Part of the ‘flurane’ family, sevoflurane, is an inhalation anesthetic and is used for general anesthesia ¹. Sevoflurane (SEV) has been reported to generate oxygen-derived free radicals, which can be one of the major causes for the impairment of nitric oxide (NOx)-induced relaxation of vascular smooth muscle ². It has also been reported that SEV reduced endothelium-dependent vasorelaxation by generating superoxide anion ³. Recently, several studies have shown that ROS exist in sevoflurane induced cardioprotection ^4,5. It is now commonly recognized that reactive oxygen species (ROS) are involved in a variety of physiological and pathological processes, including cellular signal transduction, cell prolifeation and differentiation, apoptosis as well as ischemiareperfusion injury, inflammation and many degenerative diseases ^6-9. Especially, ROS mediated lipid peroxidation and DNA damage is associated with a variety of chronic health problems, such as cancer, ageing and atherosclerosis ^10-12.

Tea is a rich source of polyphenolic compounds, particularly flavonoids. Flavonoids are phenols that are widely distributed in plants; more than 4000 have been identified ¹³. The main flavonoids present in green tea include catechins; among them, epigallocatechin 3-gallate (EGCg) has the highest antioxidant capacity. In addition, EGCg has many biological functions, including antioxidant activity ¹⁴, antimutagenic ¹⁵ and anticarcinogenic effects ¹⁶, and inhibitory action on tumour invasion and angiogenesis ¹⁷. Green tea is one of the most popular beverages in the world and has biologically important polyphenols. EGCg is the most active major polyphenol of green tea and primarily responsible for the green tea effect. It has been proved EGCg shows a potent antioxidant property ^18,19. EGCg possesses two triphenolic groups in its structure, which have been reported to be important for its potent antioxidant activity ²⁰.

The aim of the study is to investigate the effects of sevoflurane on oxidants and antioxidant status of rat heart 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 (NOx), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in cardiac tissue.

Animals, diets, experimental design and data collection
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. Wistar–Albino rats were randomly assigned to one of tree groups: Group 1 (n:6): sevoflurane; Group 2 (n:6): sevoflurane plus EGCg; Group 3 (n:6): 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% oxygen and 50% N₂O mixture (4L/minute). The gas mixture was administered for two hours in a continuous flow chamber, breathing spontaneously without any surgical maneuver. 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. 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.

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

Tissue homogenisation and determination of tissue NOx, MDA, GSH-Px, CAT and SOD level
Heart was quickly removed, the blood being washed out with ice-cold 0.9% saline solution. For the determination, 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. ²¹. Plasma NOx levels were measured in triplicate after conversion of nitrate to nitrite by nitrate reductase, and nitrite was measured by using the Griess reaction, as described previously ²². The results of tissue were expressed as nmol/g wet tissue and the results of serum were expresses as nmol/ml. MDA, which is the end product of lipid peroxidation, was measured spectrophotometrically as described by Ohkawa et al ²³.

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. ²⁴. GSH-Px activity levels were measured using the method of Paglia and Valentine ²⁵ 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 catalase (CAT) activity was determined by measuring the decomposition of hydrogen peroxide at 240 nm, according to the method of Aebi ²⁶.

Statistical Analyses
All values were presented as mean ± standard deviation (SD). Statistical evaluation of each value was performed using oneway 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.

Tissue MDA, NOx, GSH-Px, CAT and SOD levels are shown in Table 2. Sevoflurane exposure increased MDA, SOD and GSH-Px activities (p<0.05, p<0.0001, p<0.001, respectively) while non-significant changes occurred in CAT and NOx activities when sevoflurane was exposured compared with control group. Supplementation of EGCg (group 2) decreased the MDA, SOD and GSH-Px activities whereas, the NOx activities increase with EGCg supplementation compared with group 1. There were no significant differences among groups in CAT activities.

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

Free radicals such as superoxide radical or hydroxyl radical are constantly produced as a normal consequence of aerobic metabolism ²⁷. Oxidative stress results from an imbalance between radical generating and radical-scavenging systems leading to cell membrane impairment or DNA damage ²⁷. MDA is a reflection of lipid peroxidation, whereas SOD and GSH-Px are important antioxidant defenses. These enzymes are involved in the clearance of superoxide and hydrogen peroxide (H₂O₂) to maintain the structure and function of biological membranes ²⁷. SOD dismutases superoxide H₂O₂ and this compound is catabolized by catalase and GSH-Px. In higher organisms, GSH-Px appears to have largely supplanted the need for catalase membranes ²⁷. Thus, our findings support the existence of oxidative stress from mechanically ventilated animals during exposure to sevoflurane. Moreover, anesthesia conducted with EGCg reduced oxidative stress and enhanced antioxidant defense mechanisms expressed by larger concentrations of free radical scavengers.

Oxidative stress leads to the accumulation of lipid peroxidation products MDA in the heart, and causes impaired cell function, while antioxidant enzyme SOD and GSH-Px play great roles in cellular defense against oxidative stress. In this study, to confirm the presence of increased oxidative stress in cardiac tissue from mechanically ventilated animals during exposure to sevoflurane, we quantified myocardial levels of MDA, NO content, and SOD, GSH-Px and CAT activities. The results showed that the level of MDA, GSH-Px and SOD in cardiac tissue increased, while the activities of NO and CAT was not changed vs the sham-operated control, indicating a significant oxidative stress. The treatment with EGCg almost completely prevented the Sevoflurane effects in SOD and GSH-Px levels, and MDA formation both in cardiac tissue. These results suggest that the protective effects of EGCg on cardiac tissue were correlated. EGCg can generate H2O2 ^28,29, and H₂O₂ can lead to eNOS activation and vasorelaxation in aortic rings ²⁷. In a recent study ³⁰ 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 cardiac tissue incresed with EGCg supplementation.

Impairment of non-enzymatic antioxidant scavenging activity is related to elevation of toxic metabolites such as superoxide free radicals (O2.−), hydrogen peroxide (H₂O₂), or hydroxyl radicals (•OH). Elevation of O2.−, which is the substrate of SOD might be the reason of the higher enzyme activity. Also H₂O₂ which is produced by SOD might be the cause of CAT and GSH-Px production and higher activity of this enzyme with the same mechanism. In this study, we observed an elevation of SOD and GSH-Px levels in group I. Elevation of SOD and GSH-Px activities due to increased production of free radicals such as O2 .− and •OH were also observed. However these compansatory mechanisms were not able to prevent cellular lipid peroxidation. Therefore, these findings might be as a result of more production of radicals due to sevoflurane administration. Another finding supporting this hypothesis is MDA levels which have the highest values in group 1 Enzymatic findings may be important signs of effects of administration of sevoflurane and EGCg at the cellular level.

The volatile anaesthetic sevoflurane protects the heart against ischaemia-induced adenosine triphosphate (ATP) depletion, Ca²⁺ overload and oxidative stres through activation of protein kinase C (PKC), opening of mitochondrial K⁺ ATP channels (mitoK⁺ATP) and the production of ROS (4, 31-33). But Sevoflurane ³⁴ have been proposed to cause formation of ROS directly in cardiac tissues. It may be possible that an anesthetic can cause free radical release from itself or the tissue with which it interacts.

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 intravenous EGCg (40 mg/kg/d) significantly protected cardiac tissue. These conclusions were supported by the improved reduced levels of MDA.