Protection by ozone preconditioning is mediated by the antioxidant system in cisplatin-induced nephrotoxicity in rats.

BACKGROUND: Acute renal failure is a dose-limiting factor of cisplatin chemotherapy. Here, we show the protective effect of ozone oxidative preconditioning against cisplatin-induced renal dysfunction in rats. Ozone oxidative preconditioning is a prophylactic approach, which favors the antioxidant-pro-oxidant balance for preservation of the cell redox state by increasing antioxidant endogenous systems in various in vivo and in vitro experimental models. AIMS: To analyze the protective role of ozone oxidative preconditioning against cisplatin-induced nephrotoxicity. METHODS: Male Sprague-Dawley rats were pretreated with 15 intrarectal applications of ozone/oxygen mixture at 0.36, 0.72, 1.1, 1.8 and 2.5 mg/kg before cisplatin intraperitoneal injection (6 mg/kg). Serum and kidneys were extracted and analyzed 5 days after cisplatin treatment for determinations of the renal content of glutathione, thiobarbituric acid-reactive substances, renal concentration and enzymatic activities of catalase, superoxide dismutase and glutathione peroxidase. RESULTS: Ozone pretreatment prevented the increase in serum creatinine levels, the glutathione depletion and the inhibition of superoxide dismutase, catalase and glutathione peroxidase activities induced by cisplatin in the rat kidney. Also, the renal content of thiobarbituric acid-reactive substances was decreased by ozone therapy. These protective effects of ozone were dose dependent. CONCLUSIONS: Intrarectal ozone therapy prevented effectively the renal antioxidant unbalance induced by cisplatin treatment.


Introduction
Cisplatin (CDDP) is an effective chemotherapeutic agent commonly used in the treatment of a variety of solid-organs cancers, including those of the head, neck, testis, ovary and breast. 1 Unfortunately, 28Á/36% of patients receiving a dose of CDDP develop acute renal failure. 1 Therefore, its pathogenesis has been the focus of many investigations, which have shown that acute renal failure induced by CDDP is accompanied by the reduced renal blood flow associated with increased renal vascular resistance and histological damage to the proximal tubular cells. 2,3 Additionally, evidence is available that the cellular events in CDDP-mediated nephrotoxicity, including apoptosis induction, decreased protein synthesis, membrane peroxidation, mitochondrial dysfunction and DNA injury, are a consequence of reactive oxygen species (ROS) generation, which produces oxidative renal damage. 4 Á 9 Thus, administration of CDDP causes depletion of renal reduced glutathione (GSH) and an increase of lipid peroxidation accompanied with a decrease in the activity of enzymes that protect against lipid peroxidation in the kidney, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). 4 Á 9 In agreement with this, administration of known antioxidant molecules like vitamin E, ascorbic acid, ebselen, lipoic acid, GSH and its esters, CAT and the SOD mimetic orgotein, among others, have shown nephroprotective effects in CDDP-treated rats. 10 Á 16 Recently it was reported that oxidative preconditioning with ozone prevents renal damage in rats submitted to warm ischemia, 17 suggesting that oxidative preconditioning with ozone may provide a prophylactic approach for minimizing renal damage before transplantation, which is also mediated by ROS production. Taking into account these findings, we decided to evaluate the role of ozone oxidative preconditioning in CDDP-induced acute renal damage in rats and to determine its potential protective effects on some important constituents of the antioxidant system in the kidney.

Chemicals
Serum creatinine (Cr) was measured spectrophotometrically with the Cr assay kits purchased from Biological Products Enterprise 'Carlos J Finlay' (Havana, Cuba). All reagents used in determinations of GSH, SOD, CAT, GSH-Px, thiobarbituric acidreactive substances (TBARS) and cisplatin were purchased from Sigma Chemicals (St Louis, MO, USA). Other reagents of analytical grade were obtained from normal commercial sources. The animals were divided into 10 groups of seven rats each: (1) non-treated control rats, (2) rats treated with O 2 , (3) rats treated with O 3 /O 2 mixture (1.1 mg/ kg), (4) rats treated with O 2 '/CDDP, (5) rats treated only with CDDP, (6) rats treated with O 3 /O 2 mixture (10 mg of ozone/ml of mixture at a dose of 0.36 mg/ kg)'/CDDP, (7) rats treated with O 3 /O 2 mixture (20 mg of ozone/ml of mixture at a dose of 0.72 mg/kg)'/CDDP, (8) rats treated with O 3 /O 2 mixture (30 mg of ozone/ml of mixture at a dose of 1.1 mg/ kg)'/CDDP, (9) rats treated with O 3 /O 2 mixture (50 mg of ozone/ml of mixture at a dose of 1.8 mg/ kg)'/CDDP, and (10) rats treated with O 3 O 2 mixture (70 mg of ozone/ml of mixture at a dose of 2.5 mg/ kg)'/CDDP. Rats were killed 5 days after CDDP injection by an overdose of ether. The blood was collected and serum was separated by centrifugation for Cr analysis. The kidneys were dissected and immediately frozen at Á/208C until analysis could be completed.

