Rutin trihydrate attenuated cisplatin- induced cardiac toxicity in isolated perfused rat’s hearts

The present study aims to investigate the protective effect of rutin against cisplatin induced toxic effects on the mechanical performance of the myocardium, histopathology, and oxidative stress in isolated perfused rat hearts. Cardiotoxicity of cisplatin was assessed at three dosage levels (1, 7, and 14 mg/l) in the isolated perfused rat hearts. The toxic effect of cisplarin was assessed on left ventricular pressure (LVP), heart rate (HR), dp/dt(max), dp/dt (min), perfusion pressure, pressure-time index, contractility index and duration of diastole. Measurements were carried out one minute before perfusion of cisplatin and 60 minutes after perfusion. pressure (LVP), heart rate (HR), dp/dt (max), dp/dt (min), perfusion pressure, pressure-time contractility index and duration of diastole. Results were analyzed using Labchart pro 7 software.


Abstract Background
The present study aims to investigate the protective effect of rutin against cisplatin induced toxic effects on the mechanical performance of the myocardium, histopathology, and oxidative stress in isolated perfused rat hearts.

Methods
Cardiotoxicity of cisplatin was assessed at three dosage levels (1,7, and 14 mg/l) in the isolated perfused rat hearts. The toxic effect of cisplarin was assessed on left ventricular pressure (LVP), heart rate (HR), dp/dt(max), dp/dt (min), perfusion pressure, pressure-time index, contractility index and duration of diastole. Measurements were carried out one minute before perfusion of cisplatin and 60 minutes after perfusion.

Results
Cisplatin reduced signi cantly (p < 0.05) in a dose-dependent manner LVP, dp/dt(max), dp/dt(min) and pressure-time index. Perfusion of rutin trihydrate (1 µM/l), 10 minutes before administration of cisplatin and throughout the experiment signi cantly (p < 0.05) attenuated the detrimental effects of cisplatin on cardiac parameters. Cisplatin caused degeneration and necrosis of cardiac muscle cells, while rutin reduced these changes and restored normal heart histology. Moreover, cisplatin reduced the myocardium concentration of reduced glutathione and increased the level of malondialdehyde, whereas rutin almost reversed these changes.

Conclusion
Cisplatin-induced dose-dependent impairment of several parameters of cardiac function and produced histopathological alterations in isolated rat hearts. These harmful effects of cisplatin were ameliorated by rutin trihydrate. These ndings suggest the potential protective effects of rutin trihydrate against cisplatin-induced cardiotoxicity.

Background
Cisplatin is a broad spectrum anti malignant drug. It is widely used in in the management of testicular, ovarian, non-small cell lung cancer, gastric and esophageal cancers, and hematological malignancies [1].
The clinical use of cisplatin is signi cantly limited by its adverse effects and toxicities affecting the gastrointestinal, renal, neurological, and hematological systems [2].
Increase in oxidative stress and decrease in antioxidant enzymes are linked with cardio-toxic effects of cisplatin [11]. Rutin has been reported for bene cial protective effects against variety of drug induced toxicities including doxorubicin and cisplatin induced cardiac toxicity and memory de cits [12][13][14].
These protective effects of rutin are related to its profound antioxidant and anticancer properties.
Considering this hypothesis, the e cacy of rutin against cisplatin-induced cardiac toxicity was investigated. It is also an established fact that therapeutic effect of cisplatin is signi cantly increased with the increased dose [15], but at the cost of severe adverse effects [16]. Keeping this in mind the dose dependent cardiotoxicity of cisplatin was explored. The data related to prophylactic effect of rutin against dose dependent cisplatin-induced cardiotoxicity is scarce. Therefore, the focus of our study was to evaluate the protective effect of rutin on cisplatin induced derangements on mechanical performance of the myocardium, histopathology, and oxidative stress in isolated perfused rat hearts. via a bridge ampli er (ADInstruments, Sydney Australia). All hearts were allowed to perfuse for 15 minutes to achieve stabilization. Following the stabilization period, the parameters recorded are Left ventricular pressure (LVP), heart rate (HR), dp/dt (max), dp/dt (min), perfusion pressure, pressure-time index, contractility index and duration of diastole. Results were analyzed using Labchart pro 7 software. Measurements were carried out at one minute before perfusion of cisplatin (Ebewe Pharma, Austria) and 60 minutes after perfusion. In a separate group, rutin trihydrate (Sigma-Aldrich GmbH, Germany) was perfused for 10 minutes before administration of cisplatin and throughout the experiment. A third sham group was perfused with Krebs-Henseleit solution for 60 minutes. Responses obtained 60 minutes after perfusion were expressed as percentages in relation to those obtained at one minute before perfusion of cisplatin.

