Melatonin affords protection against myocardial ischemia-induced cerebral mitochondrial...

Journal of Pharmacy Research Vol.9 Issue 2.February 2015

Auroma Ghoshet al. / Journal of Pharmacy Research 2015,9(2),105-118

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Research ArticleISSN: 0974-6943

Available online throughwww.jpronline.info

*Corresponding author.Dr. Debasish BandyopadhyayProfessorOxidative Stress and Free Radical Biology LaboratoryDepartment of Physiology, University of Calcutta92, APC Road, Kolkata 700 009, IndiaPrincipal Investigator, Center with Potential for Excellence in aParticular Area (CPEPA)University of Calcutta, 92 APC Road, Kolkata 700 009, India

Melatonin affords protection against myocardial ischemia-induced cerebral mitochondrial dysfunction: an in vivo study.

Auroma Ghosh1, Mousumi Dutta1,2, Arnab Kumar Ghosh1, Aindrila Chattopadhyay2, Debajit Bhowmick3 and Debasish Bandyopadyay1*1Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata 700 009, India

2Department of Physiology, Vidyasagar College, 39 Sankar Ghosh Lane, Kolkata 700 006, India3Acharaya Prafulla Chandra Sikhsha Prangan, University of Calcutta, JD-2, Sector-III, Salt Lake City, Kolkata 700 098, India

Received on:28-12-2014; Revised on: 17-01-2015; Accepted on:23-02-2015

ABSTRACTBackground: Ischemic Heart Disease (IHD) is a health problem of global concern. The studies on myocardial ischemia induced changes inbrain particularly cerebrum portion are scanty. Lacunae exist in the knowledge whether such changes can be protected by antioxidant(s). Thepresent work was carried out to explore the ameliorating potential of melatonin against isoproterenol induced changes in tissue and mito-chondria isolated from heart and brain of male Wistar rats. Methods: The adverse changes were induced by administering isoproterenolbitartrate subcutaneously at a dose of 25mg/kg body weight. Protective effects of melatonin was examined by administering melatonin at thedose of 40mg / kg body weight. After the treatment period, biomarkers of organ damage and oxidative stress biomarkers, activities ofmitochondrial antioxidant as well as Krebs cycle enzymes, and tissue and mitochondrial morphology was studied. Results: Isoproterenoladministration caused remarkable deleterious changes in oxidative stress biomarkers like lipid peroxidation level, reduced glutathione andprotein carbonyl content of tissue and mitochondria. Moreover, isoproterenol unfavorably altered the activity of antioxidant enzymes likeMn-SOD, glutathione peroxidase (GPx) and glutathione reductase (GR) in mitochondria thus confirming the generation of oxidative stress inmitochondria. Altered activity of some of the important Krebs cycle enzymes indicated the disturbance in energy metabolism. The degenera-tion of mitochondria was observed by scanning electron microscopy and fluorescence confocal microscopy. Melatonin pre-treatmentsuccessfully restored all the parameters to normal level at a dose of 40mg/kg body weight. Conclusion: Isoproterenol causes deleteriouschanges in cardiac and cerebrum portion of the brain tissue as well as the mitochondria and melatonin pre-treatment protects against suchchanges. Thus, therapeutic use of melatonin may be considered in treating cardiovascular diseases along with subsequent brain damages asthere may be strong relationship between cardiovascular diseases and cognitive functioning of brain.

