|Year : 2014 | Volume
| Issue : 2 | Page : 274-278
Means of evaluation and protection from doxorubicin-induced cardiotoxicity and hepatotoxicity in rats
Issam Salouege1, Ridha Ben Ali1, Dorra Ben Saïd2, Noomen Elkadri3, Nadia Kourda4, Mohamed Lakhal2, Anis Klouz1
1 Service de Pharmacologie Clinique, Centre National de Pharmacovigilance; Unité d'Expérimentation Animale, Faculté de Médecine de Tunis, Tunis, Tunisia
2 Service de Pharmacologie Clinique, Centre National de Pharmacovigilance, Tunis, Tunisia
3 Service de Médecine Nucléaire, Hôpital Militaire de Tunis, Tunisia
4 Service d'Anatomie Pathologique, Hôpital Charles Nicolle de Tunis, Tunisia
|Date of Web Publication||14-Jul-2014|
Centre National de Pharmacovigilance, 9 Rue Dr Zouhair Essafi-1006 Tunis
Source of Support: None, Conflict of Interest: None
Objectives: This work is aimed on the study of doxorubicin cardiotoxicity and hepatotoxicity in rats and the evaluation of protective effect of trimetazidine administrated concomitantly with doxorubicin for 3 days.
Materials and Methods: Male Wistar rats used were subjected to different types of treatment (3 days); A: Control, B: Doxorubicin treatment and C: Trimetazidine and doxorubicin treatment. After sacrifice, tissular distribution of doxorubicin, cardiac scintigraphy, histological examination of the myocardium, and evaluation of liver function were assessed.
Results: Obtained results show that doxorubicin has a high affinity to tissues especially the heart. It causes hepatotoxicity and cardiotoxicity marked by a significant increase of aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT) levels and drop of the left ventricular ejection fraction (EF LV ) by scintigraphy. Histological examination showed general alteration of myocardium structure. Concomitant administration of trimetazidine attenuates significantly the cardiotoxicity and hepatotoxity induced by doxorubicin.
Conclusion: We have evaluated the protective effect of trimetazidine on an animal model of doxorubicin-induced cardiotoxicity and hepatotoxicity. The evaluation of these effects were assessed by several means; tissular distribution of doxorubicin, histological examination, assessment of liver function, and EF LV by scintigraphy that characterizes the originality of this study.
Keywords: Cardiac scintigraphy, cardiotoxicity, doxorubicin, hepatotoxicity, trimetazidine
|How to cite this article:|
Salouege I, Ali RB, Saïd DB, Elkadri N, Kourda N, Lakhal M, Klouz A. Means of evaluation and protection from doxorubicin-induced cardiotoxicity and hepatotoxicity in rats. J Can Res Ther 2014;10:274-8
|How to cite this URL:|
Salouege I, Ali RB, Saïd DB, Elkadri N, Kourda N, Lakhal M, Klouz A. Means of evaluation and protection from doxorubicin-induced cardiotoxicity and hepatotoxicity in rats. J Can Res Ther [serial online] 2014 [cited 2019 Nov 20];10:274-8. Available from: http://www.cancerjournal.net/text.asp?2014/10/2/274/136557
| > Introduction|| |
Doxorubicin is an anthracycline antibiotic. It is effective in treatment of acute leukemia, sarcomas, and malignant lymphoma as well as in solid tumors (breast cancer and ovarian cancer). However, the clinical use of doxorubicin is limited by dose-dependent chronic cardiotoxicity that is characterized by an irreversible dilated cardiomyopathy and congestive heart failure. Discontinuation of doxorubicin therapy at a total dose of 550 mg/m 2 has been generally advised to reduce the incidence of this cardiotoxicity. Unfortunately, this attitude prevents administration of doxorubicin to patients who might further benefit from the antitumor effect of this drug. ,
Cardiac injury has been related to the impairment of mitochondrial functions. Numerous mechanisms for inactivation of the cardiac mitochondrial respiratory chain by doxorubicin have been proposed; such as generation of free radicals, interaction with mitochondrial DNA, modification of cardiac gene expression, alteration of calcium exchange, lipid peroxidation inducing a mitochondrial membrane dysfunction, and cardiomyocyte apoptosis. ,,,
Several cardioprotective agents such as dexrazoxane have been used to reduce cardiotoxic effects of doxorubicin. , In our study, we have chosen trimetazidine for its antioxidant activity and cellular protective effects to reduce the cardiac mitochondrial injury in experimental model. ,
We report in this study, the cardiotoxicity and hepatotoxicity of doxorubicin in male Wistar rat and we evaluate the protective effect of the coadministration of trimetazidine in the alleviation of doxorubicin toxicity. The evaluation concerned mainly; tissular distribution of doxorubicin, histological examination of the myocardium, and evaluation of liver metabolic capacity.
| > Materials and methods|| |
Doxorubicin was provided by Sigma, which was dissolved in sterile saline at a final concentration of 1.45 mg/ml. Trimetazidine (20 mg/ml) was a gift from Servier Laboratory. This solution was diluted in sterile saline to a final concentration of 5 mg/ml.
