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CARDIOPROTECTIVE EFFECT OF SIMVASTATIN ON DOXORUBICIN - INDUCED OXIDATIVE CARDIOTOXICITY IN RATS.

Byline: Naiyra A. Abd Elbaky*, Azza A. Ali** and Raeesa A. Ahmed***

ABSTRACT

The usefulness of doxorubicin (DOX), a highly effective antitumor drug, is limited by the risk of developing cardiomyopathy. Subcellular changes leading to this toxicity are suggested to be mediated through a drug-induced increase in free radicals and lipid peroxidation. The present study was undertaken to investigate the possible protection that simvastatin (SIM), a lipid-lowering drug, can offer against DOX induced cardiotoxicity. DOX was administered to male Swiss albino rats in 6 equal interaperitoneal injections over a period of 2 weeks (cumulative dose, 15 mg/kg). Protection from doxorubicin-oxidative injury was investigated by administration of SIM (cumulative dose, 60 mg /kg) in 12 equal oral doses over a period of 4 weeks (2 weeks before and 2 weeks concurrent with doxorubicin). At the end of treatment, ECG was performed; myocardial antioxidants and lipid peroxidation were assayed. In addition, body weight changes during the experiment, mortality %, and histopathological examination of the heart tissue were performed. DOX treatment increased peritoneal fluid, myocardial oxidative damage and decreased survival. Myocardial antioxidants including reduced glutathion(GSH), glutathione-S -transferase (GST), and DT-diaphorase activities were reduced ,while lipid peroxidation was increased. Administration of SIM before and concurrent with DOX significantly reduced the oxidative myocardial changes-induced by DOX treatment. It also decreased mortality, and eliminated ascites. Histopathological observations were in correlation with the biochemical parameters. This indicates that SIM provide protection from DOX-induced cardiac injury in terms of oxidative stress.

Keywards: Doxorubicin, Oxidative stress ,Cardiotoxicity, Simvastatin, Antioxidant enzymes, DT diaphorase.

INTRODUCTION

Doxorubicin (DOX) is a naturally occurring anthracycline that is widely used in the treatment of a variety of human malignancies including breast cancer, small cell carcinoma of the lung and acute leukemia's (Blum and Carter, 1974). However, like most of the anticancer drugs, DOX causes various toxic effects, the commonest of which is the dose-dependent cardiotoxicity which leads to cardiomyopathy and eventually congestive heart failure (Koima et al., 1993) and (Lefrak et al., 1973). Careful monitoring of patients, and keeping the cumulative dose under the recommended limit (550 mg/m2 body surface area) (Lefrak et al., 1973) has significantly reduced the incidence of DOX-induced cardiomyopathy. However, smaller doses can compromise function that is manifested when other treatments or drugs used in combination with DOX (Billingham et al., 1977) and (Watts, 1991). Clearly, myocardial protection during DOX 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. DOX-induced myocardial injury has been believed to be mediated through different mechanisms, (Doroshow; 1983), (Olson et al., 1974)

(Singal and Pierce 1986) and (Singal et al.,1984). A common denominator to most of the proposed mechanisms is the formation of an iron-anthracycline complex that generates free radicals, which in turn, causes severe damage to the plasma membrane, and interferes with the cytoskeletal structure (Billingham et al., 1978). Due to the presence of a less developed antioxidant defense mechanism, heart is particularly vulnerable to injury by anthracycline-induced reactive oxygen species (Aebi, 1974). Therapeutic strategies, designed to augment cellular endogenous defense systems as antioxidants have been identified as a promising approach to combat against DOX toxicity (Steare and Yellon , 1995).