KEYWORDS: Brain, heart, isoproterenol, melatonin, mitochondria, oxidative damage

INTRODUCTION:A large number of people are still suffering from myocardial ischemiain spite of modern health care facilities and well known therapeuticmeasures1. So, it is a real concern for researchers, medical and healthprofessionals to counteract the insults on heart arising from myocar-dial ischemia. In myocardial ischemia there is prolonged insufficientoxygenated blood supply to heart leading to the generation of reac-

tive oxygen species (ROS) and reactive nitrogen species (RNS) whichultimately give rise to oxidative stress and myocardial damage2, 3. Dis-ruption in normal functioning of heart can trigger a potential threat toother organs. Many recent studies indicated that there may be strongrelationship between cardiovascular diseases and cognitive impair-ment4 though further investigations are needed as cognitive changesare multi-factorial. According to elementary physiological knowledgeit is apprehensible that insufficient blood supply to brain due to myo-cardial ischemia may cause oxidative damages of brain but specificphysiological and biochemical pathways involved in this associationare yet to be determined.

Oxidative stress is one of the prime reasons for damage of differentorgans5. It occurs when there is a disproportion between the antioxi-



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dant defense mechanisms and free radical generation. Excessive freeradical accumulation leads to distortion of different vital moleculeslike DNA, proteins, lipids etc. Mitochondria being the energy genera-tor of the cell is highly susceptible to oxidative stress. During ATP

Isoproterenol (ISO) is a synthetic catecholamine and also known asa beta adrenergic receptor agonist. It is widely used to produce myo-cardial ischemia and myocardial infarction in experimental animalmodels. Many studies have indicated that in rat heart ISO can pro-duce similar changes that are generally seen in human heart aftermyocardial infarction. It has been reported that ISO initiates oxidativestress which ultimately produces myocardial ischemia and myocar-dial injury2, 7.

It is well evidenced that melatonin (N-acetyl-5- methoxytryptamine)is a potent antioxidant and free radical scavenger8. Many studieshave demonstrated the protective effects of melatonin against myo-cardial infarction2, 9. It gives protection by diminishing the ROS levelby donating electrons to free radicals10. The present study has at-tempted to explore not only the extent of protection that melatoninaffords against isoproterenol induced myocardial injury but also todetermine the effect of myocardial ischemia on cerebrum portion ofthe brain at the level of mitochondria and has also investigated andgauged the efficiency of melatonin to favorably protect the effects ofmyocardial ischemia on brain.

METHODS AND MATERIALS:

Chemicals and reagents:Melatonin and isoproterenol bitartrate were procured from SigmaAldrich,USA. The other chemicals and reagents used in this studywere of analytical grade and procured from Sisco Research Laborato

synthesis electrons are transported through different complexes ofelectron transport chain (ETC) present in inner membrane of mito-chondria. These electrons are finally transported to oxygen to pro-duce water and if sufficient amount of oxygen is not available thenthis transportation of electron will generate reactive intermediates

which will cause damage to biomacromolecules. During this electron

transport single electron leaks out and reacts with oxygen molecule

thus generating ROS 6. Excessive amount of ROS will be accumulatedin the system if they are not neutralized and/ or scavenged immedi-

ately by antioxidant defense mechanisms of cell. Reactive oxygen

species, particularly, hydrogen peroxide (H2O2) can easily react with

iron to produce extremely reactive hydroxyl radical (OH) which in

turn destroys the essential macromolecules like proteins, lipids and

nucleic acids2..

ries (SRL), Mumbai, India, Qualigens (India/Germany), Merck Lim-ited, Delhi, India and SD fine chemicals ,India.

Animals:The study protocol had been approved under the Centre with Poten-tial for Excellence in Particular Area (CPEPA) Scheme by the Institu-tional Animal Ethics Committee (IAEC) of the Department of Physiol-ogy, University of Calcutta. The CPCSEA registered animal supplierwas selected for procurement of male wistar rats, weighing 150-190g.The guidelines of the Committee for the Purpose of Control and Su-pervision of Experiments on Animals (CPCSEA) for animal handlingwere strictly followed during the experiment tenure.

Experimental Design:All rats were supplied with water ad libitum and food during theexperiment and quarantine periods. The rats were divided into 4groups:

1. Control: This group comprised of vehicle treated rats.2. Positive Control: The rats in this group were injected i.p.

with melatonin at the dose of 40mg/kg body weight, oneinjection 24 hour apart, i.e., for 2 days.

