Research Paper Mediators of Inflammation, 7, 289–294 (1998)

CA Corresponding Author Te l: (+53) 7 219264 or 7 219537 Fax : (+53) 7 210233 Email: ozono@infomed.sld.cu THERE is s om e anecdotal evidence that ox ygen -ozone therapy m ay be beneficial in som e hum an diseas es . How ever so far only a few biochem ical and pharm acodynam ic m echanism s have been elucidated. On the basis of pre lim in ary data we postulated that con trolled ozone adm inis tration would prom ote an ox idative preconditionin g preven ting the hepatocellular dam age m ediated by fr ee radicals . Six groups of rats w ere class ified as follow s: (1) negative con trol, usin g in traperitoneal sun flow er oil; (2) positive con trol us in g carbon tetrach loride (CCl4 ) as an ox idative challenge; (3) ox ygen-ozone, pretreatm ent via r ectal in sufflation (15 s ess ions) and afte r it, CCl4 ; (4) ox ygen , as group 3 but using ox ygen on ly; (5) con trol ox ygen -ozone, as group 3, but w ithout CCl4 ; group (6) con trol ox ygen, as group 5, but usin g ox ygen only. We have evaluated critical biochem ical param eter s such as leve ls of tran sam in ase , cholinesterase , super ox ide dism utase, catalase , phospholipase A, calcium dependen t ATPase, r educed glutathione , glucos e 6 phosphate dehydrogenase and lipid perox idation. In terestingly, in spite of CCl4 adm inis tration, group 3 did not differ fr om group 1, w hile groups 2 and 4 show ed s ign ifican t diffe rence s from groups 1 and 3 and displayed hepatic dam age . To our knowledge the se are the firs t ex perim ental r esults show ing th at repeated adm in is tration of ozone in atox ic doses is able to in duce an adaptation to ox idative s tr ess thus enabling th e an im als to m aintain hepatocellular in tegrity afte r CCl4 poison ing.


Introduction
Ozone (O 3 ) has be en used as a the rape utical agent for the treatme nt of diffe re nt, app arently nonre late d diseases and be ne ficial effe cts have be e n obse rved in ce re brovasc ular is chae mia, 1 chronic ulcers , 2 arte riosclerosis oblite rans, 3 re tinitis p igmentosa, 4 humoral immunity de fic ie nc y, 5 hep atic ste atosis , 6 and he art ischae mia. 7 In spite of these enc ouraging re sults obtain ed w ith ozonetherap y, its clinic al use re mains controversial due to the scarc e know ledge of the biochemic al and p harmac odynamic me chanisms w hich underlie its therapeutic ac tion and the efficacy in such hete roge ne ous pathologie s. Last but not least, O 3 has be en associated w ith e nvironmental pollutions and to differe nt pathologie s. 8 -10 These fac tors have contribute d to sce ptic is m and pre judice of offic ial me dic al authorities, delaying the acc eptanc e of ozone the rap y. In order to provide sc ie ntific support to the afore mentioned clinical data, some ex perimental strate gie s have be en developed in orde r to inc re ase our know ledge conce rning its probable me chanisms of action. On the basis of the ox idant prope rties of O 3 and on the possibility that spec ific ce ll se nsors activated by lipid ox idation products (LOP) may upre gulate the antiox idant syste m, w e postulate that O 3 may induce an adaptation to ox idativ e stre ss. More ove r, not only O 3 could induce tolerance to itse lf but it could pre pare the host to fac e physiopathologic al conditions mediated by re active ox yge n specie s (ROS). With the aim to demonstrate the c apability of O 3 to promote an ox idativ e pre c onditioning proce ss, w e induced hepatoce llular damage w ith a single dose of c arbon te trachloride (CCl 4 ), w hich is a re c ognized organic age nt able to produc e a c ellular injury through gene ration of free radic als. 1 1

