Dehydroepiandrosterone Improves Hepatic Antioxidant Systems after Renal Ischemia-Reperfusion Injury in Rabbits

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1 Annals of Clinical & Laboratory Science, vol. 33, no. 4, Dehydroepiandrosterone Improves Hepatic Antioxidant Systems after Renal Ischemia-Reperfusion Injury in Rabbits Abdulkadir Yildirim, 1 Metehan Gumus, 2 Sebahattin Dalga, 2 Y. Nuri Sahin, 1 and Fatih Akcay 1 1 Biochemistry and 2 General Surgery Departments, Ataturk University Medical School, Erzurum, Turkey Abstract. This study evaluated the effects of dehydroepiandrosterone (DHEA) on the oxidant [malondialdehyde (MDA)] and antioxidant [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione (GSH)] systems in liver after renal ischemia-reperfusion (IR) injury in rabbits. Thirty rabbits were randomly assigned to 3 groups of 10: group I (sham operation), group II (renal IR group), and group III (DHEA, 25 mg/kg, sc, 15 min pre-ischemia). Renal IR injury in group II caused a decrease of SOD (25 %), GPx (36 %), and CAT (26 %) activities and GSH levels (32 %), and increases of MDA (30 %) in liver and of ALT and AST activities in serum, compared to group I. DHEA administration decreased the hepatic MDA level (19 %) and serum ALT activity (30 %) (p <0.01 and p <0.05, respectively), and considerably increased hepatic GSH levels and GPx activities (p <0.01 for both) in group III, compared to group II. These results suggest that DHEA treatment has beneficial effects on antioxidant defenses against hepatic injury after renal IR in rabbits, possibly by augmenting GSH levels and lowering MDA production. (received 13 April 2003; accepted 18 June 2003) Keywords: ischemia-reperfusion injury; antioxidant enzymes; dehydroepiandrosterone, oxygen free radicals Introduction The superoxide (O 2.- ), hydroxyl (HO. ), perhydroxyl radicals (HO 2.- ), and nitric oxide (NO. ) are the most important free radicals generally derived from oxygen. These reactive oxygen species (ROS) occur both during normal metabolic reactions and following exposures to various factors, such as smoking, atmospheric pollutants, UV light, ionizing radiation, and xenobiotics. In the body, the ROS serve as signaling and regulatory molecules at physiologic levels, and as highly deleterious and cytotoxic oxidants at pathologic levels [1]. Although most free radicals have a short half-life ( 10-6 sec) [2], they have a tendency to interact with numerous biological molecules, eg, proteins, lipids, and nucleic acids [3]. HO., the most reactive oxidant, and other Address correspondence to Abdulkadir Yildirim, M.D., Department of Biochemistry, School of Medicine, Ataturk University, Erzurum, Turkey; tel /2329; fax ; kadir@atauni.edu.tr. This study was presented at the 15th European Congress of Clinical Chemistry and Laboratory Medicine (Barcelona 2003). ROS may cleave covalent bonds in proteins and lipids, and lead to cell membrane injury [3]. These disturbances of cells cause numerous pathological processes. The extracellular environment and cells have different antioxidant systems, including enzymatic and non-enzymatic antioxidant molecules. SOD catalyzes the dismutation of O 2.- into hydrogen peroxide (H 2 O 2 ), which can be transformed into water and molecular oxygen by CAT. GPx also reduces H 2 O 2, as well as lipid hydroperoxides, and may therefore help to prevent damage from lipid peroxidation. GSH, the most abundant thiol containing substance in the intracellular compartment, functions directly as an antioxidant [2]. Following ischemia, O 2.- is produced particularly during the reperfusion phase. Studies have demonstrated that ROS have an important role in IR injury, which has a deteriorating effect in transplanted and intact organs [4,5]. DHEA is the principal C-19 steroid produced by the adrenal gland of humans. In addition to its protective effects on various disease processes (eg, Alzheimer s disease, atherosclerosis) [6,7], DHEA /03/0400/0459, $ by the Association of Clinical Scientists, Inc.