3. Treated Group: Isoproterenol bitartrate (ISO) was injectedsub-cutaneously to this group of rats at the dose of 25 mg/kg body weight, one injection 24 hour apart, i.e, for 2 days.

4. Protected Group: Rats in this group were injected i.p. with40 mg/kg body weight melatonin, 30 min prior to ISO injec-tion (25 mg/ kg body weight, s.c.); two injections of each 24hour apart, i.e., for 2 days.

Animal sacrifice and collection of blood and tissue samples:The animals were sacrificed through cervical dislocation followingmild ether anesthesia after the stipulated time period that is 24 hr afterthe last ISO injection. The chest cavity was surgically opened. Thenblood was collected carefully through cardiac puncture for serumpreparation and after blood collection the heart was surgically extir-pated. The whole brain is collected by carefully opening the cranialcavity and cerebrum portion was separated. The rinsing of the tis-sues was done in cold saline. After rinsing, the tissues were soakedproperly with blotting paper and stored at -200C in sterile plastic vialsfor biochemical analysis. A small portion of the tissue was fixed inappropriate fixative for tissue morphological studies.

Measurement of level of activity of SGOT:Serum glutamate oxaloacetate transaminase (SGOT) was measured

The animals were kept in a room of animal house where temperature(251 0C), humidity (50 10%) and 12 hr light/dark cycle were main-tained. The tenure of the treatment was 2 days.



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by applying standard protocols. The obtained values were expressedas IU/L.11

Preparation of heart and brain mitochondria:The procedure of Dutta et al.12 was followed to separate mitochondriafrom heart and brain tissue homogenates. Briefly, 10% cardiac andbrain tissue homogenates were prepared separately in a PotterElvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA) byusing ice cold sucrose buffer containing 0.25 (M) sucrose, 0.001(M)EDTA, 0.05(M) Tris-HCl (pH 7.8) at 250C. The homogenates werethen centrifuged at 500 g for 10 mins under cold condition i.e at 40C.The supernatants, thus obtained, were again centrifuged at 12000 gfor 15mins under cold condition. The obtained pellet containing mito-chondria was re-suspended in sucrose buffer and stored at -200C forbiochemical assays.

Measurement of lipid peroxidation (LPO) level, reduced glutathione(GSH) and protein carbonyl (PCO) content of cardiac and brain tis-sues and mitochondrial fraction:Ten percent tissue homogenates were prepared in ice-cold 0.9% sa-line. Lipid peroxides in the whole homogenates and mitochondrialsamples were estimated as thiobarbituric acid reactive substances(TBARS) by following the method of Buege and Aust 13 with somemodification as adopted by Bandyopadhyay et al.14. Thiobarbituricacid-trichloro acetic acid (TBA-TCA) reagent was mixed to measuredamount of tissue homogenates and mitochondrial samples separatelywith thorough shaking and then the mixtures were heated at 800C for20 min. The samples were then cooled to room temperature and cen-trifuged. The absorbance of the pink chromogen present in the su-pernatant was recorded spectrophotometrically at 532 nm using a UVVIS spectrophotometer (Shimadzu). Values were determined asnmoles/mg of protein.

To estimate reduced glutathione (GSH) content, the method of Sedlacand Lindsay 15 along with some modifications 14 was used (i.e., byusing Ellmans reagent). Briefly, 10% tissue homogenates were pre-pared by using 2mM ice cold ethylene diamine-tetraacetic acid(EDTA). Tris-HCl buffer (pH 9.0) was then mixed with thehomogenates and previously prepared mitochondrial samples. Fi-nally, DTNB was added to the above samples for color developmentand the absorbance was noted spectrophotometrically at 412 nm.Reduced glutathione content of the samples were expressed as nmoles/mg protein.

