ORIGINAL PAPER

Protective role of atorvastatin against doxorubicin-inducedcardiotoxicity and testicular toxicity in mice

Ramanjaneyulu SVVS & P. P. Trivedi &S. Kushwaha & A. Vikram & G. B. Jena

Received: 27 September 2012 /Accepted: 21 January 2013# University of Navarra 2013

Abstract Doxorubicin (DOX), a potent chemothera-peutic agent, is widely used for the treatment of variousmalignancies. However, its clinical uses are limited dueto its dose-dependent adverse effects particularly cardiacand testicular toxicities. DOX-induced toxicity is main-ly due to the induction of oxidative stress. Atorvastatin(ATV), a 3-hydroxy 3-methyl glutaryl coenzyme A

reductase inhibitor, with lipid-lowering activity, acts asan antioxidant at lower doses. It possesses pleiotropiceffects independent of cholesterol-lowering propertyusually shown at lower doses, which include antioxidantand anti-inflammatory activities. The present study wasaimed to investigate the possible protection exerted byatorvastatin against oxidative stress and DNA damageinduced by DOX in the heart and testes of mice. Theprotective role of ATV in the heart and testes of DOX-treated mice was evident from the amelioration of oxi-dative stress, DNA and cellular damage. The presentstudy clearly indicates that ATV offers a significantprotection against DOX-induced oxidative stress andDNA damage in the heart and testes of mice.

Keywords Atorvastatin . Doxorubicin . Oxidativestress . Genotoxicity . Mice

AbbreviationsDOX DoxorubicinATV AtorvastatinHMG-CoA 3-Hydroxy 3-methyl glutaryl

coenzyme APBMN Peripheral blood micronucleusLDL Low-density lipoproteinCMC Carboxy methyl celluloseH&E Hematoxylin and eosinDMSO DimethylsulphoxideNMPA Normal melting point agaroseLMPA Low melting point agaroseEDTA Ethylenediamine tetraacetic acid

J Physiol BiochemDOI 10.1007/s13105-013-0240-0

P. P. Trivedi : S. Kushwaha :G. B. Jena (*)Facility for Risk Assessment & Intervention Studies,Department of Pharmacology and Toxicology, NationalInstitute of Pharmaceutical Education and Research,Sector-67, S.A.S. Nagar,Punjab 160 062, Indiae-mail: gbjena@gmail.com

G. B. Jenae-mail: gbjena@yahoo.com

P. P. Trivedie-mail: priyankatrvd2@gmail.com

S. Kushwahae-mail: sapna.pharm@gmail.com

R. SVVSDepartment of Toxicology,NCER&D, Piramal Healthcare Limited,Goregaon East,Mumbai, Maharashtra 400 063, Indiae-mail: ramanji.niper@gmail.com

A. VikramHeart and Vascular Institute, University of Pittsburgh,Pittsburgh, PA 15213, USAe-mail: ajitvikram@gmail.com

HBSS Hank’s balanced salt solutionMDA MalondialdehydeGSH Reduced glutathioneTL Tail lengthTM Tail momentOTM Olive tail moment% DNA % DNA in tailHDL High-density lipoprotein

Introduction

Doxorubicin (DOX), an anthracycline antibiotic, is awidely used anticancer drug for the treatment of acuteleukemia, malignant lymphomas, and for some solidtumors such as breast, ovarian, and endometrial cancers[21, 40, 47]. The cytotoxic effects of DOX appear to bemediated through an inhibition of an enzyme, topoiso-merase II (a DNA gyrase), the activity of which is mark-edly increased in the proliferating cells. The clinicaleffectiveness of DOX is limited due to its dose-dependent toxicities such as cardiac and testicular toxic-ities [28, 29]. The exact mechanisms of DOX-inducedcardiac and testicular toxicities are not fully explored, butthere are a few reports which indicate that oxidative stressgenerated by the virtue of quinone groups in its structuremainly contributes to the majority of its toxicities [4, 7].