Adult male Wistar rats weighing 250-300 g were used. All rats were housed in cage and had free access to standard diet and tap water according to the standard of International Council for Laboratory Animal Science (ICLAS).
The protocol of this study was reviewed and approved by the ethics committee of animal experimentation (Tunis, Tunisia).
Rats were randomly and equally divided into three groups (eight rats per group) and were subjected to different types of treatment-by intraperitoneal injection (IP) for 3 days-as follows:
Group A: Control group receiving only sterile saline.
Group B: Treated group with doxorubicin at a dose of 3.7 mg/kg/day; corresponding to 2.5 ml/kg body weight from the doxorubicin solution already prepared.
Group C: Treated group with the same dose of doxorubicin in association with trimetazidine at a dose of 10 mg/kg/day (2 ml/kg body weight from the trimetazidine solution).
At the third day of treatment and 30 min after the last IP injection, rats were anesthetized by urethane 40% (250 μl/100 g of body weight) administered by IP route. Median thoracotomy was performed and blood samples were directly taken from the left ventricle and conserved in dry tubes containing ethylenediaminetetraacetic acid (EDTA). Blood samples were used for biochemical and doxorubicin plasmatic monitoring. After sacrifice, heart was removed and a fragment of the left ventricle was used for a histological examination, the rest served for doxorubicin monitoring in cardiac tissue. Finally, a laparotomy was performed to remove liver, kidneys, and spleen. These organs were weighed and were subjected to doxorubicin tissue monitoring. We expressed the different organ weight in their relative weight determined as follow:
Relative weight (‰) = Organ weight (g) × 1000/Animal weight (g)
Plasma samples (400 μl) were already deproteinized by acetonitrile (400 μl), then a liquid-liquid extraction with 1 ml of chloroform and isopropanol mixture (4/1, v/v) was performed. Organic phase was separated and was evaporated at 60°C under nitrogen stream. The residue was then dissolved in 100 μl of the mobile phase and 50 μl of the aliquot was injected into the HPLC (high performance liquid chromatography) column.
Liver, heart, kidneys, and spleen were mixed in chloroform and isopropanol mixture (80/20, v/v). After centrifugation, the supernatant was dried at 60°C under nitrogen stream. The residue was reconstituted in 2 ml of mobile phase and then extracted using the same method described below.
Doxorubicin and daunorubicin (internal standard) were separated by HPLC. On a LiChrospher Column RP-8 (5 μm, 250 × 4 mm; Merck, D-6100, Darmstad, F.R. Germany) maintained at 30°C using a Merck model L-6000 solvent pump with isocratic elution mode, model L-4250 variable wave length UV detector set at 250 nm. The separation was performed using a mobile phase, containing 65% sodium acetate buffer and 35% acetonitrile, pumped at a flow of 1 ml/min. Retention time of doxorubicin and daunorubicin was 4.8 and 7.5 min, respectively. ,
The cardiac scintigraphy was realized (at the third day) 30 min after the last treatment. The protocol consists of an intravenous (i.v.) injection of 1 ml of pyrophosphate and 20 mCi of TcO4. The acquisition was made by gamma camera synchronized to electrocardiogram (ECG): 1800 cycles, 16 images by cycle, zoom 1, matrices =64 * 64, collimator Pinhole 1.1 mm, 7 min. From the pictures obtained we calculated the left ventricular ejection fraction (EF LV ).
The left ventricular fragments were fixed for 24 h in formalin (10%) and embedded in paraffin. Sections of 5 μm were stained with hematoxylin and eosin (H and E). The histological examination was achieved with a photonic microscope (×400). For each analyses a score was attributed according to Billingham's classification. 
The monitoring of aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), and gamma-glutamyl trasferase (GGT) activities in blood samples was carried out with an automat (COBAS® Integra 800) using an enzymatic technique enzyme-multiplied immunoassay technique (EMIT).