Animals and sample preparation
Adult female Sprague -Daw le y rats (220-250 g) w ere used for the se studie s. Rats w ere maintain ed in an air filtere d and te mpe rature c onditione d (20 -22°C) room w ith a re lative humidity of 50-52%. Rats w ere fed w ith standard commerc ial p ellets and w ater ad libitum. O 3 w as gene rate d by an OZOMED equipme nt manufac tured by the Ozone Rese arch Ce nte r (Cuba) and w as administe re d by re c tal insufflation. O 3 obtain ed from me dic al grade ox ygen w as use d imme diately and it re pre sente d only about 3% of the gas (O 2 + O 3 ) mix ture. The O 3 conc entration is me asured by using an UV spec trophotomete r at 254 nm and is very pre c ise. The ozone dose is the product of the O 3 c oncentration (ex pre ssed as mg/l) by the gas (O 2 + O 3 ) volume (l). By know ing the body w eight of the rat the O 3 dose is c alc ulated as 1 mg/kg. Rats re ce ived 15 ozone treatments, one per day, 4.4-5.0 ml w ith O 3 c onc entration of 50 m g/ml be fore challe nge w ith CCl 4 . After the last ozone treatme nt, rats re ceived CCl 4 (1 ml/kg) by intrape ritone al administration of a solution of 10% CCl 4 in sunflow er oil. The animals w ere euthanized by e the r anaesthesia, 24 h after re c eiving CCl 4 . Imme diate ly afte r, blood samples w ere obtained from the abdominal aorta and mix e d w ith 3.8% sodium c itrate, used as an anticoagulant, for biochemical dete rminations. Afterw ards, some re pre se ntativ e sample s of differe nt liver portions w e re take n for histopathologic al studies and tissue homoge nates. Liver homoge nates w ere obtain ed using a tissue homoge nator Edmund Bulher LBMA at 4°C. The homogenates w e re pre pared by using a 50 mM KCl/histidine buffe r pH 7.4, 1:10 (w /v) and w ere sp un dow n w ith a Sigma Ce ntrifuge 2K15, at 4°C and 8500 3 g for 20 min. The supe rnatants w ere take n for biochemical dete rminations.

Treatment schedule
The protocol c onsiste d of six ex perimental groups (n = 60). (1) ne gative control group treate d only w ith sunflow er oil by intra peritone al route; (2) positive control group using 1 ml/kg of 10% CCl 4 solution;

Biochemical determinations
The biochemical parame te rs w ere dete rmined by spe ctrophotome tric methods using an Ultraspe ct Plus Spe ctrophotome te r from Pharmac ia LKB. Aspartic alanine transaminase (ASAT) and cholineste rase (CHEase) levels w e re me as ure d in plasma using standard c ommercial kits produce d by Boehringer Mannheim. In liver, homogenate s w e re assaye d for total supe rox ide dismutase s (Cu/Zn and Mn SODs) activity dete rmining the capac ity of the enzyme in inhibiting the autox idation of p yrogallol by 50%. 1 2 The catalase c onc entration w as measured through the catalytic activity w hich promotes the re duction of hydrogen perox ide (H 2 O 2 ) to ox yge n and w ater. 1 3 The phospholipase A activity w as dete rmine d ac cording to a standard proc edure . 14 Lipid pe rox idation w as asse sse d by re ading thiobarbituric acid-re active substanc es (TBARM). 1 5 The dete rmination of the ac tivity of calcium-depende nt ATPase (Ca-ATPase) 1 6,17 and gluc ose-6-phosphate dehydroge nase (G6PD) 1 8 w ere carried out as desc ribed. After pre c ipitation of thiol prote ins using 10% TCA the re duce d gluthatione levels (GSH) w e re dete rmined in supernatants of 10% w /v homoge nates. 19 The prote ins w ere me asured by a standard Coomassie Blue method. 2 0

Histological study
Samples of rat live r w ere taken and fix ed in ne utral 10% formalin, proce sse d and e mbedde d in paraffin. The histologic al sec tions w ere stained w ith hae matox ylin and eosin. In addition, ne utral lipids w ere demonstrate d w ith oil re d staining in froze n sec tions. From the sec tions embe dded in paraffin and after a pre vious study of the microsc opic alte rations, the ce lls w ith ballonic dege ne ration w ere c ounted at the zone III of the Rapp aport ac ini. Te n fields w ere take n, at random, pe r animal w ith a magnific ation of 250 3 and the count made in a blind w ay by tw o pathologists. From the froze n sections stained w ith oil re d, the damage are a by lipidosis w as c alc ulated in other 10 fields per animal, using a morphome tric softw are syste m. 2 1