In order to mitigate the oxidative stress-inducedtoxicities of DOX, antioxidants such as atorvastatin(ATV) may play an important role. ATV is a syntheticpotent statin which lowers plasma cholesterol and lipo-protein levels by inhibiting the enzyme 3-hydroxy 3-methyl glutaryl coenzyme A (HMG-CoA) reductaseand cholesterol synthesis in the liver by increasing thenumber of hepatic low-density lipoprotein (LDL) recep-tors which further promote the uptake of LDL [36]. Inclinical practice, ATV has been approved to be pre-scribed at the doses of 10, 20, 40, and 80 mg/day forthe treatment of dyslipidemias. ATV has therapeuticbeneficial pleiotropic effects independent ofcholesterol-lowering activity which are proven to beshown at low doses [3, 48, 52]. These include anti-inflammatory and antioxidant properties as well as im-provement of endothelial function, increased nitric ox-ide bioavailability, cardioprotection, and antitumoractivity [11]. Keeping this in view, the present investi-gation was aimed to evaluate the possible protectiveeffects of ATV against DOX-induced cardiotoxicityand testicular toxicity. The experimental endpoints

included lipid peroxidation (MDA) and glutathione(GSH) measurement for the determination of oxidativestress. Genotoxicity and cytotoxicity end points includ-ed PBMN assay, alkaline comet assay, and histologicalexamination in the heart and testes. The present studyclearly revealed that ATV decreased DOX-induced ox-idative stress and subsequent DNA damage in the heartand testes of mice.

Materials and methods

Animals

All animal experiment protocols were approved by theInstitutional Animal Ethics Committee. Experimentswere performed on male Swiss mice (weight range, 20to 25 g) procured from the Central Animal Facility of theinstitute. The animals were kept at room temperature (22±2 °C), with 50±10 % humidity and an automaticallycontrolled cycle of 12 h light and 12 h dark. Standardlaboratory animal feed (purchased from commercial sup-plier) and water were provided ad libitum. Animals wereacclimatized to the experimental conditions for a periodof 1 week before the commencement of the experiment.

Chemicals

ATV Calcium was obtained as a gift sample from Bio-chem Pharmaceutical Industries Ltd., Mumbai, India.DOX (CAS no. 29042-30-6) was obtained as a giftsample from Intas Pharmaceuticals Ltd., Ahmedabad,India. 1,1,3,3-Tetramethoxy propane (CAS no. 102-52-3), hematoxylin and eosin (H&E), 2-thiobarbituric acid(CAS no. 504-17-6), trizma (CAS no. 77-86-1), 5,5′-dithiobis (2-nitro-benzoic acid; CAS no. 69-78-3), acri-dine orange (CAS no. 10127-02-3), and SYBR Green I(CAS no. 163795-75-3) were purchased from Sigma-Aldrich Chemicals, Saint Louis, MO, USA. Glutathionereduced, dimethylsulphoxide (DMSO), normal meltingpoint agarose, low melting point agarose, Triton-X 100,ethylenediamine tetraacetic acid (EDTA), and Hank’sbalanced salt solution (HBSS) buffer were obtainedfrom HiMedia Laboratories Ltd., Mumbai, India.

Experimental design

Animals were randomized into various groups, eachgroup consisting of five animals for both the short-

