Cardioprotective Effects of Hesperetin against Doxorubicin-Induced ...

Cardiovasc Toxicol (2011) 11:215–225 DOI 10.1007/s12012-011-9114-2

Cardioprotective Effects of Hesperetin against Doxorubicin-Induced Oxidative Stress and DNA Damage in Rat P. P. Trivedi • S. Kushwaha • D. N. Tripathi G. B. Jena

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Published online: 7 May 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Doxorubicin is a widely used chemotherapeutic agent; however, its clinical uses are limited due to its cardiotoxicity associated with an induction of oxidative stress. This study was aimed to investigate the protective effect of hesperetin against doxorubicin-induced cardiotoxicity in rats. Doxorubicin was administered at the dosage of 4 mg/kg bw/week, ip for a period of 5 consecutive weeks. Hesperetin was administered at the dosages of 25, 50 and 100 mg/kg bw, po by gavage for 5 consecutive days in a week for 5 weeks. The animals were killed 1 week after the last injection of doxorubicin. Hesperetin at the doses of 50 and 100 mg/kg bw significantly reduced MDA and increased GSH levels in the doxorubicin-treated animals. Further, hesperetin significantly reduced doxorubicin-induced DNA damage as well as apoptosis at 25, 50, and 100 mg/kg bw as evident from the comet and terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling assays, respectively. Thus, hesperetin ameliorated doxorubicin-induced cardiotoxicity by reducing oxidative stress, abnormal cellular morphology and DNA damage in rat. Moreover, nuclear factor-kappa B, p38, and caspase-3 play a role in the hesperetin-mediated protection against doxorubicin-induced cardiotoxicity. This study indicates

P. P. Trivedi S. Kushwaha D. N. Tripathi G. B. Jena (&) Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab 160062, India e-mail: [email protected]; [email protected] P. P. Trivedi e-mail: [email protected] S. Kushwaha e-mail: [email protected] D. N. Tripathi e-mail: [email protected]

the protective effect of hesperetin against doxorubicininduced cardiotoxicity. Keywords Doxorubicin Hesperetin Heart Oxidative stress Apoptosis Abbreviations DOX Doxorubicin TUNEL Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling NFjB Nuclear factor-kappa B SD Sprague–Dawley CMC Carboxy methyl cellulose H&E Hematoxylin and eosin EtBr Ethidium bromide DMSO Dimethylsulfoxide NMPA Normal melting point agarose LMPA Low melting point agarose EDTA Ethylenediamine tetraacetic acid HBSS Hank’s balanced salt solution ip Intraperitoneal TL Tail length TM Tail moment OTM Olive tail moment % DNA % DNA in comet tail MDA Malondialdehyde GSH Reduced glutathione

Introduction Majority of the toxicities induced by chemotherapeutic agents are caused due to the generation of oxidative stress. Hence, intervention with anti-oxidants may help in the

