eyes and nose, soft watery feces and enlargement of abdomen. These observations were significantly less in curcumin-treated group.

Gross anatomical changes of Dox -treated heart

The hearts of Dox-treated rats were dilated and both the atrium and the ventricle were enlarged and hypertrophic. The cardiac hypertrophy attributed to Dox treatment was almost completely prevented by curcumin pretreatment.

Heart weight, body weight and ratio of heart weight to body weight

Effect of Dox on heart weight, body weight, ratio of heart weight to body weight and mortality is shown in Table 1. The heart weight and ratio of heart weight to body weight in Dox-treated group significantly increased as compared to normal group. The heart weight and ratio of heart weight to body weight in Cur+Dox treated group was significantly less as compared to Dox.

Table 1

Effect of curcumin on body weight, heart weight, heart/body ratio and mortality in doxorubicin-treated rats

Serum enzyme biomarkers

Animals treated with Dox showed significant increase in the levels of CPK and LDH compared to normal [Table 2]. Cur + Dox treated group shown significantly lower levels of CPK and LDH as compared to Dox treated group.

Table 2

Effect of curcumin on AST, ALT, ALP, CK and LDH enzyme activities in doxorubicin-treated rats

The myocardial damage in the various treated groups was determined by estimating the activities of AST, ALT and ALP [Table 2]. These biochemical markers were significantly increased in the Dox group compared to control (P<0.01). Curcumin pretreatment group showed significant reduction in AST, ALT and ALP levels as compared to Dox-treated group.

Oxidant and antioxidant status

Effect of Dox on tissue lipid peroxidation, antioxidant and antioxidant enzymes is shown in Table 3. The MDA levels were increased. GSH, SOD and CAT levels were significantly decreased in Dox-treated group as compared to normal group. Cur+Dox treated group showed significant decrease (P<0.01) in the level of MDA and increase in the status of antioxidant and antioxidant enzymes.

Table 3

Effect of curcumin on oxidative status in doxorubicin-treated rats

The histology of the heart tissue from normal and Cur treated animals showed normal morphological appearances, whereas in Dox-treated group loss of myofibrils and vacuolization of the cytoplasm were observed. The histology of heart tissues from Cur+Dox treated group showed less loss of myofibrils and vacuolization of the cytoplasm [Figure 1].

Figure 1

(a) Photomicrograph of normal group heart showing normal myocardial fibers and architecture. (b) Doxorubicin-treated group showing loss of myocardial fibers and vacuolated cells. (c) Curcumin-treated group showing no vacuolated cells and myofibers loss and normal architecture. (d) Curcumin + doxorubicin treated group showing scanty myocardial fibers loss and vacuolated cells.

Discussion

The study reveals the cardioprotective effect of curcumin against Dox-induced cardiotoxicity in rats. Following lines of evidence can be emphasized from the present study. Curcumin, a yellow crystalline compound that has been traditionally used in medicine and culinary practices in India, possesses cardioprotective, hepatoprotective, chemopreventive effects against various cancers and lipid lowering properties. The present study is aimed to investigate the cardioprotective effects of oral administration of curcumin against Dox -induced cardiotoxicity.

In the Dox-treated group, the animal fur became scruffy and developed a pink tinge which in the later days of observation period was followed by red exudates around the eyes and nose. Necrosis was also observed at the site of Dox injection. These changes were less pronounced in case of curcumin pretreated group animals, which accounts for the effective cell protecting property of curcumin with anti-inflammatory, antioxidant and antifibrotic effect.

The Dox-treated rats showed increase in heart weight and decrease in body weight. Increase in the heart weight may be attributed to the enlarged, dilated and hypertrophic atrium and ventricles. However, all of the above changes were found inhibited in the Cur+Dox group.

The study revealed severe biochemical changes as well as oxidative damage in the cardiac tissue after the chronic treatment with Dox. Transaminases such as ALT and AST are liberated into the serum after extensive tissue injury. Because the heart muscle is rich in both (especially AST), it suggests that their increased level is an indicator of myocardial damage.

The Dox-treated group showed marked elevation in serum levels of AST and ALT as compared to vehicle-treated group. The mild elevations of AST have been associated with liver injury or myocardial infractions. Greater the injury size, higher the activity of AST. This result implies that the Dox when taken for long period of time could cause both liver and heart injury. A typical myocardial injury gives an AST/ALT ratio greater than 1. However, AST/ALT ratio less than 1 are found due to release of ALT from the affected liver.[24] Since the result showed AST / ALT ratio to be greater than 1 with higher doses over a long period of time, Dox is likely to lead myocardial damage.