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and long-term studies. For the short-term study, group1 received 0.5 % carboxy methyl cellulose (CMC) andserved as vehicle control. Group 2 received ATV(10 mg/kg bw) for 7 days and served as ATV control.Group 3 received DOX 3 mg/kg bw for 3 (on days 1,4, and 7) days. Group 4 received DOX 3 mg/kg bw for3 (on days 1, 4, and 7) days and ATV (10 mg/kg bw)for 7 days concurrently 1 h before DOX treatment. Allthe animals were sacrificed 1 day after receiving thelast treatment. For the long-term study, group 1 re-ceived 0.5 % CMC and served as vehicle control.Group 2 received ATV (20 mg/kg bw) for the first5 days in a week and served as ATV control. Group 3received DOX 5 mg/kg bw alone on day 1 in a week.Groups 4, 5, and 6 received ATV 5, 10, and 20 mg/kgbw, respectively, for 5 days in a week and DOX5 mg/kg bw (on day 1) 1 h after ATV treatment. Thedosing regimen was repeated for a total of 3 weeks.Animals were sacrificed 1 day after receiving the lasttreatment. ATV was suspended in 0.5 % CMC imme-diately before administration per oral, and DOX wasdissolved in distilled water and administered throughthe intraperitoneal route. The doses of ATV (1, 3, 10,30, 100, 300, 1,000, and 3,000 mg/kg bw) were se-lected on a semi-logarithmic scale for the determina-tion of dose-dependent DNA damage in mice (Fig. 1),and from these doses, the non-genotoxic doses wereconsidered for the protection studies against DOX.

Estimation of lipid peroxidation

Lipid peroxide in the heart and testes was measuredaccording to the method described [27] with somemodifications. The organs were rinsed in ice-coldphysiological saline, minced, and a 10 % homogenatewas prepared in phosphate buffer (pH 7.4) containingEDTA (1 mM). The sample was centrifuged at 700×gfor 10 min, and the supernatant was used for thedetermination of lipid peroxidation. The supernatant(100 μl) was added to SDS (100 μl of 8.1 %), fol-lowed by acetic acid (20 %, 750 μl,) and thiobarbituricacid (0.8 %, 750 μl). The volume was made up to 2 mlwith distilled water and heated on a water bath at 95 °Cfor 60 min. The mixture was then cooled under the tapand was centrifuged at 10,000 rpm for 10 min. Thesupernatant was removed, and the absorbance was mea-sured at 532 nm. The protein content in the tissuehomogenate supernatant was determined as described[24]. Lipid peroxidation was calculated from the

standard curve using the 1, 1, 3, 3 tetramethoxy propane(97 %) and expressed as nanomolars MDA per gramwet weight of tissue.

Estimation of glutathione

The tissue homogenate was used for the estimation ofreduced glutathione (GSH) content as described [26]with some modifications. Heart and testes were rinsedin ice-cold physiological saline, minced, and the ho-mogenate (10 %) was prepared in phosphate buffer(pH 7.4) containing 1 mM EDTA. The sample wascentrifuged at 700×g for 10 min, and the supernatantwas used for the determination of GSH content. Thesupernatant (0.5 ml) was mixed with 5 % sulphosali-cylic acid (0.5 ml) and kept in ice for 20 min for theprecipitation of the protein. After precipitation of theprotein, the resulting solution was centrifuged at10,000×g for 5 min at 4 °C, the supernatant (50 μl)was mixed with phosphate buffer (450 μl) and 0.1 mMof 5,5′-dithiobis (2-nitro benzoic acid; 1.5 ml). Theresulting solution was incubated for 10 min at 37 °Cfollowed by measurement of optical density at 412 nm,using reduced glutathione as an external standard.Protein content in tissue homogenate supernatant wasdetermined as described [24].

Peripheral blood micronucleus assay

The peripheral blood smears were prepared as de-scribed [19] with some modifications. The blood wascollected from the tail tip of mice, and the smears wereprepared on pre-cleaned slides. The smear wasallowed to dry at room temperature and fixed in abso-lute methanol for 5 min. After fixation, the slides werestained with acridine orange and were washed twicewith phosphate buffer (pH 6.8). The slides were thenscored.