The DNPH assay16 was used to determine the protein carbonyl (PCO)content of both the tissue and the mitochondria. Tissue homogenateswere prepared by same procedure as mentioned in the measurement

of LPO. Briefly, 10 mM DNPH dissolved in 2(N) HCl was added to themeasured amount of mitochondrial suspension and or tissue homo-genate. Then, the samples were kept in dark at room temperature for 1hr with occasional mixing. After the incubation period, proteins of thesamples were precipitated by the addition of 30% TCA followed bycentrifugation at 4000g for 10 mins. The excess DNPH present in thepellet was washed with ethanol: ethyl acetate (1:1,v/v) mixture. Thiswashing was done for three times and after final washing the pelletwas dissolved in 6(M) guanidine hydrochloride prepared in 20mMpotassium dihydrogen phosphate (pH2.3). The absorbance was re-corded spectrophotometrically at 370 nm. The obtained values wereexpressed as nmoles/ mg of protein.

Determination of the activities of antioxidant enzymes present inmitochondria:Pyrogallol autooxidation method 17 was used to determine the manga-nese superoxide dismutase (Mn-SOD) activity. Increase in absor-bance was noted at 420nm for 3min in a UV/VIS spectrophotometer(Smart Spec Plus; BioRad). Activity of the enzyme was expressed asunits/min/mg of protein.

Method of Paglia and Valentine 18 with some modifications adoptedby Chattopadhyay et al. 19 was applied to assess the glutathioneperoxidase (GPx) activity. Final volume of 1 ml assay mixture con-sisted of 0.05 M phosphate buffer with 2mM EDTA pH7.0, 0.025mMsodium azide, 0.15mM glutathione, 0.25mM NADPH and mitochon-drial sample as enzyme source. The H2O2 (0.36 mM) was added toinitiate the reaction. Decrease in absorbance was noted spectropho-tometrically at 340nm and the enzyme activity was expressed as nmolof NADPH produced/min/mg tissue protein.

The method of Krohne-Erich et al.20 was used to determine the activ-ity of glutathione reductase (GR).The changes in absorbance wasrecorded spectrophotometrically at 340nm.The specific activity ofthe enzyme was expressed as unit/min/mg of protein.

Determination of the activities of pyruvate dehydrogenase (PDH)and some of the Krebs cycle enzymes:Method of Chretien et al. 21 with some modifications was adopted todetermine the activity of pyruvate dehydrogenase (PDH) by measur-ing the reduction of NAD+ to NADH at 340 nm. 50mM phosphatebuffer; pH - 7.4, 0.5 mM sodium pyruvate , 0.5mM NAD+ and optimumamount of mitochondrial sample in 1ml assay mixture were used toexecute the assay. The enzyme activity was denoted as units/min/mgof protein.

The activity of isocitrate dehydrogenase (ICDH) was determined by



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recording the reduction of NAD+ to NADH spectrophotometrically at340 nm according to the method of Duncan and Fraenkel22. One mlreaction mixture consisted of 50mM phosphate buffer;pH-7.4,0.5 mMisocitrate,0.1 mM MnSO4,0.1mM NAD+ and sufficient amount ofsample containing mitochondria. The activity of this enzyme wasrepresented as unit/min/mg of protein.

Alpha-ketoglutarate dehydrogenase (a-KGDH) activity was deter-mined by estimating the reduction of 0.35 mM NAD+ to NADH at 340nm using 50 mM phosphate buffer, pH - 7.4, as the assay buffer, and0.1 mM a-ketoglutarate as the substrate according to the methoddescribed by Duncan and Fraenkel22. The activity of this enzyme wasexpressed as unit/min/mg of protein.

Succinate dehydrogenase (SDH) activity was measured according tothe method of Veeger et al. 23with some modifications12. Assay mix-ture consisted of 50mM phosphate buffer; pH - 7.4, 2%(w/v) BSA,4mM succinate, 2.5mM K3Fe(CN)6 and optimum amount of samplecontaining mitochondria as the source of enzyme. The enzyme activ-ity was expressed as unit/min/mg of protein.