Statins, a widely used group of hypocholesteremic drugs, have shown a beneficial effect in reducing cardiovascular-related morbidity and mortality in patients with or without coronary artery disease and with or without high cholesterol levels (Downs et al., 1998) and (Simes et al., 2002). Recent evidences suggest that statins exert a multiplicity of favorable effects that are not directly related to their impact on lipid metabolism such as anti-inflammatory, anti-thrombotic, anti-angiogenic, and anti-hypertropic effects (Weitz-Schmidt, 2002) and (Bonetti et al., 2003). In addition, they posses immunosuppressive activity against pathological remodeling of the heart and blood vessels (Endres and Laufs, 2006) and (Harst et al., 2006). Statins are also known to decrease free radical generation in the vascular wall (Wagner et al., 2000) and in the myocardium during ischemia reperfusion injury (Maack et al., 2003), via suppression of oxygen-derived free radicals produced upon reperfusion.

In view of these facts, and as no study has been carried out concerning the cardioprotective effects of statins on doxorubicin-induced oxidative cardiac injury yet, the present study was designed to test the hypothesis whether a commonly used statin, simvastatin (SIM), could offer protection against DOX-induced cardiotoxicity on the level of oxidant production.

MATERIALS AND METHODS

Chemicals

Simvastatin (simvastat) was produced by FARCO and Alexandria Pharmaceuticals Co., Egypt. Doxorubicin hydrochloride was used in the form of an injectable commercial product (Adriablastina, Farmitalia Carlo Erba, Milan, Italy). Ellman's reagent (5,5' -dithio-bis-(2-nitrobenzoic acid); DTNB, thiobarbituric acid (TBA), trichloroaceticacid (TCA), glutathione(GSH), 2,6-dichloropenol indophenol, 1-chloro-2, 4-dinitrobenzene (CDNB), and malondialdehyde (MDA);1,1,3,3 tetra-methoxypropane used in this study were analytically pure product of Sigma-Aldrich Chemical Co., St. Louis, MO, USA. All other chemicals used were of high analytical grade.

Animals

All the experiments were carried out with male Swiss albino rats weighing 160-170 g. The animals were housed in an animal facility that was maintained with conditioned atmosphere at 25 +- 2degC and kept on standard diet pellets (El-Nasr, Cairo, Egypt), and tap water.

Experimental protocol

Rats were divided into four groups, seven rats each. Group 1: received saline only and served as control (CONT); group 2: received simvastatin (SIM), group 3: received doxorubicin (DOX); and group 4: received simvastatin prior and concurrently with doxorubicin (SIM+DOX). DOX was injected intraperitoneally in six equal doses of (2.5 mg/kg) (Siveski-Iliskovic et al., 1994) to animals in DOX, and SIM+DOX over a period of 2 weeks for a total cumulative dose of 15 mg/kg body weight. SIM (cumulative dose 60 mg/kg body wt) was administered orally to SIM and SIM+DOX groups in 12 equal doses (each treatment containing 5mg/kg) over a period of 4 weeks, 2 weeks before DOX administration and 2 weeks alternating with DOX injections. CONT and

SIM animals were injected with saline on the same regimen as DOX group.

Mortality and general condition of the animals were observed daily throughout the whole experiment lasting 4weeks. Body weights were recorded 2 times per week during the treatment and until the end of experiment.

Electrocardiography (ECG)

ECG was recorded at the end of the treatment after the last dosing. All rats were fasted overnight but had free access to water after the last dose administration. A Chart 5.0, ADI Instruments was used to record and monitor ECG tracings. Rats from each group were anesthetized with light ether anesthesia needle electrodes were inserted under the skin for the limb lead at position II. For each ECG tracing ST, QT interval, QRS complex and heart rate were measured.

Biochemical analysis

Rats were sacrificed after ECG, hearts were removed, homogenized and stored for subsequent measurement of lipid peroxides, glutathione (GSH) contents, glutathione-S-transferase (GST), and DT-diaphorase activities.

