Similar protective effect of ischemic and ozone oxidative preconditionings in liver ischemia/reperfusion injury

Author Olga Sonia Leon Fernandez1
Author HH Ajamieh1
Author Eduardo Candelario-Jalil1
Author S Menendez3
Author Lamberto Re5
Publication 1Center for Research and Biological Evaluation (ClEB-lFAL), Univ. Havana, Havana City 10 400, Cuba
Publication 3Faculty of Pharmacy, Univ. of Barcelona, Barcelona, Spain
Publication 4Ozone Research Center PO Box 6412, Havana City, Cuba
Publication 5Department of Chemistry and Medical Biochemistry, University of Milan, Via Saldini 50-20133, Milan, ltaly
Publication 6Laboratory of Pharmacological Biotechnology, University of Ancona, 60131, Ancona, ltaly
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Similar protective effect of ischemic and ozone oxidative preconditionings in liver ischemia/reperfusion injury




Background: Many studies indicate that, after reoxygenation of the liver, oxygen free-radical formation may initiate the cascade of hepatocellular injury, necrosis/apoptosis, and subsequent infiltration of inflammatory cells. Although reactive oxygen species (ROS) can arise from a number of sources, xanthine oxidase (XO) is frequently implicated as a significant source of these toxic oxygen species. The ischemic preconditioning (lscheP) is an inducible and potent endogenous mechanism by which repeated episodes of brief ischemia/reperfusion (l/R) confer a state of protection against subsequent sustained l/R. On the other hand, it has been demonstrated that ozone is able to promote an oxidative preconditioning through the increase and preservation of antioxidant endogenous systems.


On the basis of above results we postulated that Ozone Oxidative Preconditioning (OzoneOP) has similar biochemical parameters to lscheP mechanism.


Four groups of rats were classified as follows: (1) sham-operated animals subjected to anesthesia and laparatomy, plus surgical manipulation; (2) l/R animals were subjected to 90 min of right-lobe hepatic ischemia, followed by 90 min of reperfusion; (3) lscheP, previous to the l/R period (as in group 2) animals were subjected to 10 min of ischemia and 10 min of reperfusion; (4) OzoneOP, previous to the l/R period (as in group 2) animals were treated with ozone by rectal insufflation 1 mg/kg. The rats received 15 ozone treatments, one per day. The following parameters were measured: serum transaminases (AST, ALT) and 5′-nucleotidase (5′-NT) as indicator of hepatocellular injury; total sulfhydryl groups, calcium levels and calpain activity as mediator which take part in xanthine deshydrogenase (XDH) conversion to XO reversible and irreversible forms respectively; XO activities and malondialdehyde + 4-hydroxyalkenals (MDA+4-HDA) as indicators of increased oxidative stress.


AST, ALT were attenuated in the lscheP with regard to the l/R group while OzoneOP maintained both enzyme activities without statiscal differences in comparison with sham operated. 5′-NT activity was not different in both preconditionings with regard to sham operated. Protective effects of both preconditioning settings on the preservation of total sulfhydryl groups, calcium concentrations and calpain activity were observed. ln addition, preconditionings attenuated the increase of total XO associated to l/R injury. Generation of MDA+4-HAD was prevented by lscheP and OzoneOP without statiscal differences between the two protective procedures.


These results provide evidence that both preconditioning settings share similar biochemical mechanisms of protection in the parameters which were measured.


The liver is damaged by sustained ischaemia in liver transplantation or in liver surgical procedures, and the reperfusion after ischaemia results in further functional impairment [1]. After the first report describing protection against myocardial necrosis [2], preconditioning has been demonstrated in intestine [3], brain [4] and liver [5] indicating that it is not a mechanism restricted to the myocardium. ln spite of the fact that the ischaemic preconditioning  has been extensively studied on heart, only few studies have been performed on the model of ischaemic-reperfusion injury in the liver.