Animals
Kidney homogenates were obtained using a tissue homogenator (Ultraturrax T-25 Polytron) at 48C. The homogenates were prepared by using a 100 mM KCl buffer (pH 7) containing 0.3 mM ethylenediamine tetraacetic acid (1:10 w/v) for GSH, TBARS, GSH-Px and SOD determinations (Buffer 1). The homogenates were spun down with a centrifuge at 600 )/g for 60 min at 48C. The supernatants were taken for biochemical determinations.
Kidney homogenates for CAT enzymatic assay were carried out using 50 mM phosphate buffer (pH 7) containing 1% Triton X-100 (1:9 w/v) (Buffer 2). The homogenates were centrifuged at 600 )/g for 60 min at 48C and the supernatants were used for the CAT assay.

Determination of GSH
GSH was determined by a slightly modified version of the method of Beutler et al. , 18 using a spectrophotometer. One milliliter of the kidney homogenate, as described earlier, was mixed with 1.5 ml of 5% metaphosphoric acid and centrifuged at 3000 )/g for 10 min at room temperature. Fifty hundred microliters of this acidic supernatant was mixed with 2 ml of 0.2 M phosphate buffer and 0.25 ml of 0.04% 5, 5?-dithio-bis(-2-nitrobenzoic acid). Absorbance of the yellow solution was measured at 412 nm within 10 min. A molar extinction coefficient of 13.6 M/cm that describes the formation of the thiolate anion by the reaction of sulfhydryl groups with 5,5?-dithio-bis-2nitrobenzoic acid at 412 nm was used to quantify GSH.

Determination of SOD activity
Enzymatic activity of SOD was determined by a modified version of the method of Minami and Yoshikawa. 19 Fifty microliters of the kidney homogenate was mixed with 450 ml of cold deionized water, 125 ml of chloroform and 250 ml of ethanol. The mixture was centrifuged at 8000 )/g for 2 min at 48C. Fifty hundred microliters of the extract was added to the reaction mixture containing 500 ml of 72.4 mM Tris cacodylate buffer with 3.5 mM diethy-lene pentaacetic acid (pH 8.2), 100 ml of 16% Triton-X 100 and 250 ml of 0.9 mM nitro blue tetrazolium. The reaction mixture was incubated for 5 min at 378C before adding 10 ml of 9 mM pyrogallol (dissolved in 10 mM hydrochloric acid), and then it was incubated for exactly 5 min at 378C. The reaction was stopped with the addition of 300 ml of 2 M formic buffer (pH 3.5) containing 16% Triton-X 100. The absorbance was measured at 540 nm in the spectrophotometer. One unit of SOD enzymatic activity is equal to the amount of enzyme that diminishes the initial absorbance of nitro blue tetrazolium by 50%.

Determination of CAT activity
CAT was determined according to the method of Evans and Diplock. 20 Kidney homogenate was diluted with Buffer 2, as already described, to obtain an adequate dilution of the enzyme. Then, 2 ml of the enzyme dilution were added to the cuvette and mixed with 1 ml of 30 mM H 2 O 2 , and then the absorbance was measured at 240 nm, for 30 sec, in the spectrophotometer. Initial absorbance of the reaction mixture must be around 0.5. The enzyme activity is expressed as the first-order constant that describes the decomposition of H 2 O 2 at room temperature.

Determination of GSH-Px activity
Enzymatic activity of GSH-Px was measured using a modified version of the method of Thonson et al. 21 All reaction mixtures were dissolved in 20 mM sodium phosphate buffer containing 6 mM ethylenediamine tetraacetic acid (pH 7.0). The reaction mixture consisted of 98.8 ml of phosphate buffer, 700 ml of 2.86 mM GSH, 100 ml of 1 mM sodium azide, 100 ml of 1 mM NADPH and 4.2 ml of GSH reductase (0.5 u). Then, 10 ml of the tissue homogenate supernatant were added to the reaction mixture and incubated at room temperature for 10 Á/15 min. Afterward, 10 ml of 30 mM t -butyl hydroperoxide (dissolved in bidistilled water) was added to the reaction mixture and measured at 340 nm for 7 min in the spectrophotometer. A molar extinction coefficient of 6.22 )/10 3 M/cm was used to determine the activity of GSH-Px. The enzyme activity is expressed as international units of enzymatic activity per milligram of protein. International units are expressed as micromoles of hydroperoxides transformed per minute per milliliter of enzyme.