Aconitase activity was measured according to the method describedby Gardner et al24. Freshly isolated mitochondria were suspended in0.5 ml of buffer containing 50 mM Tris-HCl (pH 7.4) and 0.6 mMMnCl2 followed by the sonication for 2s. Aconitase activity was mea-sured spectrophotometrically at 240 nm and at 250C in terms of absor-bance of cis-aconitate gradually formed from externally added iso-citrate (20 mM). One unit was defined as the amount of enzyme nec-essary to produce 1mol of cis-aconitate per minute (e240=3.6 mM

-

1cm-1). The enzyme activity was expressed as units/min/mg of pro-tein.

Determination of the activities of mitochondrial respiratory chainenzymes:NADH-Cytochrome C oxidoreductase and cytochrome oxidase ac-tivities were measured according to the method described by Goyaland Srivastava25. The activity of NADH-Cytochrome C oxidoreduc-tase was measured spectrophotometrically by reduction of oxidizedcytochrome C at 565nm whereas activity of cytochrome oxidase wasalso determined spectrophotometrically by oxidation of reduced cy-tochrome C at 550 nm.

Measurement of di-tyrosine fluorescence intensity in mitochondria:Emission spectra of di-tyrosine, a product of tyrosine oxidation, wererecorded at 425nm (in the range 380 to 440 nm) (5 nm slit width) andexcitation wavelength was recorded at 325 nm (in the range 320 to 380nm) (5 nm slit width) 26, 27.

Determination of tryptophan fluorescence in samples containingmitochondria:The fluorescence emission spectra (from 300 to 450 nm, 5 nm slitwidth) of tryptophan were measured by excitation at 295 nm (2 nm slitwidth) according to the procedure mentioned by Dousset et al.26.

Measurement of mitochondrial swelling:To determine the mitochondrial swelling, the method of Halestrapand Davidson was followed by recording the changes in the absor-bance of the mitochondrial suspension spectrophotometrically at 520nm. The changes in values of absorption at 520nm were evaluated todetermine the swelling of mitochondria28.

Determination of mitochondrial intactness by using Janus green Bstain:Intactness of mitochondria was assessed by the procedure as fol-lowed by Dutta et al. 29. Mitochondrial smear was first prepared on aslide. Following this, few drops of Janus green stain were put on theslide and the slide was kept undisturbed in a moist chamber for 5mins. After stipulated time period, the slide was rinsed carefully withdistilled water in such a manner that the stain was not washed awaybut remained as diluted solution. The diluted stain with mitochondriawas covered with cover slip for confocal imaging. Confocal system(BD Pathway 855, USA) was used for imaging.

Scanning electron microscopy:The sample preparation for scanning electron microscopy was doneby the process as previously adopted by Dutta et al. 30with somemodifications. Images of the surfaces of the tissue and mitochondriawere captured by high definition camera attached with scanning elec-tron microscope. (SEM; Zeiss Evo 18 model EDS 8100)

Histological studies:A slice of the extirpated rat hearts and cerebrum portion of the brainwere fixed immediately in 10% formalin and embedded in paraffinfollowing routine procedure as used earlier by Mukherjee et al.31 andMitra et al.32. Left ventricular (LV) sections (5 m thick) and the sec-tions of cerebrum of brain (5 m thick) were prepared and stainedwith hematoxylin-eosin. The stained tissue sections were examinedunder Leica microscope and the images were captured with a digitalcamera attached to it.

Estimation of protein:The protein content of all samples was determined by the method ofLowry et al.33.



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Statistical evaluation:Every single experiment was repeated at least three times. Data arerepresented as mean SE. One way analysis of variances (ANOVA)followed by post hoc test (Tukeys HSD test) were done to determinethe significance of mean values of different parameters between thedifferent groups. Statistical tests were executed by using MicrocalOrigin version 7.0.