Many studies indicate that, after reoxygenation of the liver, oxygen free-radical formation may initiate the cascade of hepatocellular injury, necrosis/apoptosis, and subsequent infiltration of inflammatory cells [6,7]. Although reactive oxygen species (ROS) can arise from a number of sources, xanthine oxidase (XO) is frequently implicated as a significant source of these toxic oxygen species [8,9]. Superoxide radicals are generated by XO. Two molecules of superoxide anion react simultaneously to form hydrogen peroxide , and then, by the addition of one electron, the highly reactive hydroxyl radical is formed [10].

The ischaemic preconditioning  is  an  inducible and potent endogenous mechanism  by  which repeated episodes of brief ischaemia and reperfusion (l/R) confer a state of protection against subsequent sustained l/R injury [2]. lt has been suggested that the hepatic protective effects of the ischaemic preconditioning against the post-ischaemic injury could be partly attributed to a decrease activity of the ping-pong mechanism between the generation of reactive oxygen species and the neutrophil infiltration. Furthermore, the ischaemic preconditioning preventing the increase of neutral fatty acids seems to reduce the substrate of lipid peroxidation [2,11]. Although the mechanism of preconditioning is not yet known, some hypotheses have recently been tested. lt has been suggested that the liver ischaemic preconditioning is mediated by the inhibitory action of nitric oxide on endothelin [12] and through the balance of adenosine and xanthine levels [13].

Ozone has been used as a therapeutical agent for the treatment of different diseases  and beneficial effects have been observed [14-16]. lt has been demonstrated that low levels of ozone exposure have distinct effects within cells, they may also protect the cells against subsequent ozone exposure. This protection may contribute to the adaptation after multiple ozone exposures [17]. Moreover, ozone not only could induce tolerance to itself, it could also prepare the host to face physiopathological events mediated by ROS. lt has recently been discovered that ozone is able to induce an adaptation to oxidative stress or promote an oxidative preconditioning through the increase and preservation of antioxidant endogenous systems in animal models of hepatotoxicity, induced by carbon tetrachloride and hepatic and renal ischaemia-reperfusion [18- 21].

Taking into account the protective effects conferred by the ischaemic preconditioning and the ozone oxidative preconditioning against liver injury by l/R, the aim of this work is to establish a comparison between both of the preconditioning settings. Biochemical and histological parameters were taken into consideration in order to find out whether there were differences and/or similarities between these protecting procedures.


Materials and Methods


Adult male Wistar rats (250-300 g) were used for these studies. Rats were maintained in an air filtered and temperature conditioned (20-22 oC) room with a relative humidity of 50-52%. Rats were fed with standard commercial pellets and water ad libitum. All procedures were performed as approved by the lnstitutional Animal Care Committees (ARCA No.015) and in accordance with the European Union Guidelines for animal experimentation.


Ozone (O3) was generated by an OZOMED equipment manufactured by the Ozone Research Centre (Cuba) and was administered by rectal insufflation. Ozone was obtained from medical grade oxygen, was used immediately as generated and it represented only about 3% of the gas (O2 + O3) mixture. The ozone concentration was measured by using a build-in UV spectrophotometer set at 254 nm (accuracy, 0.002 A at 1A, repeatability 0.001 A and calibrated with internal standard). The ozone dose was the product of the ozone concentration (expressed as mg l-1 by the gas (O2 + O3) volume (l). By knowing the body weight of the rat, the ozone dose was calculated as mg kg-1 as in our previous papers [18-22]. Unless otherwise stated, all chemicals were obtained from Sigma Chemical Company (St. Louis, M.O., USA).

