Modulation of age-related changes in oxidative stress markers and energy status in the rat heart and hippocampus: a significant role for ozone therapy

Modulation of age-related changes in oxidative stress markers andenergy status in the rat heart and hippocampus: a significant role forozone therapy

Maha M. El-Sawalhi1, Hebatallah A. Darwish1*, Mohamed N. Mausouf 2 and Amira A. Shaheen1

1Biochemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt2Ozone Therapy Unit, National Cancer Institute, Cairo University, Cairo, Egypt

Oxidative stress emerges as a key player in the ageing process. Controlled ozone administration is known to promote an oxidative precondi-tioning or adaptation to oxidative stress. The present study investigated whether prophylactic ozone administration could interfere with theage-related changes in the heart and the hippocampus of rats. Four groups of rats, aged about 3months old, were used. Group 1 (Prophylacticozone group) received ozone/oxygen mixture by rectal insufflations (0.6mg/kg) twice/week for the first 3months, then once/week till the ageof 15months. Group 2 (Oxygen group) received oxygen as vehicle for ozone in a manner similar to group 1. Group 3 (Aged control group)was kept without any treatment until the age of 15months. A fourth group of rats (Adult control group) was evaluated at 3months of age toprovide baseline data. Ozone alleviated age-associated redox state imbalance as evidenced by reduction of lipid and protein oxidationmarkers, lessening of lipofuscin deposition, restoration of glutathione levels in both tissues and normalization of glutathione peroxidaseactivity in the heart tissue. Ozone also mitigated age-associated energy failure in the heart and the hippocampus, improved cardiac cytosolicCa2+ homeostasis and restored the attenuated Na+, K+-ATPase activity in the hippocampus of aged rats. These data provide new evidenceconcerning the anti-ageing potential of prophylactic ozone administration. Copyright © 2012 John Wiley & Sons, Ltd.

key words—ozone; ageing; oxidative stress; energy status; heart; hippocampus

INTRODUCTION

Ageing is one of the biological processes shared by all livingorganisms. Although the intricate causes of the ageingprocess are still a matter of extensive speculation, there issome evidence that sustained damage inflicted by endogen-ously produced oxidants contribute to the development ofthe age-related deficits.1

Consistent with the free radical theory of ageing, the continu-ous threat of oxidative damage to various macromolecules suchas DNA, proteins and lipids is rapidly accumulating in differentorgans in the course of ageing; however, unfortunately thesedamaging processes are faced by progressive impairment ofantioxidant defences.2 One of the most reproducible aspectsof cell ageing is the progressive accumulation of lipofuscin withlife as a true ageing change particularly in post-mitotic cells,such as those of the heart and the brain.3 There are strongindications that progressive lipofuscin deposition ultimatelydecreases cellular adaptability and promotes the developmentof age-related pathologies, including neurodegenerativediseases, heart failure and macular degeneration.4,5

Additionally, a number of studies have demonstrated thatmitochondrial integrity declines as a function of age. There-fore, old mitochondria appear morphologically altered andproduce more oxidants and less adenosine triphosphate(ATP).1 A decline in ATP production can consecutivelydecrease the efficiency of energy-dependent processes andATP-mediated cell signal transduction.The prevalence of cardiovascular diseases increases with

advancing age. Even though long-term exposure to cardio-vascular risk factors plays a major role in the etiopathogen-esis of these diseases, intrinsic cardiac ageing enhances thesusceptibility to developing heart pathologies in late life.6

Cardiac tissue is largely post-mitotic and relies heavily onmitochondrial oxidative energy metabolism to providehigh-energy compounds necessary for contraction, but thisexposes the myocardium to harmful reactive oxygen species(ROS) that are generated continuously as normal by-productsof the mitochondrial electron transport chain. Moreover, theheart is highly susceptible to oxidative stress because of itsinherent decreased detoxifying natural antioxidants.7 Hence,progressive decline of mitochondrial function along with aresultant increase in oxidative stress have been proposed tobe one of the key factors underlying heart senescence.8,9

Cognitive functions, such as learning and memory,decline as ageing is in process. The hippocampus, in particular,