Statistical analysis
The statistic al analysis w as started by using the OUTLIERS pre liminary te sts for dete ction of error values. Afte rw ard, the Anova me thod (Single Way) w as used follow ed by homoge ne ity varianc e te st (Bartle tt-Box ). In addition, a multiple comparison te st w as used (Duncan te st); values are ex pre ssed by the me an ± standard e rror of mean (n = 10 per group ). Diffe re nt letters indic ate a statistical signific anc e of at leas t P < 0.05.  Table 1 show s the qualitativ e re sults of the ge ne ral he patic damage (hep atoc ellular ne c rosis, ballonic degene ration, lipidosis and me senchymal re action) and the total amount of ce lls that have undergone ballonic de ge ne ration (BD). In spite of a minimal degre e of ge ne ral hepatic damage and a marke d re duction in the amount of c ells w ith BD, value s from

Discussion
In order to te st our hypothe sis that prolonged administration of judicious dose s of O 3 may promote the phenomenon of ox idativ e pre c onditioning, w e had to demonstrate that hepatocytes can be come re sis tant to the damage induce d by free radic als after CCl 4 poisoning. The re sults obtaine d in the pre se nt w ork fully support this postulation. It is w orth re me mbering that Murry e t a l. 2 2 in 1986 p ropose d the conce pt of 'is chae mic pre c onditioning' obtaine d by several c ycles of brie f c oronary oc clusion e ve ntually able to minimize myocardial damage afte r a se vere he art is chae mia. Our ex pe rimental re sults have show n that re peate d administration of a gas mix ture c omposed of O 2 -O 3 via the colorec tal route c an induc e a sort of crosstolerance to free radic als re lease d afte r one single dose of CCl 4 . On the contrary, ex perime ntal groups (2 and 4) treated w ith either CCl 4 or O 2 + CCl 4 , re spe ctively, displayed a signific ant ce llular damage. The se re sults w ere w ell c orrelated w ith the histopathologic al findings in re gard to the degre e of ballonic de gene ration and lipidosis. It must be pointe d out that administration of O 3 in rats c ould be carrie d out ne ithe r by inhalation, due to its tox icity, 2 3,2 4 nor by ozonated autohaemothe rap y for te chnical re asons. Nonetheless the colore c tal route, although somew hat empiric al, is e as y, prac tic al, atox ic and has the rationale that ozonated p roduc ts re ach the live r via portal c irc ulation. 2 5 Taking into ac count an interspe cie s factor based on the murine me tabolic rate , a correc t dose of ozone is 1 mg/kg in the rat and 0.25 mg/kg in humans, re spec tively. SODs and c atalase are re c ognized scave ngers of re active ox ygen specie s 2 6 and the y have be e n te ste d as therapeutic age nts against c ellular damage induced by ischaemia 27 ,28 in spite of the ir low bioavailability and antib ody formation. The significant stimulation of e ndogenous SODs in O 3 + CCl 4 group in c omp arison w ith CCl 4 and O 2 + CCl 4 groups, suggests c ellular prote c tion most likely through the re duc tion in the availability of superox ide anion ( O -2 ). This re sult w as somew hat ex pec te d on the basis of se veral findings 2 9 -3 1 re porting incre ase d activities of SOD, catalase and perox idases after chronic O 3 ex posure. It is note w orthy that plants c an also ex pre ss a p rote ctive re spons e to O 3 32 -3 4 suggesting that living organisms chronic ally ex pose d to O 3 have the option of e ithe r programming the ir death or to re act and survive by up re gulating the antiox idant defenc e syste m capable to re adjust the re dox balanc e. More ove r in patie nts, w e and others have found 7 ,2 5,3 5 that c alc ulate d, transie nt ox idativ e stresses such as those obtained during a cycle of ozonate d autohaemotherapy can also induc e a state of tolerance characte rized by a simultane ous ove rex pre ssion of SOD, G6PD and possibly a re duction of TBARM levels in plasma. The re ctal insufflation of O 3 (group 3) is appare ntly able to enhanc e the antiox idant syste m in a coordinate fashion be cause the incre ase d ac tivity of catalase on its ow n (groups 2 and 4) is unable to quench CCl 4 tox icity.