Lipid peroxidation assay
This assay was used to estimate TBARS levels as described by Ohkawa et al. 22 Two hundred milliliters of tissue homogenate supernatant were added to 100 ml of sodium dodecyl sulfate, 750 ml of 20% acetic acid (pH 3.5), 750 ml of 0.6% thiobarbituric acid and 300 ml of distilled water, and were incubated at 958C for 60 min. The samples were allowed to cool at room temperature. Then 2.5 ml of butanol:pyridine (15:1) and 500 ml of distilled water were added, vortexed, and centrifuged at 2000 )/g for 15 min. The absorbance of three ml of the colored layer was measured at 532 nm spectrophotometrically using 1,1,3,3tetraethoxypropane as standard.

Protein assay
Protein concentrations were determined by the method of Lowry 23 using bovine serum albumin as standard.

Histopathological assessment of renal damage
The left kidneys were quickly removed and fixed in 10% formaldehyde. Tissues were embedded in paraffin, sectioned at 3 mm, stained with hematoxylin and eosin (H/E) and evaluated by light microscopy.

Statistical analysis
Data are expressed as means9/standard error of the mean and analyzed statistically using one-way analysis of variance followed by the Duncan multiple range test for serum Cr determinations, whereas the Kruskall Á/Wallis test followed by the Mann Á/Whitney test was applied for the rest of the markers. The 0.05 level of probability was used as statistical significance.

Results
Loss of body weight in all animals was observed 5 days after CDDP injection. The mean reduction in body weight was approximately 16 g, which represents 8% of the initial body weight. This effect on body weight was not reversed in animal groups under O 3 therapy with mixtures of O 3 /O 2 administered by rectal insufflation.
The levels of Cr in the blood serum of control and experimental rats are presented in Table 1. After CDDP injection, Cr levels increased about four-fold at day 5 with respect to non-treated controls. However, there were no significant differences in the concentrations of Cr in rat serum between the non-treated controls and those only treated with O 2 . No significant differences in Cr levels were found between the group treated with CDDP alone and the other one with CDDP'/O 2 . In contrast, oxidative preconditioning with O 3 /O 2 mixture during 15 days at doses of 0.72 and 1.1 mg/kg but not 0.36 mg/kg induced a remarkable decrease in Cr levels in serum with Ozone preconditioning in nephrotoxicity respect to the higher values in CDDP group. Greater doses of 1.8 and 2.5 mg/kg did not significantly decrease Cr (Table 1).
The concentration of renal reduced GSH was significantly decreased (58% of non-treated control) in CDDP-treated rats. Ozone therapy (0.72, 1.1, 1.8 and 2.5 mg/kg i.r.) prevented renal GSH depletion induced by CDDP. This effect was greater at doses of 0.72 and 1.1 mg/kg (p B/ 0.01). The lowest dose of O 3 (0.36 mg/kg) was not effective in the prevention of GSH depletion. (Table 1).
The kidney TBARS content used as a measure of lipid peroxidation was significantly increased in rats treated with CDDP alone, as compared with nontreated controls. Oxidative preconditioning with O 3 /O 2 mixture during 15 days induced significant decrease in TBARS content at O 3 doses of 0.72 mg/kg (pB/0.05), 1.1 mg/kg (p B/ 0.001), 1.8 mg/kg (pB/0.001) and 2.5 mg/kg (pB/ 0.01) as compared with CDDP alone. The lower O 3 dose (0.36 mg/kg) did not induce any significant change on renal TBARS content. The data indicate that the induction of lipid peroxidation made by CDDP in the kidney is attenuated by O 3 therapy in a dose-dependent manner (Table 1).
SOD activity in the kidney was significantly decreased (71% of non-treated control) in CDDP-treated rats (Table 1). Pretreatment with O 3 /O 2 mixtures (0.72 and 1.1 mg/kg) significantly increased (pB/0.01) SOD activity in CDDP-treated animals, which was close to values of non-treated control. However, the lower O 3 dose (0.36 mg/kg) and the greater ones (1.8 and 2.5 mg/kg) did not avoid the decreasing in SOD activity. On the contrary, the greatest O 3 tested dose (2.5 mg/kg) induced a remarkable and significant decrease (p B/0.01) of SOD activity (3.059/0.48) versus (6.29/0.9) in the CDDP-treated control, which provides evidence of its deleterious effect on this antioxidant enzyme.
CAT activity in the rat kidney was significantly decreased (63% of non-treated control) in CDDPtreated rats. Pretreatment with O 3 (0.72, 1.1,1.8 and 2.5 mg/kg, but not 0.36 mg/kg) significantly prevented (p B/ 0.05) the inhibitory effect of CDDP on CAT activity (Table 1).
GSH-Px activity in the kidney was significantly decreased (82% of non-treated control) in CDDPtreated rats. O 3 pretreatment (0.72 and 1.1 mg/kg) significantly increased, almost twice (pB/0.01), the GSH-Px activity with respect to both CDDP-treated control rats and non-treated rats. The greater O 3 doses (1.8 and 2.5 mg/kg) induced a minor increase on the enzyme activity ( Table 1).
The histopathological changes in the kidney tissue are reported in Fig. 1. Non-treated rats exhibited cellular tumefaction, probably due to the type of euthanasia (asphyxia by ether) (Fig. 1A). Rats treated with CDDP alone revealed intense tubular necrosis, desquamation of renal tubular cells, and cast formation in the lumen, as reported in previous works (Fig. 1B). O 3 -treated rats with 1.1 mg/kg showed a moderate cellular tumefaction, revealing no significant differences between them and nontreated rats. At greater doses of 1.8 and 2.5 mg/kg doses, histopathological changes in renal tissue were quite similar to those present in CDDP-treated rats. O 3 oxidative preconditioning provided significant protection against CDDP-induced nephrotoxicity, and the majority of the treated animals showed no significant histopathological alterations (Fig. 1C).