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diminution of toxicities caused by these agents. Doxorubicin (DOX), an anticancer anthracycline antibiotic derived from the fungus Streptocococcus peuceticus var. caesius, is one of the potent components of the modern chemotherapeutic regimens. The effectiveness of DOX as an antineoplastic agent is due to its activity as a ‘topoisomerase II poison’. DOX inhibits the negative supercoiling of DNA and intercalates into DNA resulting in a change in the stereochemical conformation of its double-helical structure. Thereby, it inhibits DNA and RNA polymerases and hence interferes with DNA transcription and replication [1]. However, mechanisms leading to its antitumor activity appear to be different from those leading to its cardiotoxicity. Various mechanisms known to be involved in DOXinduced cardiotoxicity include oxidative stress, mitochondrial impairment, calcium overloading, cell necrosis and induction of apoptosis by activating both intrinsic and extrinsic pathways [2, 3]. The cardiotoxic effects produced by DOX lead to the development of cardiac dysfunction, cardiomyopathy and finally congestive heart failure [4]. Chronic administration of DOX results in dose-dependent, late-onset and irreversible cardiomyopathy [5]. DOX has been known to cause apoptosis in H9c2 cells, which results in severe cardiotoxicity in vitro [6]. It has been shown to cause cardiotoxicity in rats by inactivating mitochondrial cytochrome C oxidase [7]. It results in cardiotoxicity in mice by interfering with the glutathione redox cycling [8]. Cancer patients receiving anthracycline chemotherapy have been known to suffer from severe cardiotoxicity [9]. Due to severe cardiotoxic effects produced by DOX, patients receiving DOX are advised to undergo close monitoring of their left ventricular function [10]. Cardiovascular system is one of the major organ systems of the body, and patients suffering from cardiac insufficiency have life-threatening risks. Cardiotoxicity limits the clinical use of DOX, and hence it is of great concern for the successful management of chemotherapy. Therefore, much attention has been given nowadays to discover compounds, which can prevent detrimental cardiotoxic effects of this frontline chemotherapeutic agent [3]. Hesperetin (5, 7, 30 -trihydroxy-40 methoxy flavanone), a Chinese traditional medicine, is a bioflavonoid occurring ubiquitously in citrus fruits [11]. Hesperetin is present in the citrus fruits as hesperidin (its glycoside form), which acts as a prodrug and gets deglycosylated to hesperetin by intestinal bacteria prior to absorption [12, 13]. It has been reported that hesperetin reached the peak plasma concentration of 0.46 ± 0.07 and 1.28 ± 0.13 lmol/l after an intake of 0.5 and 1 l of orange juice, respectively, by healthy volunteers, and the urinary excretion (4.1 ± 1.2– 7.9 ± 1.7% of the intake) was nearly complete 24 h after the orange juice ingestion [14]. Hesperetin possesses multiple of biological activities such as antioxidant,

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Cardiovasc Toxicol (2011) 11:215–225

anti-inflammatory, anticarcinogenic, antihypertensive and anti-atherogenic effects [15, 16]. It has been found to show neuroprotective effects in mice, anti-convulsive effect in the rat hippocampus in vitro, prevention of DMBA-induced mammary cancer in female Sprague–Dawley rats, protection against oxidative stress in diabetic rats and suppression of the formation of aberrant crypt foci in colon cancerinduced rats [17–21]. It shows antioxidant property due to its ability to penetrate within the lipid bilayers [22]. Cardiotoxicity caused by DOX is mediated by the induction of oxidative stress. Moreover, hesperetin exerts its beneficial effects against oxidative stress-induced cellular damage. Hence, it was considered for this study in order to investigate its possible protective effects against DOX-induced cytotoxicity and genotoxicity in rat heart. Intervention with hesperetin against DOX-induced cardiotoxicity was evaluated using oxidative stress parameters, comet assay, halo assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and histopathology. Further, immunohistochemistry of the cardiac sections was carried out in order to explore the role of nuclear factor-kappa B (NFjB), p38 and caspase-3 on hesperetinmediated protection against doxorubicin-induced cardiac toxicity. This study depicts the amelioration of DOXinduced cardiotoxicity by the intervention with hesperetin.

Materials and Methods Animals All the animal experiment protocols were approved by the Institutional Animal Ethics Committee (IAEC) and the experiments on animals were performed in accordance with the CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals) guidelines. Experiments were performed on male Sprague– Dawley (SD) rats (200–220 g) procured from the Central Animal Facility (CAF) of the institute. All the animals were kept under controlled environmental conditions at room temperature (22 ± 2°C) with 50 ± 10% humidity and an automatically controlled cycle of 12 h light and 12 h dark. Standard laboratory animal feed (purchased from the commercial supplier) and water were provided ad libitum. Animals were acclimatized to the experimental conditions for a period of 1 week prior to the commencement of the experiment.

Chemicals DOX (CAS no. 29042-30-6) was obtained as a gift sample from Intas Pharmaceuticals Ltd., Ahmedabad, India.