Estimation of lipid peroxidation

Lipid peroxidation products of hearts homogenate were determined as thiobarbituric acid- reactive substances (TBARS). Hearts from control and treated rats were homogenized in ice- cold 0.9% saline to get 10% homogenate. 0.5ml of the supernatant after differential centrifugation was allowed to react with 3 ml of 1% orthophophoric acid and 1 ml of 0.6% thiobarbituric acid. The tubes were heated in a boiling water bath for 45 min, cooled, and then 4 ml of n-butanol was added to each test tube, mixed vigorously and centrifuged for 5 min at 1000 rpm. The supernatant was used for determination of TBARS at 535 nm against a reagent blank. TBARS are expressed as nmol g-1 wet weight (Uchiyama and Mihara , 1978).

Estimation of reduced glutathione (GSH)

The level of GSH was determined as non-protein sulfhydryl contents (NPSH) of heart tissues and from the standard curve with commercially available GSH according to Ellman (1959). Simply 0.5 ml of tissue homogenate were added to each tube containing 0.5 ml TCA (10%). The tubes were gently shaken intermittently for 10 min, followed by centrifugation at 3000 rpm for 5 min at room temperature. Accurately, 0.1 ml of the resulting clear supernatant was mixed with 1.8 ml of phosphate buffer (0.1 M, pH 8) in separate test tubes. At least, a duplicate was made for each sample. 0.1 ml Ellman's reagent (0.39%) was added to each tube, and then, after 5 min, the optical density was measured at 412 against a reagent blank. The data were expressed as mol/g tissue. NPSH content is expressed as umole g-1 wet weight.

Determination of DT-diaphorase activity

The activity of DT-diaphorase was assayed as described by Benson et al. (1980) which involve measurement of NADH reduction at 600 nm as the electron donor and 2,6-dichloropenol indophenol as the electron acceptor. The enzyme activity was calculated using the extinction coefficient 21 mM[?]1 cm[?]1.

Determination of glutathione-S-transferase (GST) activity

The GST activity was determined using spectrophotometry according to Habig et al. (1974) . The reaction mixture (3 ml) contained 1.0 ml of 0.3 mM phosphate buffer (pH 6.5), 0.1 ml of 30 mM 1-chloro-2, 4-dinitrobenzene (CDNB) and 1.7 ml of double distilled water. After pre-incubating the reaction mixture at 37degC for 5 min, the reaction was started by the addition of 0.1 ml of tissue homogenate and 0.1 ml of glutathione as substrate. The absorbance was followed for 3min at 340 nm. Reaction mixture without the enzyme was used as blank. The activity of GST is expressed as umoles of GSH-CDNB conjugate formed/min/mg protein using an extinction coefficient of 9.6 mM-1 cm-1.

Estimation of tissue protein content

Protein was determined according to Lowry et al. (1951), using Bovine Serum Albumin (BSA) as standard, at 660 nm.

Histopathological Examination

Cardiac muscle samples were taken from rats in different groups, fixed in 10% formol saline for one day, then washed with water. Ascending serial dilutions of ethyl alcohol were used for dehydration. Samples were cleared in xylene, then embedded in paraffin at 56 degree in hot oven for 24 Hrs. Paraffin blocks were prepared and cut at 4 microns thickness. Sections are mounted on glass slides , deparaffinized and stained by hematoxylin and eosin stains for histological examinations through the light microscope.

Statistical Analysis

The experimental data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni test for multiple comparisons. Data were expressed as mean +- S.E.M. Differences were considered significant at p value of less than 0.05.

RESULTS

General Observations and Body Weights

Within 24 hours of the last injection, no mortality was seen in any of the control, DOX, SIM, and SIM+DOX group. However, during the treatment period, the mortality rate was approximately 28.5% in the DOX group, with no deaths in the CONT, SIM, nor SIM + DOX groups (Table 1). After the completion of treatment with DOX, all animals of DOX-treated group produced characteristic signs as dyspnea, enlarged abdomen, enlarged kidneys and liver, fluid accumulation in the peritoneal cavity, and animals looked weaker and

Table 1. Effects of pre- and concurrent treatment with simvastatin on doxorubicin-induced changes in body weight, heart weight , heart/body weight %, and mortality % in rats.