RESULTS:Figure 1 depicts that there is a remarkable increase in the activity ofSGOT in the serum of the rats treated with ISO which indicates themyocardial tissue damage. The serum level of SGOT of ISO treatedrats reached the maximal value and showed significant changecompared to control (P



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As depicted in Figure 3 (A and B), it is clear that ISO treatment signifi-cantly decreased the intactness of mitochondria isolated both fromthe heart and cerebrum portion of the brain tissue. The mitochondrialintactness was found to be normal in the groups where animals werepre-treated with melatonin. However, melatonin alone has no effecton the intactness of mitochondria obtained from both the tissues.

Brai

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CON MEL ISO MEL+ISO

Hear

t (B)

CON MEL ISO MEL+ISO

Fig.3.Protective effect of melatonin against ISO induced changes inintactness of mitochondria separated from heart and cerebrum portionof brain tissue. CON: injected with vehicle,MEL: 40mg/kg b.wt mela-tonin injected, i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO:Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated withISO(25mg/kg b.wt),s.c.Figure 4A clearly shows a significant rise (P< 0.001 versus control) inLPO level in the mitochondria isolated from both the heart and braintissues of the group treated with ISO. However, the LPO level in themitochondria of the protected animals (pre-treated with melatonin 30min prior to ISO injection) was found significantly lower comparingto ISO group. Figure 4B reveals ISO induced significant decrease inthe GSH content of the mitochondria which were almost protected tonormal values when pre-treated with melatonin. Figure 4C demon-strate the PCO content of the mitochondrial fraction which was foundto be significantly increased following treatment of rats with ISO(P< 0.001 versus control).This elevated level of protein oxidation wassignificantly decreased when the rats were pre-treated with melato-nin.

02468

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Fig.4.Protective effect of melatonin against ISO induced changes in(A) Lipid Peroxidation(LPO) level (B) Reduced glutathione (GSH) and(C)Protein carbonyl (PCO) content of mitochondrial fraction sepa-rated from heart and cerebrum portion of brain tissue of rats of differ-ent groups. CON: injected with vehicle,MEL: 40mg/kg b.wt melatonininjected, i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO: Pre-treated with melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.The values are expressed as meanSE; *P



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Fig.5.Protective effect of melatonin against ISO induced changes in(A) Mn-SOD activity (B) Glutathione peroxidase activity and(C) Glu-tathione reductase activity of mitochondrial fraction separated fromheart and cerebrum portion of brain tissue of rats of different groups.CON: injected with vehicle, MEL: 40mg/kg b.wt melatonin injected,i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO: Pretreated withmelatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/kgb.wt),s.c.The values are expressed as mean SE;*P



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Fig.6.Protective effect of melatonin against ISO induced changes inPyruvate dehydrogenase and some other Krebs cycle enzyme activi-ties in mitochondrial fractions of heart and brain tissues (A) Succinatedehydrogenase (SDH) activity (B)Isocitrate dehydrogenase (ICDH)activity(C)Alpha ketoglutarate(a-KGDH) activity (D)Pyruvate dehy-drogenase (PDH) activity (E) Aconitase activity CON: injected withvehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt.ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kgb.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.The values are ex-pressed as meanSE; *P



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Fig.8.Protective effect of melatonin against ISO induced changes inthe di-tyrosine fluorescence intensity.CON: injected with vehicle,MEL:40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt. ISO injecteds.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. andtreated with ISO(25mg/kg b.wt),s.c.The values are expressed asmeanSE; *P



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CON MEL ISO MEL+ISO

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Fig.11.Representation of images of protective effect of melatonin against ISO induced changes in heart which were captured during Scanningelectron microscopy (X6000).(A)Tissue (B) Mitochondria.CON: injected with vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kgb.wt. ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.