Hesperetin (CAS no. 41001-90-5), carboxy methyl cellulose (CMC) (CAS no. 9004-32-4), hematoxylin and eosin (H&E), ethidium bromide (EtBr) (CAS no. 1239-45-8), bovine serum albumin (CAS no. 9048-46-8), dimethylsulfoxide (DMSO) (CAS no. 67-68-5), trizma (CAS no. 77-86-1), and SYBR Green 1 (CAS no. 163795-75-3) were purchased from Sigma–Aldrich Chemicals, Saint Louis, MO, USA. Normal melting point agarose (NMPA), low melting point agarose (LMPA), triton X-100, ethylenediamine-tetraacetic acid (EDTA), and Hank’s balanced salt solution (HBSS) were obtained from HiMedia Laboratories Ltd., Mumbai. Experimental Design Animals were randomized into 7 groups consisting of 5 animals in each group. A single injection of DOX at a dose of 15 mg/kg administered intraperitoneally (ip) has been reported to induce cardiotoxicity in rats [23]. DOX, at a cumulative dose of 15 mg/kg over a period of 6 weeks, in male rats has been known to induce cardiotoxicity [5]. Based on this report, it was decided to administer DOX at a dose of 4 mg/kg bw to the male SD rats once a week for a period of 5 weeks to induce cardiotoxicity. Hesperetin has been reported to exert pronounced chemopreventive effect at the dose of 20 mg/kg/day for 16 weeks in rat colon carcinogenesis model [24]. Based on this rationality, three dose regimens of hesperetin, such as 25, 50, and 100 mg/kg bw for 5 days in a week up to 5 consecutive weeks, were selected for this investigation. DOX was dissolved in isotonic saline solution and was administered through ip route. Hesperetin was suspended in 0.1% carboxymethyl cellulose (CMC) and was administered through po route. Group 1 received normal saline once in a week and served as control. Groups 2 and 3 received CMC and hesperetin (100 mg/kg bw), respectively, 5 days in a week for 5 consecutive weeks and served as vehicle control and hesperetin control, respectively. Group 4 received DOX 4 mg/ kg bw once in a week for 5 consecutive weeks. Groups 5–7 received DOX 4 mg/kg bw once in a week for 5 consecutive weeks and hesperetin 25, 50, and 100 mg/kg bw 5 days in a week for 5 consecutive weeks, respectively. Animals were killed 1 week after receiving the last injection of DOX.

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determination of MDA level in tissue samples. MDA level was estimated spectrophotometrically as an end product of lipid peroxidation using thiobarbituric acid–reactive substance method. Lipid peroxidation was calculated from the standard curve generated using 1, 1, 3, 3 tetramethoxy propane and expressed as nmol MDA/mg of protein.

Measurement of Glutathione (GSH) Content For the determination of GSH content, an equal volume of 5% sulphosalicylic acid was added to tissue homogenate and mixed. The mixture was kept for 30 min in ice bath. After centrifugation for 10 min, the supernatant was collected carefully, and GSH content was measured using Ellmann’s reagent [5, 50 -dithiobis-2-nitrobenzoic acid (DNTB) solution] according to the method described [26]. GSH levels were calculated using a standard reference curve using reduced glutathione as a standard. Results were expressed in lmol GSH/mg protein.

Determination of Protein Content Protein concentration in heart homogenate was determined as described [27] with bovine serum albumin as the standard protein.

Histological Evaluation Histological slides were prepared as previously standardized in our laboratory [28]. The heart was fixed in 10% formalin, dehydrated in increasing concentrations of ethanol and embedded in paraffin. Tissue sections (5 lm) were mounted on glass slides coated with Mayer’s albumin and dried overnight. The sections were then deparaffinized with xylene, rehydrated with alcohol and water. The rehydrated sections were stained using H&E, mounted with DPX mounting media, and examined under the microscope at both high (40 9) and low (10 9 and 20 9) magnifications (Olympus BX51 microscope, Tokyo, Japan). Heart sections from each animal were evaluated for structural changes.