Single cell gel electrophoresis (comet) assay

The comet assay was performed as described [38] withsome modifications. The blood was collected from theretro-orbital sinus of mice, and the lymphocytes wereisolated by using Ficoll histopaque solution. A finalcell-agarose suspension (100 μl) was prepared con-taining ≈1×104lymphocytes/ml in 0.5 % low meltingpoint agarose (LMPA). From the final cell-agarosesuspension, 80 μl was spread over a microscope slide,

Role of atorvastatin against doxorubicin-induced toxicity

pre-coated with 1 % NMPA to form a microgel, andallowed 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 cells were then lysed in a buffer contain-ing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris(pH 10.0) with freshly prepared 1 % Triton X-100and 10 % DMSO for 24 h at 4 °C. After lysis, theslides were rinsed three times in deionized water toremove the salt and detergent. The slides were thencoded and were placed in a specifically designed hor-izontal electrophoresis tank, and the DNAwas allowedto unwind for 20 min in an alkaline solution contain-ing 300 mM NaOH and 1 mM EDTA (pH>13) andwas then electrophoresed for 30 min at 300 mA and20 V (0.70 V/cm). The slides were then neutralizedwith 0.4 M Tris (pH 7.5), stained with SYBR Green I(1:10,000) for 1 h, and covered with cover slips.

Sample preparation for heart and testes cells

Heart and testes were collected from individual mice.The organs were chopped, and cells were isolated inHBSS containing 20 mM EDTA and 10 % DMSO.From this solution, a 5-μl sample containing approx-imately (2–5×104cells/ml) was added to 95 μl of0.5 % LMPA (in Ca2+ and Mg2+ free PBS) to preparethe final cell agarose suspension essentially as describedabove.

Data scoring and photomicrographs of comet assay

The fluorescence-labeled DNA was visualized usingan automated AXIO Imager M1 fluorescence micro-scope (Carl Zeiss, Germany), and the images were

captured with image analysis software (Comet ImagerV.2.0.0). Duplicate slides were prepared for each treat-ment and were independently coded and scored with-out the knowledge of the code. The parameters for theDNA damage analysis include: tail length (in micro-meters), TM, OTM, and % DNA. The edges of theslides, any damaged part of the gel, any debris, super-imposed comets, and comets without distinct head(“hedgehogs” or “ghost” or “clouds”) were not con-sidered for the analysis.

Histological evaluation

Histological slides were prepared as previously stan-dardized in our laboratory [9, 44]. Heart and testeswere fixed in 10 % formalin, dehydrated in increasingconcentrations of ethanol, and embedded in paraffin.Tissue sections (5 μm) were mounted on glass slidescoated with Mayer’s albumin and dried overnight. Thesections were then deparaffinized with xylene andrehydrated with alcohol and water. The rehydratedsections were stained using H&E, mounted withDPX mounting media, and examined under the micro-scope at both high (×40) and low (×10 and ×20)magnifications (Olympus BX51 microscope, Tokyo,Japan). The sections from each animal were evaluatedfor structural changes.

Sperm head morphology

After the animal sacrifice, epididymis was removedand placed in a Petri plate containing 2 ml of HBSSmedium at room temperature. The epididymis was cutinto small portions to allow the sperms to swim out.

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Fig. 1 Effect of ATV (36 h post-treatment) on micronuclei frequency in peripheral blood (i) erythrocytes (ERT) and (ii) reticulocytes(RET) of mice. All the values are expressed as mean±SEM (n=5). ***P<0.001 and **P<0.01 vs. control

R. SVVS et al.

The solution containing the sperms was centrifuged at1,000 rpm for 3 min. After centrifugation, 1 ml ofsupernatant was taken and used for sperm head mor-phology. For sperm head morphology, 0.5 ml of theabove solution containing the sperms and 0.5 ml of2 % eosin solution were mixed and kept for 1 h to stainthe sperm. Smear was prepared using two to three dropsof the above solution, air dried, and fixed with absolutemethanol for 3 min. Two hundred sperms per animalwere examined to determine the morphological abnor-malities at ×1,000 magnification [8, 22]. Sperm headmorphology was scored under the category of normal,spermwithout hook, amorphous head, banana head, andflower-shaped head essentially as described [49]. Datawere shown in terms of percentage of abnormal sperms.

Statistical analysis

Results were shown as mean±SEM for each group.Statistical analysis was performed using Jandel SigmaStat (version 2.03) statistical software. For multiple com-parisons, one-way analysis of variance (ANOVA) wasused. In cases where ANOVA showed significant differ-ences, post hoc analysis was performedwith Tukey’s test.P<0.05 was considered to be statistically significant.

