OZONE TREATMENT REDUCES MARKERS OF OXIDATIVE AND ENDOTHELIAL DAMAGE IN AN EXPERIMENTAL DIABETES MODEL IN RATS

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1 Phrmologil Reserh, Vol. 44, No. 5, 21 doi:1.16/phrs , ville online t on OZONE TREATMENT REDUCES MARKERS OF OXIDATIVE AND ENDOTHELIAL DAMAGE IN AN EXPERIMENTAL DIABETES MODEL IN RATS SAID MOHAMMED AL-DALAIN, GREGORIO MARTÍNEZ, EDUARDO CANDELARIO-JALIL, SILVIA MENÉNDEZ, LAMBERTO RE, ATTILIA GIULIANI d nd OLGA SONIA LEÓN, Center for Reserh nd Biologil Evlution (CIEB-IFAL), University of Hvn, Hvn 14, Cu, Ozone Reserh Center, Cu, University of Anon, Itly, d Deprtment of Chemistry nd Medil Biohemistry, University of Miln, Vi Sldini, Miln, Itly Aepted 17 July 21 Ozone hs een used s therpeutil gent nd enefiil effets hve een oserved. However so fr only few iohemil nd phrmodynmi mehnisms hve een eluidted. We demonstrte tht ontrolled ozone dministrtion my promote n oxidtive preonditioning or dpttion to oxidtive stress, preventing the dmge indued y retive oxygen speies (ROS). Tking into ount tht dietes is disorder ssoited with oxidtive stress, we postulte tht ozone tretment in our experimentl onditions might protet ntioxidnt systems nd mintin, t physiologil level, other mrkers of endothelil ell dmge ssoited with dieti omplitions. Five groups of rts were lssified s follows: (1) ontrol group treted only with physiologil sline solution; (2) positive ontrol group using streptozotoin (STZ) s dietes indutor; (3) ozone group, reeiving 1 tretments (1.1 mg kg 1 ), one per dy fter STZ-indued dietes; (4) oxygen group (26 mg kg 1 ), one per dy, s in group 3 ut using oxygen only; (5) ontrol ozone group, s group 3, ut without STZ. The ozone tretment improved glyemi ontrol nd prevented oxidtive stress, the inrese of ldose redutse, frutolysine ontent nd dvned oxidtion protein produts. Nitrite nd nitrte levels were mintined without hnges with regrd to non-dieti ontrol. The results of this study show tht repeted dministrtion of ozone in non-toxi doses might ply role in the ontrol of dietes nd its omplitions. 21 Ademi Press KEY WORDS: ozone, streptozotoin-indued dietes, oxidtive stress. INTRODUCTION Dietes produes lrge numer of hnges in vessels tht ffet the retivity of smooth musle nd endothelium, the prodution of vsotive sustnes y endothelium, vessel wll permeility to mromoleules, suseptiility to theroslerosis nd tivity of the thromolyti system [1 3]. These events re relted to the hroni vsulr omplitions of this disorder. The vsulr lesion in dietes onsists of mirongiopthy, distinguished y thikening of pillry sement memrnes resulting in inresed vsulr permeility. These hnges re linilly mnifested s dieti retinopthy nd/or mirongiopthy, whih onsists of theromtous involvement of lrge lood vessels. Mrongiopthy is Corresponding uthor. Center for Reserh nd Biologil Evlution (CIEB-IFAL), University of Hvn, Hvn 14, Cu. E-mil: olg@ie.sld.u morphologilly similr to non-dieti therom, ut tending to our erlier nd e more extensive [4]. Vsulr endothelium ppers to e vulnerle trget for hyperglyemi-indued metoli hnges. High gluose onentrtions promote endothelil ell dmge y different mehnisms, proly through mutul filittory intertions etween them [5]. Ativtion of polyol pthwy, non-enzymti glyosyltion of proteins nd the inrese of retive oxygen speies (ROS) ply n importnt role in dieti omplitions. Ozone, dministered y retl insuffltion in numer of ontrolled tretments, hs shown protetive effets ginst the dmge indued y ron tetrhloride nd hepti nd renl ishemi-reperfusion through prole mehnism of oxidtive preonditioning whih onfers protetion y stimultion of ntioxidnt endogenous systems, umultion of denosine nd y loking the xnthine/xnthine oxidse pthwy for ROS genertion [6 9]. In ddition, derese of lood /1/ /$35./ 21 Ademi Press