Histopathological examination
At the end of the experiment, specimens of the ventricles of control, cisplatin 14mg/l and rutin and cisplatin groups were dissected, divided into two portions, one for detecting the underlying structural changes and the other for endogenous antioxidants analysis. The rst specimens were then xed in 10% buffered formalin and processed with para n wax. For histopathological feature examination, 5 µm sections were stained with hematoxylin and eosin for the analysis using a light microscope.

Analysis of endogenous antioxidants
The other heart specimens were washed by PBS then further divided and stored at -80 o C till used for quanti cation of (GSH and MDA) the antioxidant / pro-oxidant variables. On the day of quanti cation, each specimen was weighed, homogenized in relevant speci ed buffers, centrifuged, and processed following the methodology of sample preparation for each estimate.

Reduced Glutathione (GSH) Assay
The principle is based on the reaction of reduced GSH with 5.5-dithiobis-(2-nitrobenzoic acid) (DTNB) (E. Merck Ltd., Bombay, India), according to the method of Owens and Belcher [17]. The absorbance was measured by Schimadzu double beam spectrophotometer (UV200S, Japan) at 412 nm. The amount of GSH present was calculated using a standard solution of GSH containing 1 mg of GSH/ml of 3% metaphosphoric acid. The increase in the extinction at 412 nm was proportional to the amount of GSH present and was expressed as nmol/g wet tissue.

Malondialdehyde (MDA) Assay
The principle is based on the reaction of MDA with thiobarbituric acid (TBA) (E. Merck Ltd., Bombay, India), according to the method described by Buege and Aust [18]. The concentration of the MDA-TBA complex was being quanti ed spectrophotometrically at 532 nm and expressed as nmol/g wet tissue.
Statistical analysis Data reported as mean ± SEM. One-way analysis of variance followed by Tukey's multiple comparison tests as a post hoc test was used to analyze the data. A p-value of 0.05 or less was taken as a criterion for statistically signi cant differences. Data were analyzed using Graph Pad Prism 5 software.