2 460 Annals of Clinical & Laboratory Science has been recently reported to possess antioxidant properties [8,9]. The aim of this study was to evaluate the effects of DHEA on the oxidant (lipid peroxides as MDA) and antioxidant (SOD, GPx, and CAT activities and total GSH levels) systems in hepatic tissue after renal IR injury in rabbits. Serum ALT and AST activities were also measured to check the effects of DHEA on these hepatic function tests. Materials and Methods DHEA and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. Crystalline DHEA, dissolved in 1 vol of 95% ethanol, was mixed with 9 vol of 16% Tween 80 in 0.9% NaCl. This study was approved by the local animal experimentation committee. Surgical procedure. Thirty adult male New Zeland white rabbits ( kg) were randomly assigned to 3 groups of 10 animals each. The rabbits were anesthetized with ketamine (100 mg/kg, im) and stabilized on a surgical stand. All surgical procedures were carried out under a heating lamp. The lumbar region was shaved with a safety razor and sterilized with povidone iodine solution. A paramedian incision was made and the left renal artery and vein of rabbits in group I (sham-operation) were found but not clamped. The rabbits in group II were subjected to ischemia by clamping the left renal artery and vein with a nontraumatic vascular clamp for 60 min, and then reperfused for 60 min. The rabbits in group III underwent the identical surgical procedure as group II, but received DHEA (25 mg/ kg, sc, 15 min before the left renal ischemia. At the end of each experimental procedure, blood samples were collected in Vacutainers without additives. The samples were centrifuged (3000 x g, 5 min) to obtain serum. After rabbits were sacrificed, hepatic tissue was rapidly excised, palpated to remove blood, and washed with cold 0.9% NaCl solution. Liver and serum samples were stored at -80 C prior to assays. Tissue processing and biochemical assays. Liver samples were kept at 4 C throughout preparation. For all assays except GSH and MDA, a portion of each liver was homogenized in 0.9% NaCl solution (to make 10%, w/v, homogenate) using an OMNI- TH International homogenizer. Tissue homogenates were centrifuged (18,000 x g, 15 min) and the supernatants were removed for analyses. For GSH assay, liver samples were homogenized in 1.5 N perchloric acid (to make 10%, w/v, homogenate) and centrifuged (3,000 x g, 5 min). For MDA assays, liver samples were homogenized, so that each g of tissue had a 1.15 % KCl tamponade (9 ml). Protein concentrations in homogenates were determined by the Bradford method [10]. Photometry was performed with a DU 530 spectrophotometer, (Beckman Instruments, Fullerton, CA). Serum ALT and AST activities were determined by standard laboratory methods using an automatic analyzer (ADVIA 1650 analyzer, Advia Corp, Japan). GPx assay. GPx activity was measured by the method of Paglia and Valentine [11]. Tissue supernatant (0.5 ml) was mixed with 0.5 ml of double strength Drabkin s reagent. Then, 50 µl of this mixture was combined with 100 µl of 8 mm NADPH, 100 µl of 150 mm glutathione (reduced form), 20 µl of glutathione reductase (GSH-RD) (30 units/ml), 20 µl of 0.12 M sodium azide solution, and 2.65 ml of 50 mm potassium phosphate buffer (ph 7.0, 5 mm EDTA) and the tubes incubated (30 min, 37 C). The reaction was initiated by addition of 100 µl of 2 mm H 2 O 2 solution, mixed rapidly by inversion, and the conversion of NADPH to NADP was measured spectrophotometrically for 5 min at 340 nm. The enzyme activity was expressed as units per mg protein using an extinction coefficient for NADPH at 340 nm of 6.22 x CAT assay. CAT activity was measured by the method by Aebi [12]. Supernatant (10 µl) was placed in a quartz cuvette and the reaction initiated by adding 2.99 ml of freshly prepared 30 mm H 2 O 2 in phosphate buffer (50 mm, ph 7.0). After rapid mixing, the rate of H 2 O 2 decomposition was determined from absorbance changes at 15 and 30 sec at 240 nm. CAT activity was expressed as k/mg of tissue protein, where k is the first order rate constant. SOD assay. Cu,Zn-SOD activity was assayed by the method of Sun et al [13]. To 2.45 ml of assay reagent