Measurement of Lipid Peroxidation Single-Cell Gel Electrophoresis (SCGE) Assay The lipid peroxide level in tissue homogenate was measured according to the method previously described [25] with some modifications. The heart was homogenized in ice-cold phosphate buffer (pH 7.4) for the determination of lipid peroxidation level. After homogenization and centrifugation, the supernatant was collected for the

A small piece of heart was placed in 1 ml cold Hank’s balanced salt solution (HBSS) containing 20 mM EDTA and 10% DMSO and minced into fine pieces. Five microliters of this was mixed with 95 ll of low melting point agarose (LMPA) and layered over the surface of a frosted

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slide [pre-coated with 1% normal melting point agarose (NMPA)] to form a microgel and allowed to set at 4°C for 5 min. A second layer of 1% LMPA was added and allowed to set at 4°C for 5–10 min. The slides were then immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris–HCl buffer (pH 10.0), 1% Triton X-100 and 10% DMSO) at 4°C for 24 h. After 24 h, the slides were washed with chilled water, then coded and placed in a specifically designed horizontal electrophoresis tank, and DNA was allowed to unwind for 20 min in alkaline solution containing 300 mM NaOH and 1 mM EDTA (pH [13.0). Electrophoresis was conducted at 0.6 V/cm, 300 mA for 30 min in a horizontal electrophoresis unit (SCIE-PLAS Ltd., UK, Max volts 1,000 V, Max current 500 mA). After neutralization, the slides were washed with chilled water and stained with SYBR Green I (1:10,000 dilution). Slides were rinsed briefly with double-distilled water and cover slips were placed before image analysis. The fluorescent labeled DNA was visualized using an AXIO Imager M1 fluorescence microscope (Carl Zeiss, Germany), and the resulting images were captured on a computer and processed with image analysis software (Metasysyem software, Comet Imager V.2.0.0) [29, 30]. Duplicate slides were prepared for each treatment and were independently coded and scored without the knowledge of codes. The parameters for the DNA damage analysis include the following: tail length (TL, in lm), tail moment (TM), olive tail moment (OTM), and % DNA (% DNA) in comet tail. The edges of the slides, any damaged part of the gel, any debris, superimposed comets, and comets without distinct head (‘hedgehogs’ or ‘ghost’ or ‘clouds’) were not considered for the analysis. Halo Assay The halo assay was performed essentially as described with some modifications [31]. A small piece of heart was placed in 1 ml cold HBSS containing 20 mM EDTA and 10% DMSO, minced into fine pieces, and 5 ll of this was suspended in 95 ll of 0.5% LMPA and layered over the surface of a frosted slide (pre-coated with 1% NMPA) to form a microgel and allowed to set at 4°C for 5 min. A second layer of 1% LMPA was added and allowed to set at 4°C for 5–10 min. The slides were immersed in freshly prepared lysis solution (2.5 M NaCl, 2 mM EDTA, 10 mM Tris (pH 10.0), 1% Triton X-100) for 24 h at 4°C. Following lysis, the slides were incubated with alkaline medium (0.3 M NaOH) for 20 min and stained using EtBr. Samples were run in duplicate, and 100 cells were randomly examined per slide under the microscope (Olympus BX51, Tokyo, Japan). The damaged cells were categorized as mild, moderate, and extensive.

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Quantification of Apoptotic Cardiomyocytes by TUNEL Assay TUNEL assay was used to study DNA fragmentation (Calbiochem, Oncogene Research Product, USA) according to the manufacturer’s instructions. TUNEL-positive cells and total cells were observed under fluorescent microscope. The TUNEL-positive cells were expressed as percentage (%) of total cells.