Treatment schedule

The  protocol was consisted   of   five   experimental  groups (n = 32). Group 1. Sham-operated (n

= 8): animals subjected to anaesthesia and laparatomy, plus surgical manipulation (including the isolation of the right hepatic artery and vein vs. the left hepatic artery and vein without the induction of hepatic ischaemia). Group 2. l/R (n = 8): animals were subjected to 90 min of right- lobe hepatic ischaemia, followed by 90 min of reperfusion. Group 3. lschaemic Preconditioning (lschP) (n = 8): previous to the l/R period (as in group 2), animals were subjected to 10 min of ischaemia and 10 min of reperfusion as in the previous experiments [13]. Group 4. Ozone Oxidative Preconditioning (OzoneOP) (n = 8): previous to the l/R period (as in group 2), animals were treated with ozone by rectal insufflation 1 mg kg-1. The rats received 15 ozone treatments, one per day, of 5-5.5 ml at an ozone concentration of 50 µg ml-1. Control experiments were performed with the vehicle (O2) used for the ozone administration. The sham-operated animals (as in group 1) were subjected to the previous ozone treatment.

Surgical procedure

The study was performed as in our previous papers [20,21]. ln brief, all animals (including the sham-operated) were anaesthetised with urethane (10 mg kg-1, i.p.) and placed in a supine position on a heating pad in order to maintain body temperature between 36 oC and 37 oC. To induce hepatic ischaemia, laparatomy was performed and the blood supply to the right lobe of the liver was interrupted by placement of a bulldog clamp at the level of the hepatic artery and portal vein. Reflow was initiated by removing the clamp [20].

Sample preparation

Blood samples (4 ml) were obtained from the abdominal aorta for biochemical determinations of hepatic injury. Afterwards, some representative samples of different liver portions from right ischaemic lobe were taken for histopathological studies and tissue homogenates. Liver homogenates were obtained using a tissue homogenator Edmund Bulher LBMA at 4 oC. The homogenates for calcium, calpain activity, total sulfhydryl groups and 4-Hidroxyalkenals determinations were prepared by using a 50 mM KCl/histidine buffer pH 7.4, 1:10 (w/v) and were centrifuged with a Sigma Centrifuge 2K15, at 4 oC and 8500 x g for 20 min. The supernatants were taken for biochemical determinations. Preparation of enzyme fraction for evaluation of total (XDH + XO) and XO activities was  performed as in the previous report [23].  Livers were removed quickly in freeze-clamped way, washed and homogenised in 50 mM phosphate buffer, pH 7.4 containing 1 mM EDTA (1:5 w/v); the conversion of XDH to XO during handling was minimised by adding 10 mM 2-mercaptoethanol, trypsin inhibitor (5 mg ml-1; type ll-S Sigma Chemical Co., Poole, U.K.) and leupeptin (0.5 mg l-1) to the buffer before use. lnstead of dithiothreitol (DTT), 2-mercaptoethanol was added to the buffer as a thiol group protector as it prevents XDH to XO transformation without promoting the conversion of XO to XDH. The homogenates was centrifuged at 1500 g for 10 min and then at 105 000 g for 60 min at 4 oC. The supernatant was dialysed for at least 4 hours against the same homogenization buffer at 4 oC.

Biochemical determinations

Marker of Hepatic injury: Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and 5′-Nucleotidase (5′-NT) were measured using commercial kit from Boehringer Mannheim (Munchen, Germany) and Sigma (St. Louis, MO, USA) respectively.

Xanthine Deshydrogenase (XDH)  and Xanthine Oxidase  (XO) activities: Measurements were carried out within the first hour after the isolation of the supernatant. For the evaluation of total enzyme activity defined as XDH + XO, aliquots (0.2 ml) of dialyzed enzyme fraction were preincubated for 30 min at 37 oC in the presence of 10 mM DTT, diluted aliquots (50 µg protein) were then incubated  after  the  addition  of 60 µM xanthine and 0.67 mM NAD+ for 10 min at  25 oC  (total volume 0.1 ml).  DTT   preincubation   was   carried   out  to transform the XO reversible (XOr) into XDH. XO irreversible (XOi)  and total XO (XOr + XOi) activities  were determined in the absence of NAD+; DTT activation was avoided in evaluating the total XO activity.

Values of XOr activity were obtained by subtracting the XOi value from that of total XO. lncubation was stopped by the addition of ethanol (1 ml), the samples centrifuged at 1000 g for 5 min and the supernatants dried under nitrogen flow; the residues were resuspended in 0.1 mM NH4H2PO4, pH 7 (0.3 ml). Activities were determined by using HPLC method on the basis of uric acid formation at 292 nm. Each activity was expressed as µmol min-1 mg protein-1 [23].