Groups###Intial Body###Final Body Weight###Heart Weight###Heart/Body###Mortality %###

###Weight (g)###(g)###(g)###Weigh %

CONT###164.0+- 0.68###213.9+-1.7###0.708+-0.01###0.335+-0.004###0###

SIM###165.0+-3.3###216.0+-3.1* * *###0.655+-0.014* * *###0.320+-0.001###0###

DOX###162.3+- 0.52###143.8+-5.5a###0.493+-0.021a###0.290+-0.011b###28.5###

SIM+DOX###161.7+-1.93###169.5+-5.6a* *+ +###0.573+-0.042 a* +###0.316+-0.006###0

Values are means of 7 data points [?] S.E.M.###aP/0.001, bP/0.01###compared to control, *P/0.05, **P/0.01 ,***P/0.001 compared to DOX group,###

++P/0.001, +P/0.05 compared to SIM group, respectively, using ANOVA followed by Bonferroni as a post ANOVA test.###

Table 2. Effects of pre- and concurrent treatment with simvastatin on ECG parameters of rats treated with doxorubicin

Treatment###QT(ms)###ST(ms)###QRS( ms)###HR(b/min)

CONT###78.3+- 4.09###9.1+- 0.346###20.8+- 0.92###370.5+- 14.1

SIM###89.3+- 4.21###9.3+- 0.727 * *###18.3+- 0.71###366.6+- 3.5*

DOX###97.6+-2.94b###12.5 +- 0.166a###19.2+- 1.4###444+- 13.3 b

DOX+SIM###89.6+- 4.08###7.1+- 0.51** *###20.6+- 1.17###324+- 19.74* *

Values are means of 7 experiments +- S.E.M. aP<0.01 , bP<0.05compared to control, *P<0.05, **P<0.01, ***P<0.001 compared to DOX group, respectively, using ANOVA followed by Bonferroni as a post ANOVA test.

lethargic. However, in SIM+DOX group, animals showed minimal amount of peritoneal fluid.

DOX-treatment induced a progressive reduction in body weight gain, starting from the first week of treatment, as compared to CONT or SIM-treated animals. Rats receiving SIM prior and concurrent with DOX-treatment showed a significant increase in their body weight gain as compared to DOX-treated group (Table 1, Fig. 1). At the end of experiment, DOX-treated rats showed a significant loss in their heart weights and in the heart to body weight ratio. SIM pre - and concurrent treatment significantly suppressed the reduction in heart weight as well as the decrease in the heart/body weight ratio in DOX induced rats as compared to DOX-treated group (Table 1).

Evaluation of ECG alterations

Table 2 shows the influence of SIM and /or DOX treatment on rat ECG parameters. At the end of treatment, no significant changes were observed in rats receiving SIM alone. Meanwhile, DOX treatment showed significant changes in the repolarization phase of the ECG: DOX induced a significant prolongation in ST segment, QT interval, with no significant effect on QRS complex as compared to control group. In addition, a significant increase in heart rate of DOX treated rats was observed as compared to normal group. Pretreatment with SIM significantly altered these changes in ST and QT intervals, as well as the heart rate as compared to DOX-induced group. Therefore, SIM completely prevented the changes in ECG repolarization parameters induced by DOX.

The effect of simvastatin on tissue MDA, GSH levels and antioxidant enzymes

Interstingly, treatment with SIM alone significantly reduced TBARS content and significantly increased DT-diaphorase activity in the heart tissue of normal rats. DOX administration caused a significant increase in myocardial MDA level, (80.2 +- 0.57 nmol g[?]1; p < 0.001) when compared to that of control (55.75 +- 2.7 nmol g[?]1). The pre- and concurrent treatment with SIM significantly reduced this effect (46.6 +- 2.1 nmol g[?]1; p < 0.001) compared to DOX treated group. Moreover, SIM pretreatment significantly suppressed the reduction in endogenous GSH content of the heart due to DOX injection (2.11 +- 0.02 vs 1.65 +- 0.03 umol g[?]1 in DOX group; p < 0.001). On the other hand, a marked reduction in myocardial GST (1.39 +- 0.03 vs 2.47 +- 0.13 umol min- 1 g[?]1 in control group; p < 0.001) and DT-diaphorase (0.15 +- 0.01 vs 0.74 +- 0.04 umol min1 mg[?]1protein in control group; p < 0.001) activities were observed in