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Fig.12.Representation of images of protective effect of melatonin against ISO induced changes in brain which were captured during Scanningelectron microscopy (X6000).(A)Tissue (B) Mitochondria.CON: injected with vehicle, MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.



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The routine H & E stain of the tissues revealed that ISO at 25mg /kgbody wt, s.c. caused damage of cardiac tissue by creating myocardialfibre necrosis but produced only sporadic edema in brain tissue.However, when the rats were pre-treated with melatonin, these changesin the cardiac and cerebral tissue morphology were found to be al-most completely protected from being taken place.

Hea

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CON MEL ISO MEL+ISO

Fig. 13. (A) Changes of rat cardiac tissue morphology (H and Estained, 40X magnification) (B) Changes in tissue morphology ofcerebrum portion of brain tissue. (H and E stained, 40X magnifica-tion)

DISCUSSION:Many studies revealed that melatonin efficiently protects the heartfrom myocardial injury resulted from oxidative stress8, 2. It is wellevident that oxidative stress generated by isoproterenol (ISO) notonly affects the tissue but also the mitochondria of myocytes34. Astudy also showed that melatonin favorably changes the energymetabolizing enzymes primarily altered by isoproterenol31. But to in-vestigate the protection at mitochondrial level provided by melatoninagainst oxidative stress in details we performed different biochemicalassays to determine the oxidative stress biomarkers, antioxidant en-zyme status, activity of some of the Krebs cycle enzymes and alsosome morphological and fluorescence studies in isolated mitochon-dria. The results clearly confirmed that melatonin not only benefi-cially changes the activity of mitochondrial enzymes but also pre-serves the mitochondrial integrity thus helping in the proper func-tioning of mitochondria and assisting in the recuperation from oxida-tive damages. Excess accumulation of reactive oxygen species (ROS)in a system topples the antioxidant defense mechanisms thus oxidiz-ing the essential biological marcomolecules like lipids, proteins, car-bohydrates and DNA and it is well evident fact that these ROS along

with oxidized bio-molecules initiates inflammatory responses andvascular or endothelial dysfunction leading to different degenerativediseases like cardiovascular diseases, Alzheimers disease, diabetesetc35, 36. After administration of isoproterenol in the living system theoxidation of this compound generates quinones which upon furtheroxidation form superoxide anion and hydrogen peroxide. The super-oxide anion reduces tissue ferritin and thus produces hydroxyl radi-cal and hydrogen peroxide. This hydroxyl radical is extremely reac-tive species and it starts the lipid peroxidation reaction2. One of theaim of this study was to detect if isoproterenol itself or isoproterenolinduced myocardial injury can bring changes in the cerebral corticalportion of the brain tissue and its mitochondria. We have found el-evated level of LPO in the tissue and mitochondrial fraction of ratcardiac and cerebrum portion of the brain tissue after administrationof isoproterenol. Melatonin pre-treatment was found to be effectivein decreasing the LPO level to normal in both the mitochondrial frac-tion and tissue through its free radical scavenging activity37.

Elevated level of reduced glutathione (GSH) strengthens the antioxi-dant capacity of a system thus preventing the generation of oxidativestress38. Remarkable reduction in GSH content of both the tissue andmitochondria occurred to elevate the level of oxidative stress gener-ated by ISO. Melatonin pre-treatment elevated the GSH pool of boththe tissue and mitochondria by regulating the activity of two impor-tant enzymes such as glutathione peroxidase (GPx) and glutathionereductase (GR) involved in the metabolism of reduced glutathione(GSH) and oxidized glutathione (GSSG) 39.

The increased activity of Mn-SOD found in ISO treated group helpsin the alleviation of the oxidative stress 2 by quickly neutralizing su-peroxide anion. Pre-treatment with melatonin brought back the activ-ity of Mn-SOD to normal level as melatonin detoxifies the superoxideanion40.