Discussion
The aim of this work was to determine the effect of O 3 oxidative preconditioning on the severity of CDDP-induced nephrotoxicity. In the present study, CDDP-induced acute kidney damage was character-  18 Enzymatic activity of SOD was determined by a version of the method of Minami and Yoshikawa 19 (one unit of SOD enzymatic activity is equal to the amount of enzyme that diminishes the initial absorbance of nitro blue tetrazolium by 50%). CAT was determined according to the method of Evans and Diplock 20 (the enzyme activity is expressed as the first-order constant that describes the decomposition of hydrogen peroxide at room temperature). Enzymatic activity of GSH-Px was measured using a modified version of the method of Thonson et al. 21 (the enzyme activity is expressed as international units of enzymatic activity per milligram of protein). TBARS levels were estimated by Ohkawa et al. 22 Protein concentrations were determined by the method of Lowry. 23 * Significant differences with CDDP-treated control groups (p B/0.05).
ized by a significant increase in serum Cr, decreases in the kidney GSH content and GSH-Px, SOD and CAT activities, and an increase in TBARS production in comparison with the non-treated controls. Histopathological examination of the kidneys of CDDPtreated rats revealed marked tubular necrosis and desquamation of renal tubular cells. These results are in accordance with previous reports. 4 Á 16 The mechanisms underlying CDDP-induced nephrotoxicity have not been fully elucidated. Several investigators have shown that ROS are closely related to the nephrotoxicity induced by CDDP. 5 Á 7,14 It has also been reported that the inordinate generation of ROS contributes to the initiation and/or maintenance of acute tubular necrosis. 5 Á 7,14 Indeed, several antioxidants such as SOD, ebselen, vitamin E and vitamin C are reported to attenuate cisplatin-induced renal toxicity. 10 Á 16 Several reports demonstrate different mechanisms involved in the CDDP-induced nephrotoxicity including protein kinase C activation, 24 elevation of caspase-3 activity resulting in renal cell apoptosis, 8 enhancement of intrarenal synthesis of thromboxane A 2 25 and dyslipidemia. 26 Oxidative preconditioning is analogous to other phenomena such as ischemic preconditioning, 27 thermal preconditioning 28 and chemical precondi-tioning. 29 All of these processes have in common that a repeated and controlled stress is able to protect against a prolonged and severe stress.
Intrarectal O 3 therapy has conferred protection against hepatic ischemia/reperfusion injury by the adenosine accumulation and by blocking the xanthine/xanthine oxidase pathway, decreasing ROS generation after reperfusion. 30 Á 32 Recently, it was demonstrated that intrarectal application of an O 3 /O 2 mixture reduced ROS by stimulation and/or preservation of the endogenous antioxidant systems in experimental models of liver and renal ischemiareperfusion, respectively. 30 Á 32 Also, there is evidence of an increase in the activity of antioxidant enzymes such as SOD and GSH-Px and a decrease of malondialdehyde after O 3 preconditioning in patients with cardiopathy and in rats suffering ischemia/ reperfusion damage. 17,33 Our experimental results clearly demonstrate that oxidative preconditioning with O 3 exerts protective effects in CDDP-induced acute nephrotoxicity in rats. Furthermore, the data provide strong evidence that i.r. O 3 therapy effectively prevented a decrease in the renal antioxidant defense system and certainly avoided the deleterious effect of CDDP on it. Thus, the pretreatment with O 3 /O 2 mixture prevented GSH depletion and the decrease of SOD, CAT and GSH-Px activities induced by CDDP in rat kidney. Also, renal TBARS content was diminished by O 3 therapy. These protective effects of O 3 were dose dependent. The lowest dose of 0.36 mg/kg did not induce any significant change in the former antioxidant markers. In contrast, the doses of 0.72 and 1.1 mg/kg significantly reduced serum Cr and TBARS levels and preserved some constituents of the antioxidant defense, such as GSH content, SOD, CAT and GSH-PX activities, which were levels close to or even greater than those of non treated controls (Table 1).