*Correspondence to: HebatallahA.Darwish, BiochemistryDepartment, Facultyof Pharmacy, Cairo University, Kasr Al-Aini Street, Cairo, 11562, Egypt.E-mail: [email protected]

Received 25 September 2012Accepted 24 October 2012Copyright © 2012 John Wiley & Sons, Ltd.

cell biochemistry and functionCell Biochem Funct 2013; 31: 518–525.Published online 21 November 2012 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/cbf.2930

shows a greater vulnerability to ageing.10 The agedhippocampus undergoes considerable biochemical andstructural modifications related to oxidative events thatare known to markedly compromise its function and toaccount for the most part of well-described learning andmemory deficits.11,12 For instance, enhanced lipid perox-idation,13 higher lipofuscin deposition in rat hippocampalCA3 pyramidal cells14 and increased protein carbonyls15

accompanied by disturbed glutathione redox state16 wereconsistently observed in the hippocampus of agedanimals. Interestingly, similar observations have beenfound in the same hippocampal neuronal population ofelderly individuals.17

In recent years, a great deal of research has been devotedto the development of strategies that can delay or evenreverse age-related impairments. Several lines of experi-mental and clinical evidence have confirmed advantageouseffects of ozone therapy as an adjuvant therapeutic modalityin pathophysiological conditions with an underlying oxida-tive burden. These include hepatic ischaemia–reperfusioninjury,18 renal injury19, endotoxic shock,20 diabetic nephro-pathy in rats21 and cardiovascular pathologies includingcardiopathy, atherosclerosis and experimental acute myocar-dial infarction.22,23

Controlled ozone administration could bring about a stateof ozone oxidative preconditioning (OzoneOP) or adapta-tion to oxidative stress so as to prepare the host to facepathophysiological events mediated by ROS.22 In light ofmore recent pharmacological knowledge, ozone could beconsidered as a pro-drug that, at certain non-toxic doses,can induce a rearrangement of the biochemical pathwayswith the activation of a second messenger in a cascade witha multiple system action.24 This can be seen in modulationof antioxidant defence systems, improvement of circulation,oxygen delivery and trophic processes in tissues as well asenhancement of autacoids, growth factors and cytokinesrelease.25

Given that, there are no data available concerning theeffects of ozone on the ageing process in the heart andhippocampus, in particular. The present study aimed atevaluating whether and, if so, via what mechanism theprophylactic administration of ozone could interfere withcertain age-related changes in these vital tissues.

MATERIALS AND METHODS

Chemicals

All biochemical reagents, substrates, standard enzymes andcoenzymes were purchased from Sigma-Aldrich ChemicalCo. (St. Louis, MO). Other chemicals and organic solventswere of the highest purity and analytical grade.

Ozone was generated from medical-grade oxygen byozone generator system [EXT 120-T, Longevity ResourcesInc., Canada]. It is a high quality oxygen-fed ozone gener-ator for ultra-pure medical applications. The ozone concen-tration is precisely measured by using a built-in UVspectrophotometer set at 254 nm.

Experimental design

Adult male albino Wistar rats (age, about 3months; weight,180–220 g) were used in the present study. Animals werekept under controlled environmental conditions and wereallowed free access to water and pelleted standard rat chowdiet. Animal care was supervised and approved by theResearch Ethical Committee of the Faculty of Pharmacy, CairoUniversity (Cairo, Egypt). After 1week of acclimatization, therats were randomly divided into three groups. Group 1:Prophylactic ozone group animals were given ozone/oxygenmixture by rectal insufflations at a dose of 0.6mg/kg bodyweight, administered twice weekly for the first 3months, thenonce per week till the age of 15months. The volume of insuf-flated mixture was approximately 5ml. Group 2: Oxygencontrol group rats were treated with oxygen only as the vehiclefor ozone in a manner similar to group 1. Group 3: Agedcontrol group rats were kept without any treatment untilthe age of 15months. In addition, Group 4, a group of normal3-month-old rats was obtained 1week before sacrifice andserved as an Adult control group for the aforementioned agedgroups.