The prime role of GPx is to protect the system from oxidative damageby the free radicals41. The GR which is another important enzyme inantioxidant defense system helps to regenerate GSH which is furtherused to counteract the oxidative stress 42. The reduced activity of GPxand GR were observed in the mitochondrial fraction of ISO treatedgroup. Melatonin pre-treatment was found to be effective in protect-ing the activities of these two enzymes as melatonin may spare theseenzymes by detoxifying the free radicals generated during oxidativestress.

The reduced activity of Krebs cycle enzymes in ISO treated groupwere found due to oxidative stress particularly hydrogen peroxidestress which is directly formed upon the oxidation of ISO 31,43. It is



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seen that aconitase is the most sensitive enzyme in Krebs cycle as itis readily inhibited by even the presence of small amount of hydro-gen peroxide while inhibition of a ketoglutarate activity is seen inhigher concentration of hydrogen peroxide43. Decreased activities ofother TCA cycle enzymes were found when ISO was administered.The main function of succinate dehydrogenase (SDH) enzyme is theconversion of succinate to fumarate thereby transferring the elec-trons to electron transport chain. Thus, decreased activity of SDHindicates limitation in electron flow and oxidative stress44. Decreasedactivity of respiratory chain enzymes were also observed in bothbrain and heart. Inhibited activity of Krebs cycle and respiratorychain enzymes decrease the efficiency of electron transport chainand facilitates the electron leakage thereby generating hydroxyl radi-cal which further disrupts the mitochondrial membrane 31. Melatoninpre-treatment protected the activities of these enzymes from beingaltered thereby helping in the amelioration of oxidative stress in-duced changes due to ISO. The scanning electron microscopy of themitochondrial surface confirmed the disruption of mitochondrial sur-face in ISO treated group where as melatonin pre-treatment protectedthe surface of this cell organelle from being damaged.

Mitochondria are the most important organelles present in the cell.They are called the power house of the cell as energy productionoccurred within the mitochondria. ATPs are generated by mitochon-dria when electrons are transported through the TCA cycle and respi-ratory chain complexes and thereby are very much susceptible tooxidative stress.

The study clearly shows that melatonin is a highly efficient moleculein curbing the oxidative damage 45not only in tissue level but also inmitochondrial level. Some recent studies indicated that there may bean association between ischemic heart disease and cognitive impair-ment46. From this study it can be concluded that either ISO directlyacts on brain like heart causing oxidative damage or ISO administra-tion first causes myocardial infarction and ischemia which in turndamages the brain. Further studies are essential to predict the director indirect effect of ISO on the brain.

CONCLUSION:It is clear from the study that melatonin protects both the mitochon-dria and the tissue of brain and heart against the oxidative damagesthrough its antioxidant mechanisms. So, the therapeutic use of mela-tonin may have beneficial effects on the people suffering from is-chemic heart disease with brain damages.

ACKNOWLEDGEMENT:This study was funded by a Major Research Project under CPEPA

scheme of UGC, Govt. of India, at University of Calcutta. AG, receiverof UGC JRF fellowship, gratefully acknowledges UGC. MD is sup-ported by Women Scientists Scheme A (WOS A), DST, Govt. ofIndia. Dr.AKG is the grantee of extended SRF under DST- PURSEProgram, Govt of India at University of Calcutta. Dr. AC is supportedfrom the funds available to her from Women Scientists Scheme A(WOS A), DST, Govt. of India. DB is assisted by the funds availableto him from BD Bio-Sciences, Kolkata, India. We sincerely acknowl-edge and thank Sri Pratyush Sengupta for his technical help in re-spect of Scanning Electron Microscopy. We also acknowledge withthanks the help provided to us by Center for Research in Nanoscienceand Nanotechnology (CRNN), University of Calcutta in respect ofusing sophisticated instruments. Prof. DB thankfully acknowledgesthe award of a Major Research Project under CPEPA Scheme of UGC,Govt. of India, at University of Calcutta.

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Source of support: UGC, Govt. of India, Conflict of interest: None

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