Calcium levels, total sulfhydryl groups, calpain activity and lipid peroxidation in supernatants of liver homogenates: These parameters were determined by spectrophotometric methods using an Ultrospec Plus Spectrophotometer from Pharmacia LKB. Calcium levels were measured using standard commercial kit produced by Sigma (St. Louis, MO, USA). Determination of the total sulfhydryl groups was performed according to the method of Sedlak and Lindsay [24] with Ellman’s reagent. Calpain activity was measured as the release of trichloroacetic acid-soluble peptides from azocasein (Sigma, St. Louis, MO, USA) in an assay mixture (1.5 ml) containing 20 mM Tris-HCl buffer pH 7.4, 4 mg ml-1 azocasein, 20 mM β-mercaptoethanol, 5 mM Ca2+, 1% Triton X-100 and 0.3 ml samples at 25 oC as previously reported [25]. Lipid peroxidation was assessed by measuring the concentration of malonaldehyde (MDA) and 4-Hydroxyalkenals using the Bioxytech LPO-586 kit. The assay was conducted according to the manufacturer’s instruction.

Protein measurement

Total protein concentration in the supernatant of liver homogenates was determined using a commercial kit from Bio-Rad (Munich, Germany)


Statistical analysis

The statistical analysis was started by using the OUTLlERS preliminary tests for detection of error values. The homogeneity variance test (Bartlett-Box) was used afterwards, followed by Anova method (One Way). ln addition, a multiple comparison test was used (Duncan test); values are expressed by the mean ± standard error of mean (n = 8 per group). Different letters indicate a statistical significance of at least P < 0.05.


Results and discussion

Effects of ischaemic and ozone oxidative preconditionings on hepatic injury

As shown in Figure 1 (A,B) the degree of hepatic damage evaluated by the serum levels of AST, ALT and 5′-Nucleotidase (5′-NT) induced after 90 minutes of ischaemia and 90 minutes of reperfusion increased in the group subjected to l/R. lschP attenuated transaminase activities while OzoneOP was able to maintain AST and ALT at the level of the sham-operated. The ischaemic and ozone oxidative preconditionings prevented 5′-NT increase observed in l/R group.

Role of lschP and OzoneOP on XDH and XO activities

The effects of both the preconditioning settings on calcium concentrations, calpain activity and total sulfhydryl groups were evaluated in liver homogenates (Table 1).


Fig.1. Enzymatic serum markers of liver damage.  AST, ALT (A) and 5′- Nucleotidase (B) activities (U l-1) in the following experimental groups. Sham-operated: animals subjected to anesthesia and laparotomy, plus surgical manipulation; llR: 90 minutes of ischaemia followed by 90 minutes of reperfusion; lschP+llR: lschaemia preconditioning + llR; OzoneOP+llR: Ozone oxidative preconditioning + llR. Different letters indicate a statistical significance of at least p< 0.05. Results are expressed as the means ± SEM from eight rats.

Table 1. Calcium, total sulfhydryl groups concentrations, calpain, XDH and total XO in liver lschemia reperfusion


Note: Each value is the mean ± SEM from eight rats. Different superscript letters  indicate a stastical significance of at least p<0.05. (1) One unit activity was defined as an increase in A440 of 1.0 per ml sample per hour. (2) The values of U mg /protein correspond to µmol/min /mg protein.

l/R increased total XO activity. ln correspondence, a significant increase (p<0.05) was found in calcium levels, calpain activity and decreased total sulfhydryl groups. ln the  sham-operated animals, XO represented 10% of the total eanzymatic activity (XDH + XO). After l/R, this proportion was increased to 53%. Both the ischaemic and ozone oxidative precondiotinings attenuated this conversion since XO represented 45.5% and 46.6% of total enzymatic activity respectively compared to XDH + XO. Both of the preconditionings caused a reduction in total XO; however enzyme concentrations were still raised above that seen on the sham-operated rats. Total XO was only ameliorated by the ischaemic and ozone oxidative preconditionings but calcium, calpain activity and total sufhydryl groups were maintained to the concentrations of the sham-operated. ln order to know the contribution of reversible and irreversible XO activities to total XO, the XO forms were evaluated. The results are shown in Table 1.