Tissue sampling

At the end of the experimental period, animals were sacri-ficed by decapitation. Hearts and brains were immediatelytaken out on ice, washed with ice-cold saline and blotteddry with filter paper, and the brains were then cooled in adeep freezer to facilitate its dissection. The hippocampi fromthe left and right sides of the brain were rapidly dissectedout on an ice-cold plate and pooled together to make onesample. Each assay was performed on six animals (n = 6).The isolated hearts and hippocampi were weighed andhomogenized in nine volumes of ice-cold saline using anUltra Turrax electric homogenizer and ice-cooled glasshomogenizer for hearts and hippocampi, respectively. Theresultant homogenates were appropriately prepared and usedfor determination of cardiac and hippocampal oxidativestress markers: malondialdehyde (MDA), protein carbonyls(Pr Co), lipofuscin and glutathione (GSH) levels as well asglutathione peroxidase (GPx) activity. Cardiac and hippo-campal energy status was assessed by measuring ATP andadenosine diphosphate (ADP) contents. In addition, cardiaccytosolic Ca2+ level and hippocampal sodium and potassiumadenosine triphosphatase activity (Na+, K+-ATPase) werealso determined.

Biochemical investigations

Malondialdehyde. An aliquot of each homogenate wasmixed with ice-cold 2.3% KCl (1:1) and centrifuged at600 g at 4�C for 15min, and the supernatant was used forestimation of MDA levels as an indicator of lipid peroxidationaccording to the method of Mihara and Uchiyama.26 In brief,MDA reacts with thiobarbituric acid, giving a colouredcomplex that can be measured spectrophotometrically.

519ageing and ozone therapy

Copyright © 2012 John Wiley & Sons, Ltd. Cell Biochem Funct 2013; 31: 518–525.

Protein carbonyls. Another portion of each homogenatewas mixed with an equal volume of ice-cold 100mMphosphate buffer pH7.4 and centrifuged at 11 000 g at 4�Cfor 20min using a Sorvall Combiplus ultracentrifuge (Du PontCompany, USA). Protein carbonyls were measured in the re-sultant supernatants using the 2,4-dinitrophenylhydrazinemethod of Reznick and Packer27 as modified by Liu et al.28

This method is based on the spectrophotometric detection ofprotein hydrazones following reaction with 2,4-dinitrophenyl-hydrazine. Results were expressed as nanomoles of carbonylsper milligram of protein. Because about 10–15% of proteinsare lost in the reaction procedure, the protein levels werequantified in the final pellets by reading the absorption at280 nm. The amount of proteins was calculated from a bovineserum albumin standard treated in the same way.15

Lipofuscin. Spectrofluorimetric determination of lipofuscincontent was carried out in the crude homogenates accordingto the method of Tappel et al.29 The fluorescence intensityof the chloroform–methanol tissue extracts was measuredwith a Shimadzu spectroflourimeter at an excitation wave-length of 350 nm and emission wavelength of 435 nm.Results were expressed in relative fluorescence units usingquinine sulphate as a standard.

Glutathione. Suitable portions of cardiac and hippocampalhomogenates were mixed with ice-cold 5% sulfosalicylic acid(1:2) and centrifuged at 1800 g at 4�C for 15min. The GSHcontent was measured using 5,50-dithiobis-(2-nitrobenzoicacid) (Ellman’s reagent), which produces a stable yellowcolour that can be measured colorimetrically.30

Preparation of cytosolic fractions. An aliquot of each hom-ogenate was mixed with equal volume of ice-cold Tris–EDTAbuffer pH7.6 (100mMTris and 0.2mMEDTA) and ultracen-trifuged at 105 000 g at 4�C for 20min. The separatedcytosolic fractions were used for the determination of GPxactivity in the heart and hippocampus, whereas, Ca2+ levelwas determined in the cardiac cytosolic fraction only.

Glutathione peroxidase. Glutathione peroxidase activitywas assessed according to themethod of Paglia and Valentine31

by following the rate of reduced nicotinamide adeninedinucleotide phosphate (NADPH) oxidation in the presenceof hydrogen peroxide, GSH and glutathione reductase as adecrease in absorbance at 340 nm. Enzyme activity wasexpressed as units per milligram protein, where one unit isdefined as the amount of enzyme that oxidizes 1mmolNADPH per min at 25�C.