The liver biotransformation capacity was determined by measurement of the metabolic transformation of lidocaine and its metabolite monoethylglycinexylidide (MEGX); 2.5 g of rat liver was homogenized in 5 ml of NaCl 0.9% and incubated with 2.13 mM of lidocaine in a rotatory mixer at 37°C for 2 h. The reaction was stopped by addition of 6 ml of acetonitrile and the medium was mixed for 1 min. The organic phase was separated by centrifugation at 1,700 g for 5 min and was evaporated under nitrogen stream at 60°C, in order to concentrate the sample. The resulting sample was mixed again and centrifuged at 1,700 g for 5 min. The supernatant was extracted by chloroform and was evaporated at 60°C under nitrogen stream. The residue was then dissolved in 200 ml of the mobile phase (0.05 M KH 2 PO 4 , pH 4, methanol 90%:10%), and 50 ml of the aliquot was injected into the HPLC column. Lidocaine and its major metabolite MEGX were separated by HPLC on a LiChrospher Column RP-8 (5 μm, 250 × 4 mm; Merck, D-6100, Darmstadt, F.R. Germany) maintained at 80°C, using a Merck model L-6000 solvent pump, a model D-2500 integrator, and a model L-4250 variable-wavelength UV detector set at 210 nm. The separation was performed using the mobile phase pumped at a flow of 1.2 ml/min. The results were expressed as the ratio of lidocaine biotransformation and the quantity of MEGX. ,,
Statistical study was performed using BIOSTAT software. Values are expressed as means ± standard error of the means (M ± SEM). Statistical significance was evaluated using Student's t-test. Differences between means were statistically significant for P < 0.05.
| > Results|| |
The comparison between the control group and treated groups at third-day of the treatment did not show significant differences in relative organ weight (P > 0.05) [Table 1].
|Table 1: Relative organ weight and doxorubicin levels in different organs|
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The plasma doxorubicin concentration was 351.25 ± 175.19 ng/ml. This indicates the good absorption of the doxorubicin administered by IP injection.
A maximum of tissular doxorubicin concentration was obtained in the heart (707.25 ± 227.16 ng/g), which suggests the high affinity of anthracycline antibiotic to cardiomyocytes.
The statistical analysis using the Student's t-test to compare the plasmatic concentrations of the doxorubicin as well as the tissular concentrations (heart, liver, kidneys, and spleen) in doxorubicin treated group versus protected group by trimetazidine, shows a nonsignificant increase in doxorubicin heart concentration in protected group. Moreover, a nonsignificant difference has been found in the rest of tissular and plasmatic concentrations in the two treated groups. This indicates that the coadministration of trimetazidine and doxorubicin does not modify the doxorubicin absorption and distribution [Table 1].
The scintigraphy results founded show a significant decline of EF LV after 3 days of the doxorubicin treatment; from 76.6 to 62%. The coadministration of trimetazidine protects the cardiomyocyte and significantly ameliorates the EF LV [Table 2].
|Table 2: Plasmatic activities of transaminases, percentage of transformed lidocaine, formed MEGX and other metabolites, histological study, and evaluation of FELV by scintigraphy|
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Photonic microscope observation of the left ventricles slices in control group shows streaky muscular cells arranged in bundles with conservation of the striation. The cellular cores are lengthened and regular [Figure 1]a.
|Figure 1: Histological study. Heart sections from different treated groups. (a) Section from control group showing normal architecture. (b) Altered structure with apparent apoptosis and necrosis area were observed in doxorubicin-treated group. (c) Trimetazidine treatment preserved myocytes structure. (H and E, ×400)|
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In doxorubicin treated group, several lesions have been observed which concerned the majority of the myocardium. These lesions consist on a modification of the normal architecture with modification of the sarcoplasma, muscular fibre dissociation, and a striation loss. On the slices, we observed necrotic and apoptotic areas. In addition, zones of inflammatory reaction were observed associated to a lymphoid infiltration in one case [Figure 1]b.
Injuries observed in group C (treated by doxorubicin and trimetazidine) were less severe than those observed in group B (treated by doxorubicin). The vacuolization is discreet and the striation is preserved in most areas. The apoptotic and inflammatory infiltrated areas were less extensive [Figure 1]c.
We found a significant increase in the histological score (number of apoptotic areas and altered myocytes) in treated groups versus control group. Even, we observed a reduction of Billingham score in group C versus group B; this difference remains statistically nonsignificant [Table 2].
Hepatic toxicity was evaluated by measurement of AST, ALT, and GGT levels. The AST and ALT levels in control group were respectively 247 ± 54 and 58.18 ± 11.89 IU/l. Using the Student's t-test two-tailed comparisons between control group (group A) and treated groups (group B, C) we found a significant decrease of plasmatic concentrations of the AST in the group cotreated by trimetazidine versus control group and the treated group by doxorubicin.
A significant increase on the ALT level has been found in treated groups (B and C) when compared to control group, but this difference remains nonsignificant between group B and group C [Table 2].