Immunohistochemistry for Detection of NFjB, p38, and Caspase-3 Paraffin-embedded cardiac tissues of the control and the treatment groups were cut into 5-lm-thick sections, placed on poly-L-lysine–coated slides, deparaffinized in xylene, and rehydrated with graded alcohol. Tissue sections were incubated in citrate buffer (pH 6.0) at 95–100°C for 20 min in water bath for antigen retrieval. NovoLink Polymer detection system kit (Leica microsystem: product no. RE7140-K) was used, and further steps were performed according to the manufacturer’s instructions. Tissue sections were incubated with primary NFjB, p38, or caspase-3 antibody (Rabbit polyclonal IgG, Santa Cruz Biotechnology Inc., USA) at 4°C overnight in a humidified chamber. The sections were finally counterstained with hematoxylin, dehydrated with alcohol, cleared in xylene, and mounted with DPX. Statistical Analysis Results were shown as mean ± standard error of mean (SEM) for each group. Statistical analysis was performed using Jandel Sigma Stat (Version 2.03) statistical software. For multiple comparisons, one-way analysis of variance (ANOVA) was used. In case ANOVA showed significant differences, post hoc analysis was performed with Tukey’s test. P \ 0.05 was considered to be statistically significant.

Results Effect of Hesperetin Treatment on DOX-Induced Oxidative Stress DOX treatment led to significant increase in the MDA level and decrease in the GSH level in heart as compared to the control group. Hesperetin treatment along with DOX treatment reduced DOX-induced oxidative stress as observed by decrease in MDA and increase in GSH levels in heart (Fig. 1).


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Fig. 1 Effect of DOX and/or hesperetin treatment on (i) MDA and (ii) GSH levels in rat heart. All the values are expressed as mean ± SEM, (n = 5), **P \ 0.01 and *P \ 0.05, a versus control and b versus DOX treatment

Fig. 2 Representative photomicrographs of heart sections stained with hematoxylin and eosin (H&E) from control (a), DOX-treated (b) and DOX ? hesperetin-treated rat (c)

Effect of Hesperetin Treatment on Cardiac Histology Light microscopic investigations revealed that DOX treatment induced morphological alterations such as disorganization of the cellular structure and vacuolization in the heart. Hesperetin treatment along with DOX treatment decreased damage in the cardiac cellular morphology (Fig. 2). Effect of Hesperetin Treatment on DOX-Induced DNA Damage Comet assay is reliable and sensitive for the detection of DNA damage. DOX treatment led to DNA damage in the cardiomyocytes as evident from significant increase in the comet parameters (TL, TM, OTM, and % DNA) as compared to the control group. Hesperetin treatment along with DOX treatment significantly reduced the DOX-induced cardiomyocyte DNA damage as apparent from the analysis of various comet assay parameters (Fig. 3). Effect of Hesperetin Treatment on DOX-Induced Cytotoxicity as Evident from Halo Assay Halo assay is used for the detection of apoptosis. DOX treatment led to significant increase in the % of mild, moderate, and extensive damage in heart cells as compared to the control group. Hesperetin treatment along with DOX treatment resulted in significant decrease in the % of

damaged cells as compared to the DOX-treated group (Fig. 4). Effect of Hesperetin Treatment on the Frequency of TUNEL-Positive Cells TUNEL assay efficiently detects the fragmented DNA generated due to apoptosis. Significant increase in the % of TUNEL-positive cells was observed in the cardiac sections of DOX-treated animals as compared to the control group. Hesperetin treatment along with DOX treatment led to significant decrease in the % of TUNEL-positive cells as compared to the DOX-treated group (Fig. 5). Effect of Hesperetin Treatment on NFjB, p38, and Caspase-3 Expression Higher intensity of immunostaining for NFjB, p38, and caspase-3 was observed in the heart of animals treated with DOX as compared to the control group. Intervention with hesperetin ameliorated the intensity of immunostaining for NFjB, p38, and caspase-3 (Fig. 6).