Adenine nucleotides (adenosine triphosphate and adenosinediphosphate). An appropriate volume of each homogenatewas mixed with ice-cold 4.8M perchloric acid centrifugedat 600 g at 4�C for 15min. One ml of the supernatant wasneutralized with 0.33ml of 2M KHCO3 and centrifuged at600 g at 4�C for 10min.32 The concentrations of ATP andADP in the supernatant were assayed using high-performanceliquid chromatography according to a modified technique of

Teerlink et al.33 The analysis was performed by reversed-phasechromatography on a C18 column containing 3mm particles,employing gradient elution and UV detection at 245 nm(Shimadzu CR501 Chromatopac integrator).

Cardiac cytosolic Ca2+. It was assessed in the cytosolicfraction of the heart by the atomic absorption technique34

using a SOLAAR system Unicam 939/959 atomic absorptionspectrometer.

Hippocampal Na+, K+-ATPase. An aliquot of hippocampalhomogenate was mixed (1:2) with ice-cold 0.435M sucroseand centrifuged at 600 g at 4�C for 15min. Na+, K+-ATPaseactivity was determined in the supernatant according to themethod described by Rangaraj and Kalant.35 It is based onmeasuring the difference between the rate of release of inor-ganic phosphate (Pi) from ATP in the absence and presenceof ouabain. The liberated Pi was estimated using the methodofWeidman.36 Enzyme activity was expressed as micromolesof Pi per hour per milligram protein.

Protein. The protein content of the cytosolic fractions andthe supernatant for hippocampal Na+, K+-ATPase activitywas determined according to the method of Lowry et al.37

using Folin–Ciocalteu reagent with bovine serum albuminas a standard.

Statistical analysis. The results were expressed as the mean� SEM, and statistical comparisons were carried out usingone-way analysis of variance followed by Tukey–Kramer’smultiple comparisons test. The minimal level of significancewas identified at p< 0.05.

RESULTS

Effect of prophylactic ozone administration on age-associatedchanges in the oxidative stress markers in the heart of aged rats

Compared with adult control rats, 15-month-old aged ratsshowed significant increases in cardiac levels of MDA(142%), Pr Co (213%) and the ageing pigment lipofuscin(169%). In addition, aged rat heart exhibited a significantreduction in reduced GSH level (62%) accompanied by ahigher GPx activity (139%) (Table 1). Prophylactic ozoneadministration clearly reversed the cardiac levels of MDA,GSH and GPx activity back near to their values of adultcontrol group. Moreover, ozone intervention significantlyreduced cardiac Pr Co and lipofuscin levels reaching 56%and 65% of the aged control values, demonstrating itsprotective effects. Administration of oxygen (a vehicle forozone) insignificantly reduced cardiac contents of MDA(88%), Pr Co (87%) and lipofuscin (83%) compared withthe values of aged control group (Table 1).

Effect of prophylactic ozone administration on age-associatedchanges in the oxidative stress markers in the hippocampus ofaged rats

Data presented in Table 2 indicate that ageing induced sig-nificant elevation in hippocampus MDA, Pr Co and

520 m. m. el-sawalhi ET AL.


lipofuscin levels reaching about 1.5-fold, 2.5-fold and 2-fold,respectively, relative to adult control levels. Moreover,reduced GSH level in the hippocampus of aged rats wasmarkedly lower than that of adult ones (61%), whereas agedhippocampal GPx activity was not significantly altered.Meanwhile, prophylactic ozone administration noticeablyreduced elevated MDA, Pr Co and lipofuscin levels reaching80%, 58% and 64% of the aged control values, respectively.The hippocampal level of reduced GSH in the ozone-treatedgroup was significantly higher (138%) than that of the agedcontrol group; however, it did not significantly differ fromthe values of the adult control group. On the other hand, theoxygen-treated group showed a significant decrease in hippo-campal lipofuscin (67%) levels, along with insignificantreductions in the contents of MDA (86%) and Pr Co (81%)as compared with the aged control group. In addition, neitherozone nor oxygen treatment significantly affected hippocam-pal GPx activity of the aged rats (Table 2).