3 DHEA effects on liver antioxidants in renal ischemia 461 Fig. 1. Indices of liver tissue antioxidant status in the study groups (means ± SD). * p <0.005 and p <0.05 vs group I; p <0.01 vs group II. [0.3 mm xanthine, 0.6 mm Na 2 EDTA, 0.15 mm nitroblue tetrazolium (NBT), 0.4 M Na 2 CO 3, and 1 g/l bovine serum albumin (BSA)] was added 100 µl of the tissue supernatant. Xanthine oxidase (50 µl, 167 U/L) was added to initiate the reaction and the reduction of NBT by superoxide anion radicals, which are produced by the xanthine-xanthine oxidase system, was determined by measuring the absorbance at 560 nm. Cu,Zn-SOD activity was expressed as units of SOD/mg of tissue protein, where 1 U is defined as that amount of enzyme causing half-maximal inhibition of NBT reduction. Total GSH assay. GSH in liver tissue was assayed by the method of Tietze and Anderson [14,15]. Briefly, 100 µl of tissue supernatant was placed in a 3 ml cuvette; 750 µl of 10 mm 5-5 -dithio-bis-2- nitrobenzoic acid (DTNB) solution (100 mm KH 2 PO 4 plus 5 mm Na 2 EDTA, ph 7.5 and GSH- RD, 625 U/L) was added and the mixture was incubated (3 min, room temperature). Then 150 µl of 1.47 mm β-nadph was added, mixed rapidly by inversion, and the rate of 5-thio-2-nitrobenzoic acid formation (proportional to the sum of reduced and oxidized glutathione) was measured spectrophotometrically for 2 min at 412 nm. The reference cuvette contained equal concentrations of DTNB and NADPH, but no sample; results were expressed as nmol/mg of wet tissue. MDA assay. Tissue MDA levels were determined spectrophotometrically by the method of Okhawa [16]. A mixture of 8.1% sodium dodecylsulphate (0.2 ml, Merck), 20% acetic acid (1.5 ml), and 0.9% thiobarbituric acid (1.5 ml, Merck) was added to 0.2 ml of 10% tissue homogenate. Distilled water was added to the mixture to bring the total volume to 4 ml. This mixture was incubated (95 C, 1 hr). After incubation, the tubes were placed in cold water and 1 ml of distilled water plus 5 ml of n-butanol/ pyridine (15:1, v/v) was added, followed by mixing. The samples were centrifuged (4,000 x g, 10 min). The organic phase (supernatant) was removed, and absorbances were measured with respect to a blank at 532 nm. 1,1,3,3-Tetraethoxypropane was used as the standard. Lipid peroxide levels were expressed as nmol MDA/g of wet tissue. Statistics. SPSS version 11.0 for Windows was used. Results are stated as means ± SD. The Mann- Whitney U test was used to compute p values (p <0.05 was considered significant). Results Renal IR significantly impaired the enzymatic and non-enzymatic antioxidant defense systems in hepatic tissue. As shown in Fig. 1, liver SOD, GPx, and CAT activities decreased after renal IR in group II, compared to the sham-operation group (25, 36,

4 462 Annals of Clinical & Laboratory Science Fig. 2. Liver tissue MDA levels and serum ALT and AST activities in the study groups. Bars represent mean values ± SD. ** p <0.005 vs group I, p <0.01 vs group II, * p < vs group I, p <0.05 vs group II, p >0.05 vs group II. and 26%, respectively) (Fig. 1, panels A, B, C). Renal IR in group II rabbits also caused marked decrease of hepatic GSH (32%) and increase of hepatic MDA levels (30%) and serum ALT and AST activities, compared to the sham-operation group (Fig. 2 panels A, B, C). DHEA administration (25 mg/kg sc., 15 min before renal ischemia) markedly reduced oxidative damage in hepatic tissue. Rabbits in group III (DHEA pretreatment) had significant decrease of MDA levels (19%, p <0.01) and increased GPx activities (40%) and GSH levels (35%), compared to group II rabbits (p <0.01, for both) (Fig. 2 panel A, Fig.1 panels B, D). Although DHEA caused an increase of hepatic SOD and CAT activities (8 and 11%, respectively) and a decrease of serum AST activity, the apparent differences between the groups were not significant (p >0.05, for all) (Fig. 1, panels A, C, and Fig. 2, panel C). Discussion IR injury is an important problem that affects the outcome of various surgical operations such as organ transplantation, surgical revascularization, and partial organ resection. The free radical-mediated reperfusion injury may occur not only in ischemic tissues, but also in intact tissues. Effects of IR injury in remote organs have been reported in various studies; these effects have implicated circulating cytokines and leukocytes derived from IR injury [5,17], but the effects of IR injury associated with oxidative stress in rabbit intact organs have not been well characterized. Lipid peroxidation is a free radical chain process, arising from oxidative conversion of polyunsaturated fatty acids by HO. to lipid peroxides, which, in turn, can damage biological membranes [18]. MDA is utilized as the marker of lipid peroxidation. In our study, MDA levels significantly increased in hepatic tissue of rabbits subjected to renal IR, compared to the sham-operation group. In addition, SOD, GPx, and CAT activities and GSH levels were considerably decreased by renal IR in hepatic tissue. Serteser et al [19], who have demonstrated that renal IR injury leads to release of important amounts of ROS and has damaging effects on remote tissues (liver), corroborate our results. The decreased enzymatic and non-enzymatic antioxidant defense and elevated MDA levels suggest an abnormal increase of ROS in rabbit hepatic tissue after 60 min of renal ischemia and 60 min of reperfusion. We suspect that H 2 O 2, which has high membrane permeability, originates from renal IR and induces toxic injury by conversion to HO. via the Fenton reactions in liver, which is rich in Fe +2 and Cu +1. DHEA is the major androgen or androgen precursor that is produced by the adrenal cortex. Although DHEA was identified in 1965, studies of its antioxidant properties were not emphasized until