Furthermore, the preservation of renal SOD, CAT and GSH-PX activities by O 3 pretreatment at lower doses (0.72 and 1.1 mg/kg) shows that this agent can protect these enzymes even 5 days after CDDP administration. Neither significant change of GSH and TBARS renal content nor statistic differences in CAT activity were found at the greater ozone doses of 1.8 and 2.5 mg/kg as compared with that of 1.1 mg/ kg, which indicates that the 'plateau effect' in the dose Á/response curves was reached.
Induction of CAT, SOD, and GSH-Px by O 3 therapy is probably due to hydrogen peroxide produced as result of O 3 decomposition. Hydrogen peroxide has been shown to be one of the major intermediates of O 3 decomposition along with oxygen radicals (OH + and O 2 + ). In contrast, the greatest O 3 dose (2.5 mg/kg) induced a marked and significant decrease of SOD activity (3.059/0.48 u/mg of protein) as compared with non-treated controls (8.729/1.15 u/mg of protein), which reveals deleterious effects of ozone treatment on this enzyme, which is probably due to SOD inactivation by H 2 O 2 at greater concentrations as it has been demonstrated by Marklund. 34 This author suggested that inactivation of Cu Zn SOD by H 2 O 2 appears to be due to a Fenton-type reaction of H 2 O 2 with Cu, forming a reactive intermediate that destroys an essential liganding histidine residue of the enzyme. Whiteside and Hassan also demonstrated induction and inactivation of CAT and SOD by ozone in cultures of Escherichia coli. 35 Furthermore, they showed that an increase in the activities of CAT and SOD by O 3 was due to induction of the novo enzyme synthesis rather than activation of pre-existing apoproteins. Thus, the observed induction of SOD, CAT and GSH-Px in response to O 3 pretreatment provides further evidence that there is a correlation between antioxidant enzyme biosynthesis and O 3 exposure.
Additionally, these authors also showed that the antioxidant enzymes are subjected in vivo to the damaging effects of O 3 at relatively high doses. Therefore, in concordance with our results this observation provides evidence that O 3 , besides inducing the biosynthesis of these enzymes, could also cause inactivation of them by the mechanism previously explained.
In addition, O 3 oxidative preconditioning might prevent CDDP-induced acute renal failure through attenuation of renal tubular damage and enhancement of the regenerative response of the damaged tubular cells. As shown in Fig. 1, animals treated with i.r. O 3 therapy before CDDP recovered the normal structure of its kidneys 5 days after CDDP administration, showing a moderate cellular tumefaction, in contrast with CDDP-treated rats, which showed severe acute tubular necrosis.
In conclusion, a single dose of CDDP leads to the inhibition of renal antioxidant enzyme activity, depletion of renal GSH levels, increased serum Cr and enhanced renal lipid peroxidation, which cause nephrotoxicity. Our results support the evidence that part of the mechanism of nephrotoxicity in CDDPtreated rats is related to depletion and inhibition of the antioxidant system. Therefore, the prevention of this inhibition in rats protected by i.r. O 3 therapy support the potential use of it at low doses to ameliorate CDDP-induced renal injury.