Results

Effect of ATVon DOX-induced oxidative stress in heartand testes due to short- and long-term treatment

DOX (3 mg/kg bw/day for three alternate days) treat-ment led to significant reduction in the GSH level in theheart and testes as compared to the control group.Groups receiving ATV (10 mg/kg bw) along withDOX showed significant increase in the GSH level inheart and testes as compared to animals receiving onlyDOX. Further, DOX (3mg/kg bw/day for three alternatedays in a week) treatment led to significant increase inMDA levels in heart and testes as compared to thecontrol group. Significant decrease in MDA profilewas observed in animals co-treated with ATV as com-pared to animals receiving only DOX (Fig. 2a, b).

In the long-term study, DOX (5 mg/kg bw once in aweek for three consecutive weeks) treatment led tosignificant reduction in the GSH level in the heartand testes as compared to the control group. Groupsreceiving ATV (5, 10, and 20 mg/kg bw) along with

DOX showed significant increase in the GSH level inheart and testes in a dose-dependent manner. Further,DOX (5 mg/kg bw once in a week for 3 weeks)treatment led to significant increase in MDA levelsin heart and testes as compared to the control group. Asignificant reduction in MDA profile was observed inanimals co-treated with ATV as compared to the ani-mals receiving only DOX (Fig. 2c, d).

Effect of ATV against DOX-induced micronucleiformation in the peripheral blood

DOX treatment led to a significant increase in thefrequency of MNERT in peripheral blood as comparedto the control group in both short- and long-term studies.Co-treatment with ATV significantly decreasedMNERT in peripheral blood of mice as compared tothe DOX only treated group both in the short- and thelong-term studies (Fig. 3).

Effect of ATV on DOX-induced DNA strand breaksin heart and testes

In the short-term study, DOX treatment led to DNAdamage in the heart and the testicular cells as observedfrom a significant increase in different comet parame-ters as compared to the control group. Co-treatmentwith ATV significantly decreased these parameters inboth the organs as compared to the DOX only treatedgroup (Fig. 4(i)).

In the long-term study, DOX treatment led to DNAdamage in the heart and the testicular cells as observedfrom a significant increase in TL and % DNA in tail ascompared to the control group. Treatment with ATVsignificantly decreased these parameters in both theorgans as compared to the DOX only treated group ina dose-dependent manner (Fig. 4(ii)).

Effect of ATV on DOX-induced heart and testeshistology

The animals treated with DOX alone manifested micro-scopic changes in the heart that consisted of disorgani-zation of myofibrils and associated vacuolization of themyofibers. Treatment with ATV caused a significantreduction in the frequency of the vacuolization anddisorganisation of myofibers in a dose-dependent man-ner. Testes from control and ATV group mice exhibitedtypical features of seminiferous epithelium, while DOX

Role of atorvastatin against doxorubicin-induced toxicity

Fig. 2 Effect of ATVand DOX on (i) MDA and (ii) GSH levelsin a heart and b testes of mice due to short-term treatment and in cheart and d testes of mice due to long-term treatment. All the values

are expressed as mean±SEM (n=5). ***P<0.001, **P<0.01, and*P<0.05, a vs. control, b vs. DOX

R. SVVS et al.

treatment resulted in the morphological alterations suchas reduction in the size and number of the seminiferoustubules, degeneration and vacuolization in spermatogo-nia, spermatocytes, and spermatids in comparison to thecontrol group. ATV treatment along with DOX treat-ment significantly reduced the DOX-induced testicularabnormalities in a dose-dependent manner, as character-ized by the nearly normal histological morphology inthe testicular section (Fig. 5).

Sperm head morphology

In both the short- and the long-term studies, DOXtreatment enhanced the abnormality in sperm head

morphology when compared to the control group.ATV co-treatment with DOX significantly preventedthe damage induced by DOX in a dose-dependentmanner in the long-term study and restored normalsperm head morphology. The damage was expressed aspercentage of sperms with abnormal head morphology(Fig. 6).