2 392 Phrmologil Reserh, Vol. 44, No. 5, 21 holesterol nd stimultion of ntioxidtive response in rdiopthy ptients treted with intrvenous ozone therpy hs een demonstrted [1]. Tking the view tht dietes promotes n oxidtive dmge nd ozone protets the ells in oxidtive stress situtions, we investigted the tions of ozone on streptozotoin-indued dietes, hrterizing the redox lne nd its reltion with mrkers of polyol pthwy, non-enzymti glyosyltion of proteins nd the levels of nitrtes nd nitrites, s mesure of nitri oxide (NO) prodution. MATERIALS AND METHODS Animls Mle Sprgue Dwley rts weighing g were otined from CENPALAB (Bejul, Hvn, Cu). Animls were housed in temperture- nd lightontrolled rooms nd llowed free ess to norml diet pellets nd tp wter. All proedures were performed s pproved y the Institutionl Animl Cre Committees (ARCA No. 12) nd in ordne with the Europen Union Guidelines for niml experimenttion. Indution of experimentl hyperglyemi Experimentl dietes ws indued y single intrperitonel (i.p.) injetion of 45 mg kg 1 streptozotoin (STZ) (Sigm, St Louis, MO, USA) to overnight fsted rts [11]. STZ ws dissolved in itrte uffer solution (.1 M, ph 4.5) nd freshly prepred immeditely efore injetion. Animls were onsidered hyperglyemi when non-fsting serum gluose levels were higher thn 2 mm fter 48 h of STZ injetion [12]. Blood gluose ws mesured using dignosti kit otined from Sigm (Sigm, St Louis, MO, USA) sed on olorimetri retion. Animls nd tretment The protool onsisted of five experimentl groups (n = 1 eh). (1) Control group treted only with physiologil sline solution; (2) positive ontrol group using STZ s dietes indutor; (3) ozone group, reeiving 1 tretments (1.1 mg kg 1, dose of ozone in whih the phenomenon of oxidtive preonditioning is hieved without ppreile toxiity [6 9]) one per dy fter STZ-indued dietes; (4) oxygen group, vehile of O 3 (26 mg kg 1, dose equivlent to the O 2 onentrtion present in one O 3 dose) one per dy, s in group 3 ut using oxygen only; (5) ontrol ozone group, s group 3, ut without STZ. The ozone onentrtion in the O 3 /O 2 mixture ws 5 µg ml 1. Ozone ws generted y OZOMED equipment mnuftured y the Ozone Reserh Center (Cu) nd ws dministered y retl insuffltion. Ozone ws otined from medil grde oxygen, ws used immeditely upon genertion nd represented only out 3% of the gs (O 2 + O 3 ) mixture. The ozone onentrtion is mesured y using uilt-in UV spetrophotometer set t 254 nm (ury,.2 Å t 1 Å, repetility.1 Å nd lirted with internl stndrd). The ozone dose is the produt of the ozone onentrtion (expressed s mg l 1 y the gs (O 2 + O 3 ) volume (l)). By knowing the ody weight of the rt the ozone dose is lulted s mg kg 1 s in our previous ppers [6 9]. After 11 dys of dieti indution, lood gluose ws mesured, the ody weight of the nimls ws monitored nd then they were killed y diethyl ether nesthesi. Afterwrds the pnres ws promptly removed for iohemil studies. Pnres homogentes were otined using tissue homogenizer Edmund Bühler t 4 C. The homogentes were prepred using 5 mm KCl/histidine uffer ph 7.4, 1 : 1 (w/v) nd were spun down with Sigm Centrifuge 2K15, t 4 C nd 85 g for 2 min. Superntnts were tken for iohemil determintions. Biohemil determintions The iohemil prmeters were evluted in the superntnts of pnres homogentes 11 dys fter STZindued dietes nd 24 h fter the lst tretment with ozone or oxygen, respetively. The different prmeters were determined y spetrophotometri methods using n Ultrospet Plus Spetrophotometer