Effect of prophylactic ozone administration on age-associatedchanges in the energy status and cytosolic Ca2+ levels in theheart of aged rats

As shown in Table 3, the development of ageing was asso-ciated with significant decreases in cardiac contents of

ATP, ADP and ATP :ADP ratio (78%) when compared withadult control values. Such changes in the energy status wereaccompanied by a remarkable increase in cytosolic Ca2+

level (173%) in the heart of aged rats. Prophylactic ozoneadministration caused a significant increase in the cardiacATP content (137%) along with a significant decrease incytosolic Ca2+ level (75%) as compared with those levelsin the aged control group. Furthermore, ozone treatmentconsiderably restored the reduced cardiac ATP : ADP ratioto reach 96% of the values of the adult control group.

Effect of prophylactic ozone administration on age-associatedchanges in the energy status and Na+, K+-ATPase activity inthe hippocampus of aged rats

Data in Table 4 showed marked reductions in ATP level(63%), ATP :ADP ratio (65%) and Na+, K+-ATPase activity(55%) in the hippocampus of aged rats compared with adultcontrol ones. However, age had no significant effect on theADP level in this brain region. Ozone significantly reversedthe age-associated decrease in hippocampal ATP andthe ATP : ADP ratio and restored the attenuated Na+, K+-ATPase activity, reaching 81%, 89% and 97% of the adultcontrol values, respectively.

Table 1. Effect of prophylactic ozone administration on age-associated changes in the oxidative stress markers in the heart of aged (15months old) rats

Groups Adult control Aged control Oxygen control Prophylactic ozone

ParametersMDA (nmol/g tissue) 148.8� 4.71 210.7� 5.14** 184.7� 9.3* 151.2� 9.37††

Pr Co (nmol/mg protein) 1.63� 0.205 3.47� 0.3** 3.01� 0.34* 1.93� 0.33††

Lipofuscin (RFU/g tissue) 2.64� 0.23 4.47� 0.32** 3.73� 0.31 2.92� 0.33††

GSH (mmol/g tissue) 2.14� 0.18 1.32� 0.08** 1.33� 0.09** 1.99� 0.2†

GPx (U/mg protein) 0.28� 0.024 0.39� 0.028* 0.35� 0.014 0.29� 0.013††

Values represent mean�SEM for six rats.MDA, malondialdehyde; Pr Co, protein carbonyls; GSH, glutathione; GPx, glutathione peroxidase; RFU, relative fluorescence units.*Significant difference from adult control group at p< 0.05.**Significant difference from adult control group at p< 0.01.†Significant difference from aged control group at p< 0.05.††Significant difference from aged control group at p< 0.01.

Table 2. Effect of prophylactic ozone administration on age-associated changes in the oxidative stress markers in the hippocampus of aged (15months old)rats


ParametersMDA (nmol/g tissue) 47.3� 2.65 68.8� 1.61** 58.9� 2.36 54.8� 5.32†

Pr Co (nmol/mg protein) 1.01� 0.13 2.54� 0.42** 2.07� 0.24* 1.48� 0.11†

Lipofuscin (RFU/g tissue) 1.62� 0.17 3.27� 0.33** 2.2� 0.03†† 2.08� 0.15††

GSH (mmol/g tissue) 0.80� 0.015 0.49� 0.032** 0.54� 0.039** 0.68� 0.043††

GPx (U/mg protein) 0.140� 0.009 0.163� 0.011 0.160� 0.008 0.170� 0.009

Values represent mean�SEM for six rats.MDA, malondialdehyde; Pr Co, protein carbonyls; GSH, glutathione; GPx, glutathione peroxidase; RFU, relative fluorescence units.*Significant difference from adult control group at p< 0.05.**Significant difference from adult control group at p< 0.01.†Significant difference from aged control group at p< 0.05.††Significant difference from aged control group at p< 0.01.