DOX-treated animals. This decrease in the activities of antioxidant enzymes were significantly ameloriated by

SIM pretreatment (GST; 1.9 +- 0.17 umol min-1 g[?]1) and (DT -diaphorase; 0.34 +- 0.02 umol min-1 mg[?]1protein ;p < 0.01) respectively, as compared to DOX treated rats. (Fig. 3 A,B,C,D).

Histopathological examination of cardiac tissues

Normal control cardiac muscle showed normal characteristic features of myocardium without cellular infiltration and normal vasculature. Rats received SIM alone showed also apparently normal myocardial features similar to that of normal control. Rats administered DOX showed typical myocardial toxicity in a form of myocardial muscle coagulative necrosis with focal areas of fibrosis, vascular dilatation and congestion, valves edema, and massive mononuclear cellular infiltration. Meanwhile, rats received DOX and pretreated with SIM showed few inflammatory cells infiltration, little edema and improvement of myocardial cell necrosis (Fig. 4).

DISCUSSION

The results of the present study demonstrate for the first time that SIM pre- and concurrent treatment markedly attenuated DOX-induced oxidative cardiac injury in rats as confirmed by biochemical assays and microscopic examination.

DOX-induced cardiotoxicity and oxidative stress relationship has been confirmed in many experimental models. As the role of reactive oxygen species (ROS) including hydroxyl radical in DOX-induced cardiotoxicity have been well documented (Dorr, 1996) and (Siveski-Iliskovic et al., 1994). DOX is converted in the cardiac tissue into its semiquinone form, which is a toxic, short-lived metabolite that can interacts with molecular oxygen initiating a cascade of reaction leading to ROS generation (Davies and Doroshow, 1986). Another reported mechanism of DOX-induced oxidative stress is the formation of an anthracycline-iron (Fe2+) free radical complex (Billingham et al., 1978). The latter reacts with hydrogen peroxide to produce hydroxyl (OH*) radical. ROS react with lipids, protein and other cellular constituents causing damage to mitochondria and cell membranes of the heart muscle cells (Singal et al., 2000).

In the present study, development of oxidative cardiac injury due to multiple doses (2.5 mg/kg three times a week for 2 weeks) of DOX was confirmed by the myocardial cell damage, the alteration in oxidative stress markers as the significant increase in TBARS, and the significant decrease in activities of GST and DT-diaphorase as well as the levels of reduced GSH in the heart tissue. These changes in the antioxidant parameters are in accordance with several published reports (Azza et al., 2008) and (Saad et al., 2001). In addition, 28% of the DOX treated animals died before termination of the experiment and their body weight was significantly below that of the normal control group during most of the study. DOX also caused a significant decrease in heart weight and heart/body weight ratio, which indicate loss of myofibrils and myocardial necrosis (Weinberg and Singal, 1987). This suggestion is in accordance with the histopathological observations which revealed myocardial coagulative necrosis with focal areas of fibrosis, vascular congestion, as well as mononuclear cellular infiltration. Similar observations have also been made in earlier studies on DOX-induced cardiotoxicity (Morishima et al., 1998) and (Saad et al., 2001).

Reduction of body weight, and death associated with multiple, long administration of DOX in experimental animals, are considered multifactorial. The decrease in body weight gain in this study is in accordance with other studies (Herman et al., 2000) and (Hoekman et al., 1999), and it may be attributed to reduced food intake and inhibition of protein synthesis due to DOX treatment. On the other hand, the majority of authors dealing with DOX toxicity consider the cardiomyopathy and the nephropathy as the most important contributors to the mortality in experimental animals after treatment with DOX (Herman et al., 2000) and (Rossi et al., 1994).