Discussion DOX, an anthracycline antibiotic, is widely used against a variety of neoplasms. The mechanism of action of DOX

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Fig. 3 i Representative photomicrographs of comet assay showing DNA migration pattern in the heart cells from control (a), DOXtreated (b) and DOX ? hesperetin (100 mg/kg bw)-treated rat (c). The symbols ‘‘-’’ and ‘‘?’’ represent cathode and anode, respectively, during electrophoresis of negatively charged DNA. ii Effect of

hesperetin treatment against DOX-induced DNA damage in the cardiomyocytes of heart as measured by the comet assay parameters. All the values are expressed as mean ± SEM, (n = 5), ***P \ 0.001 and *P \ 0.05, a versus control and b versus DOX treatment

is attributed to its intercalation with DNA. It acts by stabilizing a reaction intermediate in which DNA strands are cut and covalently linked to tyrosine residues of topoisomerase II. Thus, it inhibits negative supercoiling of DNA and thereby blocks further DNA transcription and replication. But its clinical use is limited due to its severe cardiotoxicity [1, 5]. It has been reported that oxidative stress from lipid peroxidation is mainly responsible for the cardiotoxicity produced by DOX [32, 33]. In the present study, DOX treatment led to significant increase in the MDA level and decrease in the GSH level in heart as compared to the control group. Hesperetin treatment along with DOX treatment led to significant reduction in the DOX-induced oxidative stress in the rat heart. It has been reported that chronic administration of DOX leads to multifocal degeneration of cardiomyocytes in rats [34]. In the present study, the cardiotoxicity of DOX was clearly depicted from the histological evaluation of heart. Cellular disorganization and vacuolization were observed in

the heart as evident from the histological evaluation. Similar cellular disorganization in heart has been reported due to acute DOX administration in rats [35]. Intervention with hesperetin led to significant protection in the cardiac cellular morphology as manifested from histological examination. It has been reported that DOX induces genetic damage in cardiomyocytes in rats [36]. Comparable genotoxic effects of DOX were clearly expressed by an increase in the comet assay parameters like TL, TM, OTM, and % DNA in comet tail in heart. Hesperetin treatment along with DOX treatment prevented the DNA damage in the heart nuclei as evident from the evaluation of various comet assay parameters. DOX has been reported to induce apoptosis in rat heart [37]. Apoptotic effect of DOX was made evident in the present study by halo assay and TUNEL assay, which substantiates the cytotoxic effect of DOX on cardiomyocytes [33]. Hesperetin treatment along with DOX treatment protected cardiac cell integrity by reducing the % of apoptotic cells

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Fig. 4 i Representative photomicrographs showing cytotoxic effect in cardiomyocytes as revealed from halo assay. Stain: EtBr; control (a), DOX treatment (b), and DOX ? hesperetin treatment (100 mg/kg bw) (c). ii Effect of hesperetin treatment against DOX-induced cytotoxicity in the cardiomyocytes of heart as depicted by the halo assay. All the values are expressed as mean ± SEM, (n = 5), ***P \ 0.001 and **P \ 0.01, a versus control and b versus DOX treatment

as apparent from the halo and the TUNEL assays. It has been reported that generation of oxidative stress is responsible for the activation of a redox-sensitive transcription factor, NFjB [38]. Induction of apoptosis in adult rat cardiomyocytes is due to the activation of NFjB, which in turn is due to DOX-induced oxidative stress in cardiomyocytes [39]. DOX has also been reported to cause apoptosis by increasing the expression of various genes like p38, p53, and caspase-3 and down-regulating the anti-apoptotic factors like Bcl-2 [40, 41]. In the present investigation, immunohistochemistry results have shown increased expression of NFjB, p38, and caspase-3 in DOX-treated cardiomyocytes as compared to the control group. Thus, it can be inferred that increased expression of NFjB due to DOX-induced oxidative stress led to an increase in the expression of various genes like p38 and caspase-3, which resulted in the induction of apoptosis in the cardiomyocytes. While, hesperetin treatment along with DOX treatment resulted in suppression of NFjB, p38 and caspase-3 in rat cardiomyocytes. Moreover, it has been reported that hesperetin modulates NFjB pathway by suppressing NFjB expression through four signal transduction pathways, i.e., NIK/IKK, ERK, p38, and JNK [42]. Thus, hesperetin suppressed DOXinduced apoptosis in rat heart. Furthermore, DOX has been reported to disrupt the myocyte sarcomere, increase the intracellular calcium level as well as loss of mitochondrial membrane potential [43, 44]. Hesperetin has