DISCUSSIONThe present study may be the first to report beneficial effectsof long-term prophylactic administration of low ozone doseon the age-associated alterations in the heart and the hippo-campus of rats. The promising anti-ageing effects of ozoneare likely mediated via a mechanism that involves alleviat-ing age-associated redox state imbalance as evidenced byreduction of lipid and protein oxidations markers, lesseningof lipofuscin deposition, restoration of GSH levels in bothtissues and normalization of GPx activity in aged hearttissue. Ozone also mitigated age-associated energy failurein the heart and the hippocampus of rats, improved cardiaccytosolic Ca2+ homoestasis and restored the attenuatedNa+, K+-ATPase activity in the hippocampus of aged rats.Oxidative damage is strongly implicated in the ageing

process by inducing striking biochemical, structural andbehavioural changes.38 The level of oxidized proteins wasprogressively increased in different organs of several experi-mental models of ageing39 and more particularly in the hippo-campus region.15 In the present investigation, significantlyelevated protein carbonyl contents were observed in the heartand the hippocampus of old rats. Age-related accumulation ofoxidatively modified proteins has been suggested to reflectboth increased rates of ROS generation and decreased antioxi-dant activities together with losses in the capacity to degrade

modified proteins by the proteasomal- and lysosomal-proteolytic pathways.40 In conformity with the presence ofoxidative events, our results showed an age-dependent in-crease in lipid peroxidation and GPx activity, along with areduction in GSH levels. In fact, ROS once generated pro-voke deleterious effects on various cellular components,among which are membrane lipids that are extensivelysubjected to peroxidation.41 Meanwhile, the observedreduction of GSH concentration might be attributed to theimbalance between reduced synthesis and increasedutilization. In the present study, lowered ATP levels in theaged group may account for the reduced GSH synthesis,as its de novo synthesis is an ATP-dependent process.42

On the other side, the increased GSH consumption couldbe related in the current work to the tendency of cardiacand hippocampal GPx activity to be increased by age. It isknown that both the heart and the hippocampus are highlysusceptible to oxidative stress because of their inherentdecreased detoxifying natural antioxidants.7,43 Thus, withregards to the fact that exposure to oxidants acts as a signalto increase the activity and expression of antioxidantenzymes,44 it seems more likely to expect an increase inantioxidant enzyme activity with age as an adaptive mech-anism to protect these antioxidant-deficient tissues fromoxidative stress.

Table 4. Effect of prophylactic ozone administration on age-associated changes in the energy status and Na+, K+-ATPase activity in the hippocampus of aged(15months old) rats


ParametersATP (mmol/g tissue) 6.24� 0.161 3.90� 0.141** 4.39� 0.325** 5.04� 0.416*†

ADP (mmol/g tissue) 4.03� 0.323 3.87� 0.279 3.75� 0.147 3.58� 0.157ATP :ADP ratio 1.58� 0.091 1.02� 0.040** 1.16� 0.056** 1.4� 0.074††

Na+, K+-ATPase(mmol Pi/hr/mg protein) 4.29� 0.38 2.38� 0.26* 2.50� 0.34* 4.16� 0.47†

Values represent mean�SEM for six rats.ATP, adenosine triphosphate; ADP, adenosine diphosphate; Na+, K+-ATPase, sodium, potassium adenosine triphosphatase.*Significant difference from adult control group at p< 0.05.**Significant difference from adult control group at p< 0.01.†Significant difference from aged control group at p< 0.05.††Significant difference from aged control group at p< 0.01.

Table 3. Effect of prophylactic ozone administration on age-associated changes in the energy status and cytosolic Ca2+ levels in the heart of aged (15monthsold) rats


ParametersATP ( mmol/g tissue) 13.49� 0.923 7.21� 0.236** 8.30� 0.489** 9.85� 0.345**†

ADP ( mmol/g tissue) 5.40� 0.287 3.80� 0.283** 4.39� 0.327 4.13� 0.264*ATP :ADP ratio 2.50� 0.108 1.94� 0.103** 1.92� 0.101** 2.40� 0.066†

Ca2+ (mg/g tissue) 114.3� 6.233 197.8� 10.965** 192.0� 16.823** 148.6� 10.837†

Values represent mean�SEM for six rats.ATP, adenosine triphosphate; ADP, adenosine diphosphate.*Significant difference from adult control group at p< 0.05.**Significant difference from adult control group at p< 0.01.†Significant difference from aged control group at p< 0.05.



