Materials and Methods: Tree groups of animals were studied. Sevoflurane 3% (v/v) in air/O2 were administered to animals in group 1 (n = 7) and sevoflurane plus EGCg was administered in group 2 (n = 7). Seven animals were allocated to control group (group 3). Malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in kidney tissue were studied. Histopathological changes were also assessed.

Results: In group I, MDA, NO, SOD and GSH-Px activities were increased while decreased CAT activities occurred when sevoflurane was exposured compared with controls. Supplementation of EGCg (group 2) decreased the MDA (P<0.05), SOD (P<0.005) and GSH-Px activities whereas, the NO (P<0.005) and CAT (P<0.005) activities increase with EGCg supplementation compared with group 1. In histopathology, sevoflurane exposure group had severe degeneration with moderate cortical necrosis in kidney tissue. Administration EGCg with sevoflurane exposure decreased degeneration.

Conclusion: The amount of lipid peroxidation and antioxidant enzyme levels were more increased in following sevoflurane administration. The administration of EGCg significantly protected kidney tissue. ©2008, Firat University, Medical Faculty

Green tea has highly reputed chemotherapeutic effects and is one of the most widely investigated herbs. Since it has been imbibed in China, Korea and Japan for thousands of years, its long-term safety is well established. Its wide safety margin makes it one of the safest herbal medicines available ⁹. The main constituents of green tea extract are catechins: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG) and (-)-epigallocatechin gallate (EGCg). The most important property of catechins is their antioxidative ability to scavenge free radicals from damaging biomolecules, chelate catalytic metal ions from free radical formation and quench singlet oxygen from activating organic molecules to form peroxides and free radicals ^10-12. Such properties prevent DNA damages by reactive oxygen species. Catechins are therefore both anti-mutagenic and anticarcinogenic ⁹. The main flavonoids present in green tea include catechins; among them, EGCg has the highest antioxidant capacity. The aim of the study is to investigate the effects of sevoflurane on oxidants and antioxidant status in the kidney tissue. In this paper, we also have performed in vitro experiments to investigate the protective effects of EGCg against sevoflurane anesthetic exposure by evaluating levels of malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in kidney tissue and by evaluating histopathology.

A total of 18 Wistar-albino rats (approximately 200 g BW) obtained from Veterinary Control and Research Institute of Elazig, Turkey were used in the study. Rats were housed in cages at room temperature and were submitted to light/dark cycle of 12 hours. The rats were randomly assigned to one of tree groups: Group 1 (n:7): sevoflurane; Group 2 (n:7): sevoflurane plus EGCg; Group 3 (n:7): control. Placebo (physiological saline, 0.9%) was given to animals in Group 1 and Group 3 by gavage and EGCg (Teavigo; 40 mg/kg/d) was given to animals in group 2 by gavage for ten days. After EGCg suplementation for 10 days, the Group 1 and Group 2 rats were exposed to an anesthetic gas mixture. Sevoflurane (3%) (v/v) was given to animals in the vaporizer in air oxygen mixture (50/50) at 4L/min for 2 h. The anesthetic gases were administered via Dräger anesthetic machine (Draeger Cato-Edition, Lübeck, Germany). This Dräger anesthetic machine made by glass and its size was 40x40x70. The anesthetic gas mixture was exposed to athmosphere air by pipe. During the study, these animals were fed ad libitum with a food including the ingredients shown in Table 1. The gas mixture was administered for two hours. Then animals were killed by cervical dislocation, blood and tissue samples were taken into ice bath until homogenisation.

Tissue homogenisation and determination of tissue NO, MDA, GSH-Px, CAT and SOD level