Results
1-Effect of cisplatin (1, 7, and 14 mg/l) on parameters of mechanical performance of the heart Table 1 depicts the effects of different concentrations of cisplatin (1, 7 and 14 mg/l) on left ventricular pressure, dp/dtmax, dp/dtmin, heart rate, contractility index and pressure-time index. Eight isolated hearts were perfused with Krebs solution containing different concentrations of cisplatin for 60 minutes. The response obtained at minute 60 is calculated as a percentage of the response obtained at the rst minute before perfusion of hearts with cisplatin. All concentrations of cisplatin-induced a statistically signi cant (p < 0.01) and dose-dependent reduction in left ventricular pressure. Cisplatin at 7 and 14 mg/l concentrations, signi cantly (p < 0.05) reduced dp/dt max and dp/dt min. Cisplatin exerted a bell-shaped concentration-response relationship on heart rates. Cisplatin at 1 mg/l increased the heart rate signi cantly (P < 0.01), while 7 mg/l slightly reduced the heart rate. All three doses of cisplatin increased signi cantly (p < 0.01) the contractility index, whereas, Cisplatin 7 and 14 mg/l signi cantly (p < 0.05) reduced pressure-time index.  Table 2 shows the effects of rutin trihydrate (1 µM) on alterations of the mechanical performance of isolated rat hearts induced by cisplatin (14 mg/l). Rutin trihydrate (1 µM) was perfused 10 minutes before administration of cisplatin. Rutin (1 µM) signi cantly (p < 0.05) reversed the reduction of left ventricular pressure induced by perfusion of hearts with 14 mg/l cisplatin for 60 minutes and attenuated the reduction in dp/dt max and dp/dt min exerted by cisplatin. There was a trend for the heart rate to increase, when hearts were perfused for 60 minutes with cisplatin (14 mg/l), but this increase did not attain statistical signi cance. Perfusion of hearts with rutin (1 µM) signi cantly (p < 0.05) reduced heart rate. After 60 minutes of perfusion, cisplatin (14 mg/l) signi cantly (p < 0.05) increased contractility index. Perfusion of hearts with rutin (1 µM) signi cantly (p < 0.01) reversed this increase in contractility index and signi cantly (p < 0.01) reversed the reduction in pressure-time index induced by cisplatin. When hearts were perfused for 60 minutes with cisplatin(14 mg/l), there was a tendency for the duration of diastole to decrease, but perfusion of hearts with rutin (1 µM) signi cantly (p < 0.05) increased period of diastole. There was a trend for the perfusion pressure to increase, when hearts were perfused for 60 minutes with cisplatin (14 mg/l), but this increase was statistically non-signi cant. Perfusion of hearts with rutin (1 µM) signi cantly (p < 0.01) reduced perfusion pressure. Histological Examination Figure 1: A specimen of the control group showed single, oval, and centrally located nuclei of cardiomyocytes with regularly arranged cardiac myo bers and no histopathological lesion observed in the control group. While cisplatin group showed degenerated muscle cells, necrosis of cardiac myo bers and dissolution of nuclei. The cardiac myo bres in this group were found to be in a disarrayed pattern compared to the control muscle (Fig. 2). Another specimen of cisplatin specimen showed degenerated muscle cells with interstitial edema (Fig. 3). Addition of rutin to hearts perfused with cisplatin 14 mg/l showed a reduction in the toxic changes and the normal heart histology was signi cantly restored ( Fig. 4).

Antioxidants Analysis
Perfusion of hearts with cisplatin 14 mg/l signi cantly (p < 0.01) reduced the concentration of glutathione, while perfusion of hearts with both rutin and cisplatin approximately restored the concentration of the reduced form of glutathione (Fig. 5). Whereas, perfusion of hearts with cisplatin greatly and signi cantly (p < 0.01) increased the concentration of malondialdehyde. Addition of Rutin to the perfusate containing cisplatin greatly reduced the tissue concentration of malondialdehyde (Fig. 6).