Oxidatively modified protein and lipid peroxidationproducts constitute the bulk of lipofuscin granules. Thisundegradable pigment gradually accumulates within thelysosomal compartment of post-mitotic cells and is consid-ered a hallmark of normal ageing.3,4 In harmony, the presentdata showed that the level of lipofuscin was significantlyincreased in aged rats relative to the adult ones. This appar-ent lipofuscin accumulation represents a prominent andstable structural marker of cellular oxidative damage,45

being linked to decreased antioxidative defence systems,lysosomal iron overload and mitochondrial dysfunction.4,45

The widespread use of medical-grade ozone at well-definedand safe protocols has highlighted its potential benefits as atherapeutic agent.24,46 In the present study, prophylactic lowozone dose administration for 12months clearly reversed theage-associated increases in cardiac and hippocampal proteincarbonyls, lipid peroxides, namely, MDA and lipofuscinlevels of aged rats back near to their normal values of the adultcontrol group. Additionally, ozone intervention elevated GSHlevels in the heart and the hippocampus and normalized GPxactivity in cardiac tissue. Previous studies have demonstratedthat ozone therapy effectively reduced oxidative stressmarkers and improved antioxidant capacity in various animalmodels.18–23 These positive experimental observations couldbe explained in the light of OzoneOP, a state obtained on judi-cious and controlled use of ozone.47 The concept of OzoneOPimplies that a repeated and controlled stress is able to provideprotection against a prolonged and severe stress. Duringozone therapy, ozone dissolves in the plasmatic water andinstantaneously reacts with biomolecules, generating ROS,among which are H2O2 and lipid peroxidation products(LOPs) acting as second messengers responsible for elicitingseveral biological and therapeutic effects. ROS are actingimmediately and disappear as early and short-acting mes-sengers, whereas LOPs, via the circulation, are distributedthroughout the tissues, and their pharmacodynamics allowsthem to become late and long-lasting messengers. Thesemolecules can eventually induce an antioxidant responsecapable of reversing a chronic oxidative stress andconcomitantly preserve the cellular redox state.25,46 OzoneOPmay also involve protein synthesis, as a major mechanism ofredox homeostasis is based on the ROS-mediated inductionof redox-sensitive signal cascades that lead to increasedexpression of antioxidants.48 Indeed, Iles and Liu49 havedemonstrated that some LOPs, by inducing the expressionof glutamate cysteine ligase, cause intracellular increase ofGSH, thus increasing the detoxification capacity of the cells.This finding may account at least partially for the protectiveeffects of ozone on GSH levels in the heart and the hippocam-pus of aged rats in the current study. In the meantime, theelevated GSH levels could also be related to the ability ofOzoneOP to preserve cardiac and hippocampal ATP :ADPratio in our study, which might contribute to the de novoGSH synthesis. Additionally, ozone possibly regulates glu-cose-6-phosphate dehydrogenase22 and, therefore, facilitatesregeneration of reduced GSH via providing NADPH andenhancing the GSH redox enzyme system. It is plausible thatthe enhancement of GSH in the ozone pretreated group has in

turn provided a protection against ROS and suppressed theprocess of protein and lipid oxidations leading to decrementsof protein carbonyls and MDA levels in both the heart andthe hippocampus of these aged rats. A further explanationfor the observed effect of ozone on elevated protein carbonyllevels could be via the upregulation of heat shock protein-90,with its ability to activate the proteasome degradative path-way, thus reducing the accumulation of oxidized proteins.50

Moreover, the lessening effect of ozone therapy on lipofuscindeposition in both tissues in the present study revealed oncemore its protective ability. The decreased accumulation oflipofuscin induced by ozone may well be a consequence ofits ability to protect proteins and lipids against oxidativealterations. Likewise, normalization of cardiac GPx activityof aged rats in response to ozone therapy in the current workcould reflect the ability of OzoneOP to improve the antioxi-dant–prooxidant balance and hence to spare the compensatoryincrease in this vital antioxidant enzyme.

An age-dependent increase in the production of freeradicals in mitochondria inevitably elevates the oxidativedamage in the mitochondria as well as the entire cell, therebydecreasing mitochondrial enzyme activities and compromis-ing their ability to meet cellular energy demands.51,52 Inagreement, the present data showed significant reductions ofATP levels as well as ATP :ADP ratios in both the heartand the hippocampus, along with a significant reduction inADP level only in cardiac tissues of aged rats relative to theadult ones. Yarian et al.53 showed that mitochondrial aconi-tase and ATP synthase, in particular, are likely targets ofMDAmodification with an age-related decrease in their activ-ities in the mouse heart. Thus, the observed decrease in ATP :ADP ratio clearly indicated that the inhibitory effect on ATPsynthesis was greater than that exerted on its utilization withnet reduction in the ATP content.