ECG alterations were mointered in this study because it was demonstrated that the severity of changes in ECG are parallel to the known clinical DOX cardiotoxicity (Nousiainen et al., 1999). In the present study, there was significant increase in the heart rate, as well as significant ST and QT segment prolongation in DOX-treated rats in comparison to control animals. The increased heart rate of DOX treated rats can lead to increase in oxygen consumption and can accelerate myocardial necrosis. This suggestion is in line with the deteriorated histopathological features observed in DOX- treated rats (Fig. 4C). On the other hand, and according to Rossi et al. (1994), the reliable ECG changes noted with cumulative dose of DOX were QT prolongation and T-wave flattening. Meanwhile,Villani et al. (1990) insisted on the significance of ST interval prolongation in evaluating the ECG signs of toxicity induced in rodents with chronic administration of DOX, and all of these observations are in accordance with our result .

Glutathione is one of the essential compounds for regulation of variety of cell functions. It has a direct antioxidant function by reacting with superoxide radicals, peroxy radicals and singlet oxygen followed by the formation of oxidized glutathione (GS-SG) and other disulfides (Umalaksmi and Devaki, 1992). The depletion of GSH seems to be a prime factor that permits lipid peroxidation (Kimura et al., 2000). Glutathione S-transferase (GST) is GSH-dependent antioxidant enzyme which catalyses the conjugation of reduced glutathione via the sulfhydryl group, to electrophilic centers on a wide variety of substrates (Karthikeyan et al., 2007). This activity is useful in the detoxification of endogenous compounds such as peroxidised lipids, as well as the metabolism of xenobiotics (Valavanidisa et al., 2006). In the present study, the suppression of myocardial GSH and GST signifies that free radicals and oxidative stress are increasing due to DOX treatment, and this might be attributed to a direct action of DOX on the GSH and GST synthesis pathways, and/or an indirect function of the overwhelming response of DOX- stimulated generation of ROS. In addition, the observed decline in the level of GSH in DOX treated rat as compared to control group indicated that the depletion of GSH resulted in enhanced lipid peroxidation, and excessive lipid peroxidation caused increased GSH consumption Mohamed et al. (2000). On the other hand, DT-diaphorase is an obligatory two electron reductase enzyme, which has multiple cellular roles, many of which are poorly understood. The most important one is its action as a Phase II detoxification enzyme with the detoxifying step bypassing the formation of free radicals and so protecting tissues against mutagens, carcinogens and cytotoxics (Danson et al., 2004). Also, DT-diaphorase can recycle the membrane antioxidants ubiquinone and vitamin E. (Beyer et al., 1996) and (Siegel et al., 1997). DT-diaphorase is induced in a "stress response", along with other enzymes, including the GST which conjug te hydrophobic electrophiles and reactive oxygen species Jaiswal (2000). The fact that GST and DT-diaphorase activities were found to be reduced in DOX treated group could represent an effort, on the part of the myocardial cells, to counteract the toxic effect of DOX that finally end with the reduction in their activities. This is in line with the fact that heart tissue has limited capacity of antioxidant defense systems, including various isoforms of the enzymes DT-diaphorase and GST, that can enzymatically detoxify DOX-induced hydroxy radicals (Doroshow et al., 1980). Moreover, the reduced activity of GST in DOX treated group might be also due to decreased availability of its substrate, the reduced GSH.