Kidney was quickly removed, the blood being washed out with ice-cold 0.9% saline solution. For the determination of oxidant and antioxidants, 1 gr tissues were homogenized in 9 mL Tris/HCl (0.02mM and pH 7.4), using an all-glass homogenizer. For determination of NO and MDA, homogenate was used. After centrifugation at 800×g for 20 min, the resulting supernatant fraction was used for determination of SOD, GSH-Px and CAT levels. The protein concentration of the homogenat and supernatant were determined by the method described by Lowry et al. ¹³. Plasma NO levels were measured after conversion of nitrate to nitrite by nitrate reductase, and nitrite was measured by using the Griess reaction, as described previously ¹⁴. The results of tissue 6 were expressed as nmol/g wet tissue MDA, which is the end product of lipid peroxidation, was measured spectrophoto-metrically as described by Ohkawa et al ¹⁵. A Part of the homogenate was extracted in ethanol/ chloroform mixture (5/3 v/v) to discard the lipid fraction, which caused interferences in the activity measurements of glutathione peroxidase. After centrifugation at 10.000 × g for 60 min, the upper clear layer was removed and used for the analyses. Total (Cu-Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Durak et al. ¹⁶. GSH-Px activity levels were measured using the method of Paglia and Valentine ¹⁷ in which GSH-Px activity was coupled with the oxidation of NADPH by glutathione reductase. The oxidation of NADPH was followed spectrophotometrically at 340 nm. The tissue CAT activity was determined by measuring the decomposition of hydrogen peroxide at 240 nm, according to the method of Aebi ¹⁸.

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Table 1: Ingredients and chemical analyses of the starter and grower diets fed to quails, g/100 g

Histopathological evaluation

Kidney tissue was fixed in 10% buffered formaldehyde for histopathology evaluation. The formaldehyde-buffered kidney tissues were embedded in paraffin and at least four cross-sections were taken from kidney in 4–5 ml thickness and stained with hematoxyline–eosin (H and E). Histopathological examination was performed by a pathologist blinded to the study groups.

Statistical Analyses

All values were presented as mean ± standard deviation (SD). Statistical evaluation of each value was performed using one-way analysis of variance for multiple comparisons. Two-way ANOVA was performed to compare differences in groups. Values were considered statistically significant at P <0.05.

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Table 2: Levels of MDA, NO, SOD, GSH-Px and CAT activities in kidney tissue

Histopathology

In sevoflurane exposure group, there was severe degeneration with moderate cortical necrosis in kidney tissue. In kidney tissue, there was degeneration and desquamation with intertubular hemoragie in proximal tubulus epitel. Despite of degeneration, there were picnotic nucleuses in tubulus cells. Hyalin cylinders were shown in the lumen of tubules. Also, in the medulla of the kidney, severe congestion with intertubular hemoragie was shown (Figure 1). Administration EGCg with sevoflurane exposure decreased the degeneration and hyalin cylinder formation didn’t show in the tubuler lumen (Figure 2).

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Figure 1: After sevoflurane exposure, severe degenerative and necrotic changes in proximal tubulus epitel in the kidney (H&E, × 200).

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Figure 2: After administration EGCg with sevoflurane, mild degenerative changes (H&E, × 200).

In a recent study ²² demonstrates that endothelium-dependent vasorelaxation induced by the tea-derived catechin EGCg occurs in response to a potent, dose-dependent activation of eNOS in endothelial cells. The resulting increase in eNOS activity is observed within a few minutes, suggesting posttranslational regulation of eNOS as an underlying mechanism. In agree with this study, we also found that level of NO in kidney tissue incresed with EGCg supplementation. It has been showed that catechins chelating properties could also have a fundamental role in preventing peroxidation ²³.

Dikmen et al ²⁴ observed an elevation of SOD, GSH-Px and CAT levels in anasthesic groups they reported that elevation of SOD, CAT and GSH-Px activities due to increased production of free radicals. In the present study, our results show that animals exposed to sevoflurane had increased MDA, NO, SOD and GSH-Px concentrations in kidney tissue, but the increased level of GSH-Px was not statically significant. Conversely, EGCg supplementation decreased MDA, SOD and GSH-Px levels in animals exposed to sevoflurane. We could not find any previous report on the effects of EGCg supplementation on kidney tissue levels of MDA, NO and antioxidant enzymes in rats exposed to anasthesic agents and to compare with our results. Against all the protective enzymatic mechanisms, cellular damage that supports the elevation of lipid peroxidation was observed in group 1. Histopathological findings in group 1, there was severe degeneration with moderate cortical necrosis in kidney tissue. Administration of sevoflurane plus EGCg in rats, decreased degeneration in histopatological findings.