In the current work, the level of free cytosolic Ca2+ in theaged heart was significantly increased. Such increase maybe due to increased production of ROS and loss ofmitochondrial membrane integrity. Formerly, it has beenreported that Ca2+-ATPases are quite sensitive to exposureto oxidative stress and are inhibited by a variety of oxygenradicals. Inhibition of Ca2+-ATPases can in turn increaseintracellular concentration of Ca2+, alter the signal transduc-tion pathways and cellular fluidity, and eventually result incell death.54

Similarly, alterations in Na+K+-ATPase activity mayrepresent an important neurotoxic mechanism for neurons.55

In line with earlier studies,56,57 the activity of hippocampalNa+K+-ATPase was found to be decreased in aged rats.Decreased Na+K+-ATPase activity might be related to theprogressive increase in the lipofuscin contents in the hippo-campus, which damages the mitochondrial lipid bilayercontaining Na+K+-ATPase.56,57 Moreover, the cross-linkingof membrane proteins by MDA may lead to disturbed cellmembrane fluidity and the inactivation of importantmembrane-spanning proteins including the ion transportATPases.58

In the present study, OzoneOP effectively reversed theage-associated decrease in cardiac and hippocampal ATP



as well as the ATP :ADP ratio, significantly decreasedcardiac cytosolic Ca2+ level and restored the attenuated hip-pocampal Na+, K+-ATPase activity of aged rats. Improvedenergy status observed in our model might be explainedon the basis that ozone activates enzymes responsible forbody protection from processes linked to overproductionof superoxides, induces antioxidant enzymes and makesoxygen utilization in the mitochondrial respiratory chainmore effective.47 OzoneOP also stimulates transmembra-nous flow of oxygen47 and improves the blood flow incerebral low-perfusion conditions.59 The improved bloodflow, and hence oxygen supply to tissues, might stimulateaerobic respiration, recover energy metabolism and restorethe normal ATP content. Additionally, Re et al.24 havedemonstrated that OzoneOP significantly antagonized thedecrease in the level of ATP and GSH in cortical and thestriatal regions of rotenone-treated animals. Thus, it seemslikely that ozone could enhance mitochondrial functionsby scavenging free radicals and by increasing the reducedforms of antioxidants such as GSH that might play roles aspotent protective agents.The effect of OzoneOP on cellular Ca2+ homeostasis may

be due to ozone’s capacity to protect Ca2+-ATPases againstinactivation of oxidative challenge.47 This was in line withthe report of Ajamieh et al.60 that showed similar protectiveeffect of OzoneOP on Ca2+ levels in a liver ischaemia–reperfusion injury model. Moreover, lack of energy has beenshown to cause inhibition of Ca2+-ATPases present in thesarcoplasmic reticulum, leading to the intracellular Ca2+

overloads.61 One could speculate that energy conservationcaused by OzoneOP might result in Ca2+-ATPase activationand thereby restoration of cardiac cytosolic Ca2+ levels.Meanwhile, the efficacy of OzoneOP in restoring the

attenuated hippocampal Na+, K+-ATPase activity of aged ratscould be related to increased GSH availability, as it has beenreported that thiol level is important for maintaining thestructure and function of ATPases, which has been depletedwith advancing age.56,62 Furthermore, Na+, K+-ATPase activ-ity is known to be sensitive to lipid peroxidation.56 Thus,ozone’s antilipidperoxidative activity may be responsible forthe activation of Na+, K+-ATPase in aged rat hippocampus.Taken together, data presented in this study are indicative

of potentially positive effects caused by long-term use oflow ozone doses. Eventually, the study warrants furtherresearches and clinical trials to support the use of ozone asa prophylactic medical approach that could maintain a goodquality of life for elderly people.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

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Modulation of age-related changes in oxidative stress markers and energy status in the rat heart and hippocampus: a significant role for ozone therapy