Interestingly, this study presented evidence that SIM (5 mg/kg/ day 3days/week o.p., cumulatively 60 mg/kg) two weeks before and two weeks alternative with DOX treatment ameloriated the development of DOX cardiotoxicity. This was indicated by zero mortality, the suppression of body weight reduction, and the relative normalization in heart weight as well as heart/body weight ratio in the SIM+DOX group. In addition, the ECG alterations induced by DOX were almost totally prevented as well as the myocardial ultrastructure of treated animals. Above all, the cardioprotective effect of SIM was further confirmed by the significant decrease in oxidative stress as indicated by reduction in MDA production and restoration of the Protein-SH contents in the heart tissues. SIM also maintained the activities of GST and DT-diaphorase enzymes when compared to DOX treated group. Thus, SIM clearly improved "endogenous antioxidant reserve," and the latter has been suggested to improve myocardial structure and function. These results are in line with the previously confirmed data of Luo et al. (2002), which indicated that SIM increased catalase (CAT) and glutathione peroxidase (GSH-Px) activities and reduced TBARS content as well as angiotensin II level in hypertrophic rat myocardium. Also, SIM treatment for 4 weeks in patients with dyslipidemia significantly decreased TBARS concentrations and significantly increased the antioxidant enzyme (CAT, GSH-Px, and superoxide dismutase) activities in the isolated erythrocyte membranes (Broncel et al., 2006). In Fact a recent study which assessed the total antioxidant activity of the most prescribed statins, fluvastatin, atorvastatin, simvastatin and pravastatin, the scavenging capacity for hydroxyl radicals was found to be highest for SIM (Franzoni et al., 2003). Meaning that SIM is capable of scavenging strong free radicals as hydroxyl radicals, and that explain in part the apparent increase in myocardial GSH content as well as GST and DT-diaphorase activities with the consequent reduction in myocardial damage due to DOX treatment.

Other possible mechanism for the cardioprotective effect of SIM may be related to the enhancement of the NO level. As statins stabilize endothelial nitric oxide (NO) synthase mRNA, and increase levels of eNOS and thus promote NO bioavailability (Mital et al., 2000) . This will lead to preservation of tissue perfusion, as well as cardiac performance by reducing the coronary and vascular resistances and thus reducing myocardial afterload. Second, statins inhibit leukocyte - endothelial cell interactions and reduce the leukocyte extravasation (Lefer et al., 1999), which suppresses the release of proinflammatory cytokines and chemically active compounds that induce tissue inflammation and injury. The cardioprotective effects of SIM could be also accounted by its inhibitory effect on expression of TNF-a in particular, which is enhanced in various pathophysiological states, associated with increased oxidative stress (Kupatt et al., 1999) and (Meldrum et al.,1998). DOX was reported to increase TNF-a expression markdly in the subendocardial region, myocardial cells and intramyocardial vessel wall, and this increase was attributed to increased oxidative stress induced by DOX administration. TNF-a expression promotes the induction of various other cytotoxic substances on endothelial cells and myocytes, which has profound pathological significance, in terms of development of cardiomyopathy, myocyte apoptosis, and ventricular fibrosis as well as overall functional deterioration of heart (Bozkunt et al., 1998). This suggestion is in line with recent data that showed that 8 weeks of SIM therapy for hypercholesterolemic patients reduced monocyte expression of TNF-a and IL -1b by 49% and 35%, respectively (Ferro et al., 2000). All of these data are in accordance with our previously mentioned results, in which SIM significantly ameliorated oxidative myocardial damage induced by DOX in rats.

Statins are used at a dose range of 20-80 mg day[?]1 for the treatment of hypercholesterolemia. SIM -at the dose used in this study- did not change the lipid profile of the animals compared to control values (data are not shown). Thus, attenuation of the oxidative myocardial injury due to DOX treatment in this study may reflect the direct anti-oxidant activity of the drug that is independent of its cholesterol-lowering activity.

In conclusion, SIM is beneficial in ameloriating DOX-induced cardiac damage in rats within the therapeutic dose range. The mechanism of this cardioprotective effect may involve prevention of lipid peroxidation and tissue fibrosis, preservation of antioxidant glutathione, and antioxidant enzymes (GST and DT-diaphorase) as well as scavenging of free radicals.

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*Pharmacology Department, Faculty of Pharmacy, ***Antomy Department, Faculty of Medicine, King Saud University, Riyadh , KSA

**Department of Pharmacology and Toxicology, Faculty of Pharmacy, AL-Azhar University, Cairo, Egypt

Corresponding author: email: nayira@ksu.edu.sa, nayiraabdelbaky@yahoo.com
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