from Phrmi LKB. Ctlse tivity ws mesured y following the deomposition of hydrogen peroxide t 24 nm t 1 s intervls for 1 min [13]. Superoxide dismutse (SOD) nd glutthione peroxidse (GSH-Px) were mesured using kits supplied y Rndox Lortories Ltd., Irelnd (Ct. No. SD125 nd No. RS55). Conentrtions of mlondildehyde (MDA) were nlyzed using the LPO-586 kit otined from Cliohem (L Joll, CA,USA). In the ssy, the prodution of stle hromophore fter 4 min of inution t 45 C ws mesured t wvelength of 586 nm. For stndrds, freshly prepred solutions of mlondildehyde is [dimethyl etl] (Sigm St Louis, MO, USA) were employed nd ssyed under identil onditions [14]. Quntifition of totl hydroperoxides ws mesured y Bioxyteh H 2 O 2-56 kit (Oxis Interntionl In., Portlnd, OR, USA) using xylenol ornge to form stle olored omplex, whih n e mesured t 56 nm. Totl protein onentrtion ws determined y the method of Brdford with ovine serum lumin s stndrd [15]. After preipittion of thiol proteins using TCA 1%, the redued glutthione (GSH) ws mesured ording to the method of Sedlk nd Lindsy [16] with Ellmn s regent (5,5 dithiois (2-nitroenzoi id) 1 2 M (Sigm St Louis, MO, USA)), the sorne ws mesured t 412 nm. Nitrite/nitrte levels were determined y the Griess retion y first onverting nitrtes to nitrites using nitrte redutse (Boehringer Mnnheim Itly SpA, Miln, Itly). Then the Griess regent (1% sulphnilmide,.1% N-(1-nphthyl)- ethylenedimine dihydrohloride in.25% phosphori

3 Phrmologil Reserh, Vol. 44, No. 5, Tle I Body weight nd plsm gluose onentrtions Groups n Body weight Plsm gluose Sttistil signifine of hnges (g) (1) (mmol l 1 ) plsm gluose Strt End (2) Non-dieti ontrol ± ± ± 1.25 ns Dieti(STZ) ± ± ± 2.12 P <.1 STZ + Ozone ± ± ± 1.45 (3) P <.1 STZ + Oxygen ± ± ± 1.34 (3) P <.1 Ozone ± ± ± 1.18 ns Dt re men ± SEM. ns: non-signifint. (1) Chnges in orporl weight etween the strt nd end of the experiment. Groups with t lest ommon letter non-signifint (P >.5), (2) 1 dys fter STZ-indued dietes, (3) fter 1 tretments with ozone or oxygen in STZ-indued dieti rts s desried in Mterils nd Methods. Tle II Levels of ldose redutse, frutolysine, dvned oxidtion protein produts nd NO 2 NO 3 Groups AR (1) FA (2) AOPP (3) NO 2 /NO (4) 3 Non-dieti ontrol (NC).58 ± ± ± ±.74 Dieti (STZ) 1.38 ± ± ± ±.98 STZ + Ozone.58 ± ± ± ±.82 STZ + Oxygen 1.17 ± ± ± ± 1.27 Ozone.56 ± ± ± ±.6 (1) AR: ldose redutse (mmol gluose min 1 mg protein 1 ), (2) FA: frutolysine (reltive frutolysine ontent mg protein ), (3) AOPP: dvned oxidtion protein produts (µmol hlormine-t equivlent mg protein 1 ), (4) NO 2 /NO 3 : nitrites/nitrtes (nmol mg protein 1 ). Dt re men ± SEM. Mens hving different supersript letters indite signifint differene (P<.5) etween groups. id) ws dded [17]. Smples were inuted t room temperture for 1 min nd sorne ws mesured t 54 nm using miroplte reder. The dvned oxidtion protein produts (AOPP) were mesured through the oxidtion of iodide nion to ditomi iodine y AOPP [18]. Reltive frutolysine ontent (Amdori s produt of glyted serum protein) ws mesured y redution of the redox inditor nitroluetetrzolium (NBT) t 53 nm [19]. Aldose redutse tivity ws determined using onventionl proedure [2]. Sttistil nlysis The OUTLIERS preliminry test for detetion of error vlues ws initilly pplied for sttistil nlysis. Afterwrd, the ANOVA method (single wy) ws used followed y the homogeneity vrine test (Brtlett- Box). In ddition, multiple omprison test ws used (Dunn test). Dt were expressed s the men ± stndrd devition of 1 nimls. The level of sttistil signifine employed ws t lest P <.5 for ll experiments. RESULTS Body weights nd lood nlysis Rts treted