Effect of chronic restraint stress and alpha-lipoic acid on lipid peroxidation and antioxidant enzyme activities in rat peripheral organs

Pharmacological Research 54 (2006) 247–252

Effect of chronic restraint stress and alpha-lipoic acid on lipid peroxidation and antioxidant enzyme activities in rat peripheral organs Mehmet S¸ahin a , Gamze Sa˘gdıc¸ a , O˘guz Elmas a , Deniz Akpınar b , Narin Derin b , Mutay Aslan a , Aysel Agar c , Yakup Alıcıg¨uzel a,∗ , Piraye Yargıc¸o˘glu b a

Department of Biochemistry, Akdeniz University Medical School, 07070 Antalya, Turkey Department of Biophysics, Akdeniz University Medical School, 07070 Antalya, Turkey c Department of Physiology, Akdeniz University Medical School, 07070 Antalya, Turkey b

Accepted 12 May 2006

Abstract Objectives: The aim of this study was to evaluate the effect of chronic restraint stress and alpha-lipoic acid (LA) administration on lipid peroxidation and antioxidant enzyme activities in rat peripheral organs. Methods: Forty male wistar rats, aged 3 months were randomized to one of the following groups: control, restraint stress, LA treated and restraint stress + LA treated. Chronic restraint stress was applied for 21 days (1 h/day) and LA (100 mg/kg/day) was administered intraperitoneally for the same period. Results: Restraint stress had no statistically significant effect on lipid peroxidation, copper/zinc superoxide dismutase (Cu/Zn SOD), catalase (CAT) and glutathione peroxidase (GPx) activity in rat liver and heart, when compared to the control group. Lipid peroxidation, determined by measuring malondialdehyde (MDA) levels, was found to be increased in the kidney of restraint stress treated rats, compared to controls. Restraint stress-induced lipid peroxidation in the kidney was significantly decreased via LA treatment. Administration of LA also enhanced GPx and decreased Cu/Zn SOD activity in rat kidney, liver and heart, compared to the control group. Conclusions: The presented data shows that LA is a protective agent against restraint stress—the inducer of lipid peroxidation in the kidney. These findings also suggest that LA-induced changes in antioxidant enzyme activities in rat peripheral organs may contribute to their versatile effects observed in vivo. © 2006 Published by Elsevier Ltd. Keywords: Restraint stress; Lipoic acid; Lipid peroxidation; Antioxidant

1. Introduction Stress, both psychological and physical is common in everyday life and is known to induce circulatory diseases and ulceration of the digestive tract [1]. The term “restraint stress” involves a specific procedure that limits movement. Early restraint stress procedures were validated as “stressful” through studies of adrenocortical response [2], ulcer induction and stress-induced analgesia [3]. Two major types of experimental restraint stress models have been confirmed. The first model includes confinement, where the animal’s movement is limited by restricted space. The second model utilizes immobilization, where the limbs and body of the animal are held immobile by tape or

∗

Corresponding author. Tel.: +90 242 227 43 54; fax: +90 242 227 44 82. E-mail address: [email protected] (Y. Alıcıg¨uzel).

1043-6618/$ – see front matter © 2006 Published by Elsevier Ltd. doi:10.1016/j.phrs.2006.05.007

plaster [4]. Previous studies have shown that restraint stress increases plasma lipid peroxidation and decreases plasma protection against oxidation [5]. Furthermore, administration of reduced glutathione has been shown to be protective against stress-induced ulceration and oxidative damage [5]. In mammals, LA can be supplied by both de novo synthesis or by dietary intake. Alpha-LA is synthesized de novo from octanoic acid by the addition of two sulfur atoms to the octanoyl group. This reaction takes place in the mitochondria and is catalyzed by LA synthase [6]. Endogenously synthesized LA is used for lipoylation of LA-requiring enzyme complexes. Lipoyltransferase catalyzes the transfer of the lipoyl group to a lysine residue of specific enzyme proteins [7]. The lipoyllysine arm is then responsible for shuttling intermediates and reducing equivalents between the active sites of enzyme complexes that serve a critical role in energy metabolism [8].