In Cur+Dox treated group, AST and ALT levels significantly decreased as compared to Dox treated group; therefore, present results suggest that treatment of curcumin may inhibit myocardial damage. These findings confirm that curcumin is responsible for maintenance of normal structural and architectural integrity of cardiac myocytes, which can be accounted for membrane stabilizing property of curcumin, as evident from the near normal serum enzymatic activities of AST and ALT.

The serum ALP, LDH and CPK enzyme activities are important measures of both early and late phases of cardiac injury. It is reported that serum LDH and CPK were increased after Dox administration in mouse. The present results are in good agreement with our earlier findings.[10] ALP activity on endothelial cell surface is responsible, in part, for the conversion of adenosine nucleotides to adenosine, a potent vasodilator and anti-inflammatory mediator that can protect tissues from the ischemic damage that results from injury. This may account for the elevation of ALP in the Dox group, where tissue injury and inflammation are prominent. On the other hand, CPK and LDH are not specific for myocardial injury individually; however, evaluation of these enzymes together may be an indication of myocardial injury. In the preventive group, i.e. Cur +Dox, the ALP, LDH and CPK enzyme levels were decreased to a level near to that of control group, suggesting that curcumin may protect the myocardial tissue against Dox toxicity.

The mechanism of cardiotoxicity induced by Dox is not clearly known from the present study, although large body of evidence indicates toward the formation of oxygen free radicals, which can damage cells by lipid peroxidation. In rat treated with Dox, we found significant increase in heart tissue MDA levels suggesting increased lipid peroxidation. Cardiac tissue damage may be due to increased oxidative stress and depletion of antioxidants similarly in rats reported earlier.[25]

In our study, Dox treated rats showed increase in heart tissue MDA levels with decrease in levels of GSH, SOD and CAT, which confirms the oxidative stress and cardiac damage. Curcumin prevented the Dox-induced changes in MDA and enzyme levels. Significant increase in the GSH, SOD and CAT activity and decrease in lipid peroxidation in heart tissue of Cur+Dox treated groups was found. The study suggests the protective effect of curcumin.

It is commonly accepted that SOD protects against the free radical injury by converting O2- radical to H2O2 and prevent the formation of OH radicals through O2- driven Fenton reaction[26] and the H2O2 can be removed by catalase. Administration of curcumin improved the antioxidant status and thereby preventing the damage to the heart, mainly because of the antioxidant sparing action of curcumin.

The antioxidant mechanism of curcumin may include one or more of the following interactions. Scavenging or neutralizing of free radicals,[27] inhibition of oxidative enzymes like cytochrome P450,[28] oxygen quenching and making it less available for oxidative reaction, interacting with oxidative cascade and preventing its outcome[29] and disarming oxidative properties of metal ions such as iron.[30] Thus, in this work, curcumin effectively prevented tissue damage by decreasing the oxidative stress and restoring the antioxidant status.

Curcumin protects Dox-induced biochemical and histological changes in the cardiac tissue of rats either by restoring endogenous antioxidant activity or as antioxidant/both. A similar action of scavenging of Dox-induced free radicals by curcumin in the present study cannot be ruled out.

The vehicle-treated rats did not show any morphological changes and heart showed normal appearance. The cardiac muscle fibers were found to be of uniform size, shape, and configurations with no inflammatory cell infiltrates were present. Cardiomyopathy occurred in all rats injected with Dox as illustrated by the appearance of enlarged, swollen mitochondria and vacuoles within the cytoplasm. Dox produced massive change in the myocardium showing a varying degree of vacuolar changes in the cardiac muscle fibers mainly in the form of degeneration of myocardial tissue, vacuolization of the cardiomyocytes, myofibrillar loss, myocardial hypertrophy and fragmentation of the nuclei. Histopathological report suggest curcumin pretreated group attenuates the Dox -induced cardiac toxicity. The histopathological changes observed in the Dox -treated rats were similar to those previously reported.[10] This cardioprotection was apparent by fewer and less extensively swollen cardiac mitochondria and myofibrils loss.

Finally we conclude that the cardiotoxicity induced by Dox is related with oxidative stress. Anti-proliferative, anti-initiation and free radical scavenging properties of curcumin may boost myocardial integrity and attenuate the cardiac toxicity.

Curcumin has shown to be cardioprotective, which may be attributed to its potent antioxidant properties. Our study suggests that curcumin may be considered as a potentially useful candidate in combination with Dox to limit free radical mediated organ injury.