5 DHEA effects on liver antioxidants in renal ischemia [20]. Aragno et al [21] and Boccuzzi et al [9] reported that DHEA reduced acute hyperglycemia and copper-induced lipid peroxidation in rat tissues (liver, kidney, and brain). In other studies, DHEA was reported to have pro-oxidant properties [22,23]. In our experiment, subcutaneous DHEA administration significantly decreased MDA levels in liver of rabbits with renal IR. In addition, by DHEA administration induced meaningful increases of liver GSH levels and GPx activities following renal IR. Several mechanisms have been proposed to explain the protective effects of DHEA, but the mechanisms associated with its antioxidant properties are not fully understood. Studies suggest that DHEA decreases the NADPH level, which is a substrate necessary for the NADPH oxidase reaction to generate O 2.- from O 2 [24], by inhibiting glucose- 6-phosphate dehydrogenase (G-6-PDH) [25]. This explanation is not suitable for the findings of the present study, since the GPx activity and GSH level were both increased after DHEA pretreatment in hepatic tissue. GPx is the major scavenger of H 2 O 2 in liver subcellular compartments (eg, cytosol and mitochondria), and G-6-PDH activity is the basic pathway of NADPH production. Moreover, NADPH is required for maintaining a normal redox state in cells that convert oxidized glutathione (GSSG) to reduced glutathione. We speculate that the fact that NADPH is used more in the GSH-RD reaction for producing reduced glutathione than in the NADPH oxidase system for generating O 2.-, contributes to the diminution of oxidative damage. DHEA has rapid metabolic clearance and a short half-time (15-30 min) [26]. The antioxidant properties of DHEA may be related to its active metabolites, rendering the cell membranes more resistant to attack by ROS [27]. In addition to a reduction of lipid peroxide products (eg, MDA) with DHEA pretreatment, we observed an increase of hepatic GSH levels. DHEA has been reported to increase glutathione levels in rat hepatic tissue [28], which may explain, in part, its protective properties against oxidative stress. We believe that DHEA may protect the liver against RI by inducing glutathione synthesis in rabbit liver. The antioxidant properties of DHEA probably depend on its concentration in biological matrices. In a previous study, DHEA showed a protective effect at the 10 to 100 µm range in post-mortem human hippocampal cell cultures, but had a maximal effect at 100 µm of DHEA concentrations [30]. In another study, a low dose [35 µmol/kg body weight, ip] of DHEA was ineffective against copper-induced lipid peroxidation, but at a higher dose (175 µmol/ kg, ip) DHEA reduced the formation of thiobarbituric acid reactive substances (TBARS) in liver and brain microsomes of rats [9]. In an in vivo experiment, a high dose of DHEA (350 µmol/kg, ip) decreased lipid peroxidation induced by acute hyperglycemia in liver, kidney, and brain tissues of rats, but a low dose DHEA (35 µmol/kg bw, ip) was effective only in liver; DHEA pretreatment at any dose did not such alter antioxidant parameters as GSH and GPx [21]. In the present study, 88 µmol of DHEA (25 mg/ kg, sc) resulted in a decrease of IR-induced MDA formation and increases of GSH level and GPx activity. These results suggest that protective properties of DHEA against the harmful effects of free radicals may vary, depending on the DHEA dosage, administration route, (sc, ip, im), and experimental protocol. Therefore, clinical studies of the phenomenon must be designed with careful attention to such details. Grattagliano et al [29] reported that glutathione monoethylester pretreatment protected the liver against reperfusion injury in rats, probably by augmenting intra- and extra-cellular glutathione, and improvement of hepatocyte function as documented by reduced serum ALT activity. In group III, we observed decreased serum ALT and AST activities, compared to the elevated activities in group II, which reflect the hepatic damage after renal IR injury. In summary, our data suggest that the enzymatic and non-enzymatic antioxidant defense systems are weakened in rabbit hepatic tissue after 60 min of renal ischemia and 60 min of reperfusion, possibly because of an increase of ROS. Although the underlying mechanisms are not fully understood, DHEA pretreatment appears to have beneficial effects on antioxidant defenses against hepatic injury after renal IR in rabbit, possibly by augmenting GSH levels and lowering MDA production.

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