been shown to attenuate the elevation of intracellular calcium level as well as decrease of mitochondrial membrane potential in H2O2-induced PC12 cells [45]. These mechanisms may also be involved in the amelioration of DOX-induced cardiotoxicity by the intervention with hesperetin. Recently, it has been reported that hesperetin at a dosage of 50 mg/kg daily for 8 weeks in DMBA-induced breast cancer rat model acts as proapoptotic. However, hesperetin daily at a dose of 10 and 50 mg/kg for 7 weeks acts as an anti-apoptotic against DMBA-induced hepatic apoptosis in mice [46]. Moreover, naringin, a flavonone similar to hesperetin found in citrus species, when administered prior to DOX, acts as the dual modulator of P-gp expression, and it was hypothesized that this may be an attractive new agent for the chemosensitization of cancer cells [47]. Further, it has been reported that flavonone along with anticancer drug, DOX, showed inhibitory effect on the growth of A549 lung cancer cells as well as on Lewis lung carcinoma [48]. Hesperetin significantly induces G1-phase cell cycle arrest in human breast cancer MCF-7 cells through the regulation of CDK4 and p21 (Cip 1) pathways [49]. Flavonoids (particularly naringin, daidzein and hesperetin) are the effective drugs to inhibit the function of mutant H-Ras p21 protein, which in turn, arrests the process of cell growth and proliferation of the cancer cell [50]. Thus, hesperetin acts as a chemopreventive agent against standard carcinogens, chemoprotective agent against harmful

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Fig. 5 i Representative photomicrographs showing apoptotic cardiomyocytes having fragmented DNA as revealed from TUNEL assay. Control (a–c), DOX treatment (d–f) and DOX ? hesperetin treatment (100 mg/kg bw) (g–i). The cardiomyocytes emitting green signals are TUNELpositive apoptotic cells. ii Effect of DOX and/or hesperetin treatment on the % of TUNELpositive cells in the heart of rat. All the values are expressed as mean ± SEM, (n = 5), ***P \ 0.001, a versus control and b versus DOX treatment

effects induced by toxicants and chemosensitizer along with different chemotherapeutic agents. Such property of hesperetin depends on the types of tissue, involvement of particular molecular pathways as well as the nature of circumstances under which it is implicated. From this study, a conclusion can be drawn that hesperetin has preventive effects against DOX-induced cardiotoxicity in rat as evident from the decreased oxidative stress, DNA damage, apoptosis, and cardiac tissue damage. Moreover, hesperetin shows protection against

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cardiotoxicity produced by DOX by reducing the oxidative stress-induced DNA damage and apoptosis due to its antioxidant effect. As cardiovascular system is a key organ system of the body, protection of hesperetin against DOXinduced cardiotoxicity may improve the quality of patient’s life. The precise molecular mechanisms responsible for the protective effects of hesperetin remain to be explored. Further, clinical data will strengthen the scientific basis for the intervention with hesperetin as an efficient anti-oxidant for the prevention of DOX-induced cardiotoxicity.


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Fig. 6 Representative photomicrographs illustrating the immunohistochemical staining of NFjB [control (a), DOX-treated (b) and DOX ? hesperetin-treated (100 mg/kg bw) (c) rat], p38 [control (d),

DOX-treated (e) and DOX ? hesperetin-treated (100 mg/kg bw) (f) rat] and caspase-3 [control (g), DOX-treated (h) and DOX ? hesperetin-treated (100 mg/kg bw) (I) rat] in cardiomyocytes

Acknowledgments We wish to acknowledge the financial assistance received from National Institute of Pharmaceutical Education and Research (NIPER), Mohali, to undertake this study. The authors would also like to acknowledge Intas Pharmaceuticals Ltd., Ahmedabad, Gujarat, for benevolently granting the gift sample of doxorubicin.

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