with streptozotoin (STZ) nd STZ + O 2 were hyperglyemi nd lost weight over the experimentl period (Tle I). Ozone tretment redued hyperglyemi y 4% in omprison with STZ-treted rts. Body weight of the rts ws inresed in similr wy s for the non-dieti ontrol. Antioxidnt prooxidnt lne The O 3 + STZ tretment inresed glutthione (GSH) onentrtions with regrd to the remining groups [Fig. 1()]. The enzymes superoxide dismutse (SOD) nd tlse (CAT) showed similr trend [Fig. 1(, )]. Neither GSH nor SOD were different in the remining groups (non-dieti, STZ-indued dietes, O 2 -treted dieti or O 3 -treted rts). Tretment with ozone used redution in glutthione peroxidse with regrd to STZ (43%) nd STZ + O 2 (36%) groups; however, onentrtions in ozone-treted dieti rts were still rised ove those seen in nondieti ontrol rts [Fig. 1(d)]. Totl peroxides were redued in the ozone-treted group with regrd to ll tretments, inluding the ontrol non-dieti [Fig. 2()], wheres mlondildehyde (MDA) onentrtions were mintined t the level of the ontrol in the nimls treted with O 3 or in the group treted with O 3 + STZ (P <.5) nd signifint inrese ws noted in the tretments with STZ nd O 2 + STZ (P <.5) with respet to ontrol group. Biomrkers of the polyol pthwy, non-enzymti glyosyltion, protein oxidtion nd nitri oxide The results otined for these prmeters re shown in Tle II. Aldose redutse tivity whih tlyzes the redution of gluose to soritol nd the reltive frutolysine ontent, preursor of Advned Glytion Endproduts (AGEs) ws signifintly ( P <.5) inresed in STZ nd O 2 -STZ dieti rts. On the other hnd, there ws no signifint differenes when ompring the dieti rts treted with ozone nd

4 394 Phrmologil Reserh, Vol. 44, No. 5, 21 () GSH (ug/g tissue) () Ctlse (U/g protein) NC STZ STZ + STZ+ O 3 () 2.5 SOD (U/mg protein) GPx (U/mg protein) (d) NC STZ STZ + STZ+ O 3 Fig. 1. Behvior of ntioxidnt systems in non-dieti nd dieti rts: NC, non-dieti ontrols; STZ, dieti group indued y streptozotoin 45 mg kg 1 i.p.; STZ + O 3 /O 2, dieti group treted with ozone (1.1 mg kg 1 ), 1 tretments y retl insuffltion; STZ + O 2, dieti group treted with oxygen, vehile of ozone (26 mg kg 1 ), 1 tretments y retl insuffltion. Dt re mens ± SEM. Mens hving different supersript letters indite signifint differene (P<.5) etween groups. () glutthione (GSH); () superoxide dismutse (SOD); () tlse (CAT); (d) glutthione peroxidse (GSH-Px). () Totl Peroxides (umol/g tissue) () MDA (nmol/mg protein) Fig. 2. Levels of totl hydroperoxides () nd lipid peroxidtion produts () in non-dieti nd dieti rts. NC, non-dieti; STZ, dieti group indued y streptozotoin 45 mg kg 1 i.p.; STZ + O 3 /O 2, dieti group treted with ozone (1.1 mg kg 1 ), 1 tretments y retl insuffltion; STZ + O 2, dieti group treted with oxygen, vehile of ozone (26 mg kg 1 ), 1 tretments y retl insuffltion. Dt re mens ± SEM. Mens hving different supersript letters indite signifint differene (P<.5) etween groups. the ontrol non-dieti. The ozone group did not signifintly ( P <.5) modify the ldose redutse tivity with regrd to norml ontrol rts. A lose reltion ws found (r =.78, P <.5) etween reltive frutolysine ontent nd AOPP onentrtions. The levels of NO 2 /NO 3, in the ozone-treted group, did not differ from the ontrol group. Both groups showed signifintly higher onentrtions with regrd to STZ nd STZ + O 2. DISCUSSION Most previous studies hve foused on immedite or onurrent ftors, whih ontriute to the phenomenon of dietes-indued endothelil dysfuntion. In the present study we hve integrted some of the most importnt metoli events ssoited with the dieti endotheliopthy proess nd its ontrol y ozone tretment.