In conclusion, the amount of lipid peroxidation and antioxidant enzyme activities were more increased in following sevoflurane administration in the 3% concentration. The administration of EGCg (40 mg/kg/d) significantly protected kidney tissue. These conclusions were supported by the improved reduced levels of MDA.

1) Gonsowski CT, Laster MJ, Eger EI, Ferrell LD, Kerschmann RL. Toxicity of compound A in rats: effect of increasing duration of administration. Anesthesiology 1994; 80: 556-565.

2) Higuchi H, Arimura S, Sumikura H, Satoh T, Kanno M. Urine concentrating ability after prolonged sevoflurane anaesthesia. Br J Anaesth 1994; 73: 239-240.

3) Bito H, Ikeda K. Plasma inorganic fluoride and intracircuit degradation product concentrations in long-duration, low-flow sevoflurane anesthesia. Anesth Analg 1994; 79: 946-951.

4) Morio M, Fujii K, Satoh N, et al. Reaction of sevoflurane and its degradation products with soda lime. Anesthesiology 1992; 77: 1155-64.

5) Kandel L, Laster MJ, Eger EI, Kerschmann RL, Martin J. Nephrotoxicity in rats undergoing a one-hour exposure to compound A. Anesth Analg 1995; 81: 559-63.

6) Martin JL, Laster MJ, Kandel L, et al. Metabolism of compound A by renal cysteine-S-conjugate beta-lyase is not the mechanism of compound A-induced renal injury in the rat. Anesth Analg 1996; 82: 770-774

7) Eger EI, Gong D, Koblin DD, et al. Dose-related biochemical markers of renal injury after sevoflurane versus desflurane anesthesia in volunteers. Anesth Analg 1997; 85: 1154-1163.

8) Eger EI, Koblin DD, Bowland T, et al. Nephrotoxicity of sevoflurane versus desflurane anesthesia in volunteers. Anesth Analg 1997; 84: 160-168.

9) Mitscher LA, Jung M, Shankel D, et al. Chemoprotection: a review of the potential therapeutic antioxidant properties of green tea (Camellia sinensis) and certain of its constituents. Med. Res. Rev. 1997; 17:327-365

10) Rice-Evans C. Plant polyphenols: free radical scavengers or chain-breaking antioxidants? Biochem Soc Symp. 1995; 61: 103-116.

11) Kuo SM, Leavitt PS, Lin CP. Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells. Biol. Trace Element Res. 1998; 62: 135-53.

12) Yoshino M, Murakami K. Interaction of iron with polyphenolic compounds: application to antioxidant characterization. Anal. Biochem. 1998; 257: 40-44.

13) Lowry O, Rosenbraugh N, Farr L,Randall R. Protein measurement with folin phenol reagent. J. Biol. Chem. 1951; 182, 265–275

14) Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990; 36: 1440-1443.

15) Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Analytical Biochemistry. 1979; 95: 351-358.

16) Durak I, Yurtarslani Z, Canbolat O, Akyol O. A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium (NBT) reduction. Clin Chim Acta; 1993; 214: 103-104.

17) Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterisation of erythrocyte glutathione peroxidase. J Lab & Clin Med 1967; 70: 158-169.

18) Aebi H. Catalase In: Bergmeyer U, ed. Methods of enzymatic analysis. New York and London: Academic Press; 1974; 673-677.

19) McCord JM. The evolution of free radicals and oxidative stress. Am J Med 2000; 108: 652–659.

20) Matés JM, Pérez-Gómez C, Núñez de Castro I. Antioxidant enzymes and human diseases. Clin Biochem 1999; 32: 595–603.

21) Sivaci R, Kahraman A, Serteser M, Sahin DA, Dilek ON. Cytotoxic effects of volatile anesthetics with free radicals undergoing laparoscopic surgery. Clin Biochem. 2006; 39: 293-298.

22) Lorenz M, Wessler S, Follmann E, et al. Constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase- cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation. J Biol Chem 2004; 13: 6190-6195.

23) Frei B., Higdon J.V. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies, J. Nutr. 2003;133: 3275– 3284

24) Dikmen B, Unal Y, Pampal HK. Effects of repeated desflurane and sevoflurane anesthesia on enzymatic free radical scavanger system. Molecular and Cellular Biochemistry, 2007; 294: 31-36.