Discussion

In the present investigation, ATV at the doses of10 mg/kg bw and 5, 10, and 20 mg/kg bw in short-and long-term studies, respectively, offered significant

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Fig. 3 Effect of ATV onDOX-induced MN frequen-cy in peripheral blooderythrocytes of mice due toa short-term and b long-termtreatment. All the values areexpressed as mean±SEM(n=5). ***P<0.001,a vs. control, b vs. DOX

Role of atorvastatin against doxorubicin-induced toxicity

protection against DOX-induced toxicities in the heartand testes. In the long-term study, DOX treatment led

to severe cardiac and testicular toxicities. DOX hasbeen reported to cause severe cardiotoxicity as well as

Fig. 4 Effect of ATVon DOX-induced DNA damage in a heartand b testes of mice as measured by the comet assay parametersdue to (i) short-term and (ii) long-term treatment. All the values

are expressed as mean±SEM (n=5). ***P<0.001, **P<0.01,and *P<0.05, a vs. control, b vs. DOX

R. SVVS et al.

the testicular toxicity in human as well as animalmodels [1, 14, 41–43, 50]. In the present study, asignificant increase in the oxidative stress was foundas evident from a significant increase in the lipidperoxidation in terms of MDA and decrease in theantioxidant enzyme GSH levels in both heart and

testes after short- and long-term treatment of DOX[30]. However, ATV treatment along with DOX treat-ment led to a significant reduction in the oxidativestress as evident from a significant decrease in theMDA levels and increase in the GSH levels in theheart and testes after short- and long-term treatment.

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Fig. 5 (i) Representative photomicrographs of the heart (acontrol, b DOX-treated, and c DOX+ATV-treated mice) andtestes (d control, e DOX-treated, and f DOX+ATV-treatedmice) sections due to short-term treatment stained with hema-toxylin and eosin (H&E). (ii) Representative photomicrographs

of heart (a control, b DOX-treated, and c DOX+ATV-treatedmice) and testes (d control, e DOX-treated, and f DOX+ATV-treated mice) sections due to long-term treatment stained withhematoxylin and eosin (H&E)

Role of atorvastatin against doxorubicin-induced toxicity

This is in agreement with earlier reports where lowdose of ATV reduced oxidative stress in vitro and invivo [6, 45, 46]. The exact mechanism of ATV in theamelioration of oxidative stress is not fully explored,but a few authors suggested and proved that lipid-independent inhibition of isoprenoids and their underly-ing molecular events were responsible for ATV-mediated amelioration of oxidative stress and inflam-mation in experimental diabetic conditions [35]. Preston

Mason et al. [25] reported that in the absence of HMG-CoA reductase, the active metabolite of ATV, ortho-hydroxy ATV, blocked membrane cholesterol domainformation as a function of oxidative stress. This effect ofATV metabolite is responsible for the electron donationand proton stabilization mechanisms associated with itsphenoxy group [5], which in turn might lead to ATV-induced inhibition of lipid peroxidation in the heart andtestes in a dose-dependent manner. There is also a notion

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Fig. 6 (i) Effect of ATVon DOX-induced abnormality in spermhead morphology of mice due to a short-term and b long-termtreatment. All the values are expressed as mean±SEM (n=5).***P<0.001 and **P<0.01, a vs. control, b vs. DOX. (ii)

Representative photomicrographs of sperms. Normal murinesperm (a, b), sperm with blunt hook (c), sperm without hook(d, e), banana head sperm (f), sperm with triangular head (g),and sperm with amorphous head (h)