XOi was increased compared to the sham-operated group. Reversible XO was unaffected by l/R when rats were previously subjected to the ischaemic and ozone oxidative preconditionings.

Effects of the ischaemic and ozone oxidative preconditionings on MDA and 4-hydroxyalkenals generation.

MDA plus 4-hydroxyalkenals is an index of lipid injury. The results of these parameters are shown in Figure 2. There was a significant increase (p<0.05) in lipid peroxidation in l/R group. The ischaemic and ozone oxidative preconditionings avoided lipid damage and they maintained the concentrations of MDA + 4-hydroxyalkenals at the level of the sham-operated group.

The results of biochemical and histopathological analysis reached in sham-operated group were not different from the control group (oxygen).


Fig.2. Malondialdehyde + 4-hydroxyalkenals concentrations. Sham-operated: animals subjected to anesthesia and laparotomy,  plus surgical manipulation; llR: 90 minutes of ischaemia followed by 90 minutes of reperfusion; lschP+llR: lschemic preconditioning + llR; OzoneOP+llR: Ozone oxidative preconditioning + llR. Each value is the means ± SEM from eight rats. Different letters indicate a statistical significance of at least p< 0.05.

The mechanism underlying preconditioning remains unknown and is currently under intense investigation. lt has been suggested that protection depends on the release of substances by the organ, helping to protect it against injury. Potential mediators include adenosine [26], nitric oxide [12,27] and others.

Ozone protected against l/R injury attenuating the increase of adenosine deaminase activity in liver. This process favoured adenosine accumulation and decreased hipoxhantihe and xanthine formation, thus decreasing the ROS generation after reperfusion [21].

The biochemical markers of liver l/R injury indicate the protective effects of the ischaemic and ozone oxidative preconditionings on preventing hepatic l/R damage (Fig. 1). lt has recently been reported that Kupffer cells are activated during the early phase after reperfusion in the ischaemic areas with the generation of Reactive Oxygen Species (ROS) and proinflammatory cytokines production [28,29].

Ozone treatment might avoid the Kupffer cells activation through its preconditioning mechanism which, during ischaemia, decreases or prevents the raise of precursors of ROS such as calcium levels, xanthine oxidase activity and xanthine generation [21]

Many studies have suggested that ROS, generated at the time of reperfusion, can cause a loss of organ function [30]. ROS production in the revascularized liver involves several mechanisms: (1) ischaemic conversion of XDH to XO, in addition to hypoxanthine generation from adenosine triphosphate (ATP), resulting in ROS production within the hepatocyte [31]; (2) activation of Kupffer cells, resulting in massive ROS production within the sinosoidal lumen which enhances endothelial cell injury and leads to polymorphonuclear cell accumulation and subsequent capillary plugging [32].

Liver, intestine  and endothelial cells  contain the  highest specific activity  of XDH + XO [33]. The liver release XDH + XO into the vasculature after l/R. Circulating XDH + XO could result in direct damage to the vascular endothelium, activate oxidant-producing inflammatory cells (i.e., neutrophils), and then, via the direct production of ROS in the plasma or by activated neutrophils, extend oxidant-induced injury to tissues remote from the site of origin [34].

XDH is a homodimer with a subunit molecular mass of approximately 150 kDa [35]. Reversible conversion into XO may occur through sulfhydryl groups oxidation. lrreversible conversion takes place by calcium-dependent proteolytic processing of XDH during tissue ischaemia through cleavage of an approximately 15-20 kDa fragment and is believed to occur subsequently to the reversible conversion [35,36].