It is know n that an incre ase of intra c ellular Ca 2 + conc entration plays an important role in the damage and ce llular death, re pre se nting a critic al and early e ve nt in the development of tox icity of hepatocytes submitted to ox idativ e stress. 36 The suggestion that free radicals may affe ct the activity of the Ca-ATPase, thus c ontributing to the hepatoce llular injury, 37 is w e ll supported by our data be cause the Ca-ATPase ac tivity w as se ve re ly inhibited in groups 2 and 4 w hile the O 3 tre ate d group equalize d the control. Inde ed it has be en pre viously show n that Ca 2 + -ATPase e nzyme s have critic al thiol groups rapidly inactivate d by ROS. 3 8,3 9 Calc ium-dep endent phospholipas e A activity w as decre as ed in the O 3 + CCl 4 treatme nt group indicating that ozone ex e rted indire ctly a prote c tion against the ce llular disruption, mediated by the activation of an enzyme w hich ge ne rates lysophospholipids and other me tabolite s re sponsible for c ellular lysis . The incre ase d phosp holipase A activity in the fourth (O 2 + CCl 4 ) group sugge sts the partic ip ation of this enzyme in the he patoc ellular damage note d in the histopathologic  studies. CCl 4 poisoning on its ow n caused only a mode st inc re as e of phospholipase A activity w ith re spect to the control, probably due to the moderate charac te r of the ox idativ e challenge after one single dose of CCl 4 . The gre ater ce llular damage observe d after ox ygen administration plus the he patotox ic tre atment indicates the de lete rious effe ct of hyperox ygenation. 1 0,1 1 Sustaine d le ve ls of GSH and G6PD can be take n as re liable marke rs of antiox idant defens es in the face of an ox idativ e challe nge. Finally the de cre ase in hepatic lipid perox idation obtained in the O 3 + CCl 4 group w as in good agree ment w ith the re duction of the lipidosis obse rved in the histopathologic studies, w hile under CCl 4 and O 2 + CCl 4 tre atments lip id pe rox idation levels w ere raise d significantly. Why the O 3 treatment, w ithout the final challe nge w ith CCl 4 (Group 5), did not show a significant inc re ase of antiox idant enzymes may be ex plained by e ithe r the fairly short treatment or, more like ly, by the fac t that O 3 acts be st w hen the re is an ongoing ox idativ e insult. In c onclusion, the pre se nt study contributes to clarify an important pharmac odynamic effect after prolonge d ozonetherapy in rats. The phe nomenon can be desc ribed as an induc tion of tole ranc e to O 3 and ROS gene rate d by tox ic agents and has be en denominated as either 'ox idativ e pre conditioning', or 'ox idative stre ss adaptation'. 2 5 Ozone therap y has be e n able to pre serve liver integrity by inducing either enzymes or activating me tabolic p athw ays that maintain an equilibrate d re dox balance. High SODs and GSH levels, low p erox idation and a normal Ca 2 + home ostasis are cle ar ex ample s of the e fficacy of ozone therapy. We be lie ve that the possibility show n by appropriate ozone the rap y to upre gulate the antiox idant syste m re pre se nts a fundamental property of this compleme ntary me dic al approach and that O 3 come s to typify a unique drug. In fac t ex ogenous administration of antiox idant substances such as ascorbic acid, GSH, n -ace tyl c yste ine, SOD and the like are use ful but hardly able to dramatic ally re vers e a chronic ox idativ e stre ss. In this se nse the improveme nt of the antiox idant de fe nce is bound to be cruc ial in c ancer, 4 0 chronic viral infections 41 and ne urodegene rative disease s, 42 w he re the c ontrol of endogenous ox idation has gone aw ry w ith p rogre ssive ce ll damage . The re fore w e should most actively pursue this lead for improving the therapy of these dise as es.