Discussion
Cardiac dysfunction can be demonstrated by hemodynamic changes in various parameters of cardiac function. Parameters of cardiac function assessed in this study included left ventricular pressure, heart rate, dp/dt (max), dp/dt (min), perfusion pressure, pressure-time index, contractility index and duration of diastole. Data from this study revealed that cisplatin reduced signi cantly left ventricular pressure, dp/dt(max), dp/dt (min) and pressure -time index in the isolated rat heart in a dose-dependent manner after perfusion for 60 minutes. In the present study, histopathological ndings of heart tissues depicted that cisplatin administration induced degeneration and necrosis of cardiac muscle with the dissolution of nuclei. These observations correlate well with the alterations of hemodynamic and mechanical function induced by cisplatin. Moreover, cisplatin increased lipid peroxidation in heart tissue and reduced antioxidant activity.
Numerous injurious effects of cisplatin on the heart have been reported. Cisplatin has been shown to produce creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) leakage, a progressive reduction in total carnitine and ATP and depletion of glutathione content in cardiac tissue [10]. It is also reported to produce a dose-dependent decrease in contractile force, heart rate, and coronary ow [19] and induce oxidative and nitrosative stress [10,20]. Furthermore, it caused apoptosis [21], deterioration of diastolic cardiac function [22] oxidative stress, in ammation, and histopathological alterations [10].
Several mechanisms have been reported to explain the harmful effects of cisplatin on the myocardium. Cisplatin can generate reactive oxygen species, such as superoxide anion and hydroxyl radical [23]. These reactive oxygen species are associated with an increase in lipid peroxidation [24]. Lipid peroxidation of the cardiac membrane results in leakage of lactate dehydrogenase and creatinine kinase from cardiac myocytes [19]. Moreover, cisplatin induced a fall in plasma concentrations of various antioxidants [25]. This may lead to failure of the anti-oxidative defense mechanism against free radicalmediated organ damage. These effects are evident in the current study. Cisplatin increased lipid peroxidation, which is indicated by increased tissue concentration of malondialdehyde and reduced tissue antioxidant activity, which is indicated by reduced tissue reduced glutathione concentration. Glutathione has a direct antioxidant function by reacting with free radicals. This activity is useful in the detoxi cation of peroxidized lipids. Thus, it is likely that cisplatin-induced detrimental effect on the hemodynamic and mechanical function and histology of the myocardium, by increasing lipid peroxidation and reduction of glutathione tissue concentration.
To overcome cisplatin-induced cardiotoxicity, numerous efforts have been made in the past [26]. In the present study, rutin trihydrate reversed the harmful effects of cisplatin on left ventricular pressure, dp/dt(max), dp/dt(min), contractility index, pressure-time index, heart rate, duration of diastole and perfusion pressure. Moreover, cisplatin restored the normal histology of the myocardium, inhibited lipid peroxidation, and increased reduced glutathione concentration. Coronary blood ow is impeded during systole, the duration of diastole is an important determinant of coronary blood ow and subendocardial perfusion. Rutin trihydrate by prolonging the duration of diastole and reducing perfusion pressure could greatly improve coronary blood ow and subendocardial perfusion.
Cisplatin causes cardio-toxic effects by oxidative stress and decrease in antioxidant enzymes [11]. Rutin has been previously reported for bene cial protective effects against variety of drug induced toxicities including doxorubicin and cisplatin induced cardiac toxicity [12][13][14]. The protective effects of rutin trihydrate observed in our current study mostly likely occurred due to its profound anti-oxidant properties. This protective effect of rutin trihydrate could be attributed to its antioxidant property. Rutin trihydrate has been shown to enhance superoxide dismutase (SOD) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity [27] and reduced lipid peroxide content increased by cisplatin in kidneys of rats [28][29]. Moreover, rutin is reported to have anti-in ammatory and anti-apoptotic effects. It inhibited NFκB and TNF-α pathway mediated in ammation and caspase-3 cell apoptosis [30].

Conclusion
Cisplatin-induced dose-dependent impairment of several parameters of cardiac function, induced histopathological alterations, and reduced antioxidant activity. These harmful effects of cisplatin were ameliorated by rutin trihydrate. Cisplatin the ability of rutin to ameliorate cisplatin-induced cardiotoxicity could be attributed to its antioxidant effects.
The attenuation of the detrimental effects of cisplatin by rutin trihydrate could have eventual clinical implications in terms of more rational usage of this agent. Rutin trihydrate appears to be a potential candidate to ameliorate cardiotoxicity associated with cisplatin in rats. Hence, it would be worthwhile studying the effects of rutin trihydrate supplementation in cisplatin-treated cancer patients, in the hope of reducing cisplatin-induced cardiotoxicity. Control group showed single, oval and centrally located nuclei of cardiomyocytes with regularly arranged cardiac myo bers. No histopathological lesion observed in control group.

Figure 2
Cisplatin Group depicts degenerated muscle cells, necrosis of cardiac myo bers, and dissolution of nuclei in cardiac myo bers. The cardiac myo bers in this group were found to be in disarrayed pattern compared to the control muscle.  Rutin+cisplatin group shows reduction in the toxic changes and the normal heart histology is signi cantly restored. (Cisplatin 14mg/l, rutin 1µM).