The doxorubicin does not modify the transformed lidocaine quantity. However, a significant decrease in the quantity of its major metabolite (MEGX) and a significant increase in the other metabolites were obtained in treated groups when compared to the control group. This suggests a deviation of metabolic pathway of lidocaine biotransformation to other metabolites induced by doxorubicin. This effect is significantly accentuated by trimetazidine [Table 2].
| > Discussion|| |
The oxidative stress following the free radical generation and alteration in redox status were the possible mechanisms of doxorubicin cardiotoxicity. The voltage-dependent anion channels tend to open, leading to a mitochondrial membrane permeability transition and a release of cytochrome c (proapoptotic protein) which is able to induce apoptosis. , Also, doxorubicin causes oxidative alteration of the mitochondrial permeability transition pore. , This effect is responsible of the decrease in mitochondrial calcium loading capacity. These damages can be restored by the addition of an antioxidant. 
Doxorubicinol has compromised both systolic and diastolic function in isolated heart preparations and blocks ATPase activity in the sarcoplasmic reticulum, mitochondria, and sarcolemma. 
The administration of a cumulative dose of 11.1 mg/kg of doxorubicin (during 3 days) was used in a short-term model. In the literature, induction of acute doxorubicin cardiotoxicity was made using a cumulative dose varying between 10-12 mg/kg in a single injection. , In this work, we induced acute cardiotoxicity after 3 days of doxorubicin treatment. Thus, this model allowed us to induce toxicity of doxorubicin and to evaluate the protective effect of trimetazidine.
Doxorubicin cardiotoxicity induced in court period treatment, is the most serious complication that can be observed. The injury was evaluated by determination of aminotransferase levels, a suitable marker of structural membrane damage.
Doxorubicin is known to have a high affinity for cardiolipin and for a major phospholipids component of the mitochondrial membrane in heart cells; it was responsible for a selective accumulation of doxorubicin inside cardiac cells.  The originality of the present study is the tissular monitoring of doxorubicin. We found an important tissue fixation of doxorubicin especially in the heart. As well as a high affinity of doxorubicin for other organs was observed such as spleen, liver, and kidneys (in decreasing order). This tissue affinity can, in part, explain the cardiotoxicity of doxorubicin induced in court period treatment. 
The AST is more specific of the cardiac cytotoxicity. We did not found a significant increase on the AST levels after three days of doxorubicin treatment. Concerning the ALT, we found a significant increase of its levels in the treated group with doxorubicin which may be explained by the hepatic toxicity potency of this drug.  The changes were prevented with doxorubicin used together with trimetazidine.
When evaluating liver metabolic capacity, doxorubicin alters hepatocytes function results confirmed by the deviation of metabolic pathway of lidocaine biotransformation. This alteration is due to a mitochondrial dysfunction.
Doxorubicin has been shown to cause apoptosis either in the rat heart or in isolated myocytes or in cell culture models.  Doxorubicin cardiomyopathy was histologically characterized by cytoplasmic vacuolization due to dilatation of the sarcotubules and loss of myofibrils. In addition, the desmin can be profoundly altered even after a single injection of doxorubicin and the sarcomeres and intercalated discs were hardly visible.  In the present study, the myocardium histological examination shows a loss of the normal striation aspect, vacuolization, and the presence of several apoptotic areas and necrosis (significant increase of the Bellingham score).
In the literature, the EF LV has never been evaluated by scintigraphy. This method can sensibly evaluate the EF LV .
We found a significant decrease of the EF LV estimated to 14.6% in doxorubicin group due to reduction of the contractile capacity in the heart. It was reported that the free radical accumulation, the reduction of the energizing level, and the myocardial apoptosis are responsible for this effect. In addition, other factors are implied; as the change in the cytoskeleton and in the contractile element of the cardiomyocytes.
Trimetazidine is a cytoprotective drug, which counteracts the metabolic disorders occurring in ischemic cells and it contributes to the preservation of the energy metabolism of cardiomyocytes exposed to ischemia or hypoxia. Mechanism of action of trimetazidine has not been elucidated; may be it depends on a direct effect on mitochondrial enzymatic systems impaired by ischemic conditions. Trimetazidine has been shown to maintain cellular homeostasis, preserve electrical and contractile function. 
In this study, we used the trimetazidine to limit the tissular damages. Our results show that trimetazidine exerts a cardioprotective effect in doxorubicin treated rats. Its use leads to a reduction of AST levels that could explain conservation of good cellular metabolic function. Additionally, it induces a partial reduction of myocardium lesions. In fact, the trimetazidine is a cellular protector that acts by preservation of the mitochondrial function and maintaining the calcium mitochondrial homeostasis.  This result was confirmed by the preservation of the lidocaine metabolic pathway to the formation of MEGX as a major metabolite.
These encouraging results support the possibility to realize clinical test to better visualize the protective effect of trimetazidine which is already used in human therapeutics.
In conclusion, we have developed an animal model of doxorubicin-induced acute toxicity. This model was used to evaluate the protective effect of trimetazidine against doxorubicin injuries using enzymatic monitoring, histological examination, and scintigraphy of EF LV .
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[Table 1], [Table 2]