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that ATV-dependent antioxidant effect is mediatedthrough increase in the levels and activity of HDL andits associated antioxidant enzyme paraoxonase [18]. Anextensive literature survey revealed that several antiox-idants depict protection against DOX-induced toxicities,both in vivo and in vitro, by ameliorating the oxidativestress [4, 12, 31]. Few among these include vitamin A,vitamin C, vitamin E, coenzyme Q, carotenoids, flavo-noids, polyphenols, selenium, taurin, silymarin, lipoicacid, and green tea extracts [16, 23, 31]. Besides ATV,other statins have also been reported to depict protectionagainst DOX-induced toxicities. Rosuvastatin has beenknown to show cardioprotective effect against DOX-induced cardiotoxicity in mice by inhibiting apoptosisand improving the lipid profile altered by DOX [37].Fluvastatin pretreatment has been reported to attenuateDOX-induced cardiotoxicity via antioxidant and anti-inflammatory effects [34]. However, few statins havebeen reported to protect against DOX-induced toxicitiesthrough the mechanisms other than the amelioration ofoxidative stress. Simvastatin ameliorated DOX-inducednephropathy in rat via its anti-inflammatory actionthrough a reduction of NF-κB activation, and IL-1βand TGF-β expression [51]. Lovastatin protected thehuman endothelial cells against DOX-induced toxicityby impairing DNA strand break formation [10].

DOX has been reported to induce DNA damage inthe germ cells of rat as revealed from the sperm cometassay [41]. In the present investigation, a significantincrease in the DNA damage was observed as evidentfrom a significant rise in all the comet assay parametersin both heart and testes after short- and long-term treat-ment with DOX as compared to the control group,whereas co-treatment with ATV significantly reducedthe DNA damage as compared to DOX alone treatedgroup as evident from the comet assay. Further, DOXtreatment led to genotoxicity as evident from a signifi-cant increase in the micronuclei (MNERT) in the pe-ripheral blood erythrocytes of mice, which was furtherdecreased significantly by co-treatment with ATV inboth short- and long-term studies. Moreover, histologi-cal examination of heart and testicular tissue sectionsclearly revealed the toxicity of DOX on these organs.The animals treated with only DOX were shown toinduce microscopic changes in the heart, such as disor-ganization and vacuolization in the cardiac myofibres inthe left ventricular portion, after short- and long-termtreatment as compared to the control group. Moreover,these changes were more prominent after long-term

treatment as revealed from the degeneration of cardiactissue. Several reports in animal models show histolog-ical abnormalities in the heart due to DOX treatment [2,13, 15, 17, 20, 33, 39]. However, co-treatment withATVoffered significant amelioration of histopathologi-cal changes as observed with DOX treatment. Further,DOX treatment resulted in the morphological alterationsin the testes, such as reduction in the size and number ofseminiferous tubules as well as degeneration and vacuo-lization in the spermatogonia, spermatocytes, and sper-matids in comparison to the control group in both short-and long-term studies [31, 32]. In the long-term study,DOX treatment resulted into the diminution and degen-eration of the semeniferous tubule. In mice co-treatedwith ATV (ATV+DOX), testes of mice depicted nearlynormal histological morphology. Further, sperm headmorphological evaluation was carried out in bothshort- and long-term studies, where DOX treatment ledto significant increase in the percentage of abnormalsperms as compared to the control group, whereas co-treatment with ATV significantly reduced the abnormal-ity in the sperm head morphology as evident from thesignificant reduction in the percentage of abnormality inboth short- and long-term studies in a dose-dependentmanner.

The present study clearly indicates that ATV pos-sesses beneficial effects against DOX-induced toxicityin mice as evident from the decreased oxidative stress,DNA damage, and cellular damage. However, theprecise molecular mechanisms underlying the protec-tive effects of ATV as well as its possible clinicalintervention remain to be further explored.

Acknowledgments We wish to acknowledge the financialassistance received from National Institute of PharmaceuticalEducation and Research (NIPER), S.A.S. Nagar for this work.The authors would like to acknowledge Biochem Pharmaceuti-cal Industries Ltd., Mumbai, India and Intas PharmaceuticalsLtd., Ahmedabad, India for benevolently granting the gift sam-ples of ATV Calcium and doxorubicin, respectively.

Conflict of interest None.

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Role of atorvastatin against doxorubicin-induced toxicity