5 Phrmologil Reserh, Vol. 44, No. 5, It is of ritil importne to mintin the ntioxidnt potentil of the pnreti ell in order to ensure oth its survivl nd insulin seretory pity during times of inresed oxidtive stress. On the other hnd, the pnres is the min trget of STZ. The ntioxidnt prooxidnt lne, ssoited with the ontrol of oxidtive stress ws fvored y ozone tretment, while the group treted with oxygen (vehile of ozone) did not differ from the STZ-indued dieti rts. Ozone redued STZ-indued hyperglyemi nd it inresed the ntioxidnt defenses (GSH, SOD nd CAT levels) of the pnres [Fig. 1(,, )]. The pity of ozone to enhne ntioxidnt endogenous systems, in front of oxidtive stress y oxidtive preonditioning or dpttive mehnisms, hs een demonstrted [6]. There is evidene tht hyperglyemi n lower oth the tivity of numer of enzymes inluding SOD [21] nd GSH synthesis, presumly y glytion [22]. It is not possile t this knowledge stte to define how ozone tretment dereses hyperglyemi. However the oservtion tht dieti ptients hve lowered ntioxidnt defenses, oth enzymti (SOD, CAT, GSH-Px) nd non-enzymti (vitmin C, E or A, free rdil svengers or totl rdil-trpping ntioxidnt pity ) is lmost s well estlished s the oservtion of inresed oxidtive dmge [21]. Therefore, these results suggest tht ozone protetive effets on ntioxidnt endogenous defenses improve gluose metolism. In line with the inrese in ntioxidnt systems there ws redution of totl peroxides nd the onentrtions of MDA were t the level of the ontrol group (Fig. 2). MDA nd peroxides hve een ssoited with dietes nd its omplitions. An pproximtely three-fold inrese in ROS prodution ompnied y similr elevtion of MDA, n index of lipid peroxidtion, ws seen in rt ort fter 1 month of dietes [23]. In ddition, role for H 2 O 2 hs een demonstrted in protein rosslinking in dietes [24]. No differenes were oserved in GSH nd SOD mong non-dieti, STZ-indued dietes nd oxygentreted dieti groups. This ehvior my e due to ompensting mehnisms similr to the one whih ws found for (mrna) SOD in STZ-treted rts [24]. When nlyzed, the tretment with ozone mintined the neessry ntioxidnt prooxidnt lne. Nevertheless, endothelium integrity nd funtion depend not only on the ROS ontrol ut lso on possile modes of tion nd some potentil intertions etween the polyol pthwy, ROS prodution, dvned glytion endproduts nd NO genertion [5, 25, 26]. The onentrtions of the meditors derived from the inresed flux of gluose through the polyol pthwy (ldose redutse nd frutolysine) were redued y ozone tretment while AOPP were not inresed in the ozone tretment group. Corresponding with these results, lose reltion etween frutolysine ontents nd AOPP onentrtions ws found (r =.78, P <.5). The regultive effets of ozone on ldose redutse tivity represent nother interesting tion of this omplementry medil pproh sine ldose redutse is key enzyme of the polyol pthwy nd its inhiitors hve een used s therpeutil drugs linked to improving NO prodution or relese [27]. This is rought out through NADPH-spring tivity tht helps to replenish ntioxidnt reserves, thus hving n indiret ntioxidnt tion in mild dieti neuropthy or in preventing peripherel nd utonomi neuropthy in unffeted dieti ptients [28]. Sustntil evidene exists tht dietes results in impired endothelil dysfuntion suggesting diminished nitri oxide prodution from dieti endothelium [29]. Ozone tretment prevented depletion of NO 2 /NO 3 (Tle II). This result indites tht NO prodution hs not een ffeted y STZ-indued dietes. Thus, ozone my protet ginst the imlne in NO ROS intertions, improve NO-medited relxtion nd derese mirovessel retivity, in this experimentl model of dietes. In summry, ozone tretment improved glyemi ontrol nd prevented oxidtive stress, the inrese of ldose redutse, frutolysine ontent nd dvned oxidtion protein produts. NO 2 /NO 3 levels were mintined without hnges with regrd to non-dieti ontrol. These events re losely relted with endothelil dmge. Therefore these results suggest tht ozone, in our experimentl onditions, my hve role in the tretment of dieti omplitions. Other works studying the effets of ozone on dieti ptients with mrongiophti omplitions re in progress. ACKNOWLEDGEMENTS These studies were supported in prt y Rndox Lortories (Antrim, UK) nd the Deprtment of Chemistry nd Medil Biohemistry, University of Miln. REFERENCES 1. Rudemn NB, Willimson JR, Brownlee M. Gluose nd dieti vsulr disese. FASEB J 1992; 6: Willimson JR, Chng K, Frngos M, Hssn KS, Ido Y, Kwmur T, Nyengord JR, Vn Den Enden M, Kilo C, Tilton RG. Hyperglyemi pseudo hypoxi nd dieti omplitions. Dietes 1993; 42: Lin SJ, Hong CY, Chng MS, Ching BN, Chien S. 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