OzoneOP maintained reversible form of XO at the level of the sham-operated. lt was in correspondence with preservation of total sulfhydryl groups concentrations. lt is in line with our previous findings that ozone treatment promotes an increase in antioxidant endogenous systems reducing oxidative stress mediated by liver l/R [20].

ln both the preconditioning settings, XOi accounts for almost entire total XO activity present in liver samples (Table 1).

The inability to maintain cellular calcium homeostasis appears to be an important event during ischaemia. Loss of calcium-pumping activity during ischaemia allows intracellular calcium concentrations to rise with associated increases in calcium dependent methabolic activities and eventual cell death. The activation of calcium-dependent enzymes, such as phospholipases, leads to membrane damage due to phospholipid degradation [3,4]. Cellular calcium overload may also trigger the release of ROS and potentiate oxygen radical membrane injury through the increased conversion of XDH to XO.

There were not differences in calcium concentrations between the two preconditionings with regard to the sham-operated group (table 1). The protective effect of Ozone Oxidative Preconditioning on cellular calcium homeostasis may be due to Ozone’s capacity to protect Ca2+-ATPase activities against inactivation of oxidative challenge [18]. Ca2+-ATPase plays an important role in the transport of intracellular calcium in the liver.

Many lines of evidence suggest that calpain, a Ca2+-dependent neutral protease, is implicated in injury during ischaemia or reperfusion. lt has been demonstrated that reperfusion with EGTA (Ethylenglycol bis (β-aminoethyl-ether)-N, N, N’, N’-tetraacetic acid) suppressed the ischaemia- induced increase in calpain activity which is the evidence that Ca2+ influx is involved in calpain activation [25]. Nevertheless, the role of calpain activity in conversion of XDH into XO is not yet a clear event. lt has been shown that the cytoplasmic XDH of human liver is irreversibly cleaved to XO by mitochondrial protease and apparently not through a calcium-dependent protease (calpain). lntracellular calcium influx and subsequent mitochondrial Ca2+ overload during and after ischaemia have shown to promote the mitochondrial permeability transition. The role of excess intracellular calcium on XDH-XO conversion is thus likely to be indirect, rather than directly coupled to the activation mechanism of the protease [36].


ln line with these reports, the effects of both preconditionings on XDH conversion suggest that calpain is not the major protease which catalyse irreversible conversion XDH into XO.


Aldehyde production, as a result of ozone inhalation, could be an important mediator of ozone toxicity [37]. Although a large number of aldehydes can be formed during polyunsaturated fatty acid peroxidation, three aldehydes have been extensively studied as physiologically relevant lipid peroxidation products [4-hydroxynonenal, 4-hydroxyhexenal and MDA [38].


The contribution of 4-hydroxynonenal to ozone injury was studied on a human exposed to 0.4 ppm ozone for 1 hour with exercise. lt was reported that 4-hydroxynonenal protein adducts after ozone exposure were consistent with a potential role for 4-hydroxynonenal in the cellular toxic effects of acute ozone exposure [39].


OzoneOP similarly to lschP maintained 4-hydroxyalkenals + MDA concentrations at the sham- operated group levels (Fig. 2). Similar results were reported when the MDA levels were studied on rats previously treated with ozone and after that, subjected to liver l/R [20].


Low doses of ozone increased antioxidant endogenous systems as glutathione, superoxide dismutase and catalase [16,18,20]. Therefore, antioxidant-prooxidant balance is favoured to preservation of cell redox state and toxic aldehydes are not formed.


ln summary, lschaemic and OzoneOP protected against  liver l/R injury. This study provides evidence that both of the preconditioning settings share similar biochemical mechanisms of protection. lt is noteworthy that histological results showed a more effective protection of OzoneOP than lschP in our experimental conditions. Therefore, ozonetherapy may be considered as a potential medical approach against liver l/R damage.


These studies were supported in part by Cyted Program lV.11: “Development of Antioxidants from Natural Sources with Comercial lnterest”,  Randox Laboratories (Antrim, UK) and the Department of Chemistry and Medical Biochemistry, University of Milan.


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