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Dietary bioavailability studies have shown that 20–40% of LA from an oral dose appears in the plasma [9]. This rapid uptake of LA in the gastrointestinal system is followed by rapid transport into tissues. A major part of LA incorporated into cells is translocated into the mitochondria where LA-requiring enzyme complexes are located [10]. Approximately 98% of radiolabeled LA either fed or administered intraperitoneally to rodents is excreted via glomerular filtration and can be recovered within 24 h following administration [11]. Mitochondrial dihyrolipoamide dehydrogenase and cysolic glutathione reductase enzyme systems are reported to reduce LA to dihydrolipoic acid (DHLA) [12]. Under physiological conditions only the NAD(P)H/NAD(P)+ redox couple has a redox potential higher than that of DHLA/LA [10]. Thus, DHLA can act as a powerful reductant that can interact with a host of compounds including oxidized glutathione and other oxidized antioxidants such as Vitamins C and E [8]. Alpha-lipoic acid does not directly recycle Vitamin E. It might do so via recycling of ascorbate. More likely, the protective effect on Vitamin E is due to scavenging of free radicals. Indeed, several studies have provided evidence for the protective role of LA in a variety of diseases that have been associated with an altered redox state including diabetes [13], ischemia-reperfusion [14] and hypertension [15]. This study aimed to investigate the effect of chronic restraint stress and LA administration on lipid peroxidation and antioxidant enzyme activities in rat peripheral organs. 2. Materials and methods 2.1. Animals All experimental protocols conducted on rats were performed in accordance with the standards established by the Institutional Animal Care and Use Committee at Akdeniz University Medical School. Male albino Wistar rats aged 3 months, weighing 200–300 g were housed in stainless steel cages in groups of five rats per cage and given food and water ad libitum. Animals were maintained at 12 h light–dark cycles and a constant temperature of 23 ± 1 ◦ C at all times. Restraint stress was performed as described previously [16]. Rats were exposed to 1 h of restraint stress daily for 21 days, in which movement was limited by placing the animal in a 25 cm × 7 cm plastic bottle, fixed with plaster tape on the outside. Aeration, maintained via a 1.5 cm hole at one end of the bottle, allowed animals to breath. Rats were randomly divided into 4 groups of 10 animals each: Group 1: control (C); Group 2: rats exposed to restraint stress (RS); Group 3: rats treated with LA; Group 4: rats exposed to restraint stress and treated with LA (RS + LA). All experiments were performed between 9:00 and 12:00 a.m., daily. dl-␣-Lipoic acid (Sigma, St. Louis, MO, USA; 100 mg/kg/ day) was mixed with sterile saline in a dark bottle and l M NaOH was added until the suspension dissolved. The pH was then brought to pH 7.4 with HCl. Alpha-LA solutions were freshly prepared just prior use and administered intraperitoneally for 3 weeks prior to restraint stress, as described previously [17,18]. The effective dosage (100 mg/kg body weight/day) was selected

by us on the basis of the concentration capable of inhibiting lipid peroxidation [17,18]. Rats in the control and restraint stress treated groups received physiologic saline in a similar manner for 21 days. 2.2. Sample preparation At the end of the 21 day experimental period, animals were sacrificed 24 h after the last LA administration and restraint. Rats were anesthetized with diethyl ether and sacrificed by exsanguination via cardiac puncture. Dissected liver, kidney and heart were placed in ice cold potassium phosphate buffer (50 mM, pH 7,4) containing 1 mM EDTA and rapidly homogenized. Lipid peroxidation was measured in homogenized tissue samples stored at −80 ◦ C. For antioxidant enzyme activity measurements, tissue homogenates were centrifuged (10,000 × g, 15 min, 4 ◦ C), and supernatants were stored at −80 ◦ C for further analysis. 2.3. Measurement of lipid peroxidation Levels of MDA were measured using the thiobarbituric acid (TBA) fluorometric assay [19] with 1,1,3,3-teraethoxypropane as a standard. Briefly, 50 ␮l of tissue sample was added to 1 ml distilled water which was then mixed with equal volumes of 29 mM TBA in acetic acid. After 1 h incubation at >95 ◦ C, samples were cooled and 25 ␮l of 5 mM HCl was added. The final reaction mixture was extracted with 3.5 ml of n-butanol and the butanol phase was separated via centrifugation at 1500 × g for 5 min. MDA levels were determined fluorometrically with excitation and emission wavelengths of 532 and 547 nm, respectively. 2.4. Measurement of antioxidant enzyme activities Tissue Cu/Zn SOD (EC 1.15.1.1) activity was assayed spectrophotometrically (Shimadzu UV-1601, Kyoto, Japan) by the indirect inhibition technique of Misra and Fridovich based on the ability of SOD to inhibit the autooxidation of epinephrine to form adrenochrome at alkaline pH [20]. One unit of SOD activity is defined as the amount of SOD required to cause 50% inhibition of the oxidation of the epinephrine. Catalase (EC 1.11.1.6) activity was measured by the spectrophotometric method of Aebi using hydrogen peroxide as a substrate. The method is based on the decomposition of hydrogen peroxide which is indicated by the decrease in absorbance at 240 nm [21]. Catalase activity is reported in katal (k) which is the amount of enzyme that will cause the decomposition of 1 mol of H2 O2 per second. Glutathione peroxidase (EC 1.11.1.9) activity was measured indirectly by the coupled reaction with glutathione reductase. Oxidized glutathione (GSSG), produced upon reduction of t-butyl hydroperoxide by GPx, is recycled to its reduced state by GR and NADPH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm that is measured spectrophotometrically [22]. One unit of GPx activity is defined as the amount of enzyme that will cause the oxidation of 1 ␮mol of NADPH to NADP+ per minute.

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Table 1 Body weight measurements Group

Body weight (g) Day 1

Control Restraint stress (RS) Lipoic acid (LA) RS + LA

268.33 278.18 226.00 219.17

Day 7 ± ± ± ±

20.38 38.42 40.32 9.96

273.33 277.78 225.33 225.83

Day 14 ± ± ± ±

20.66 13.94 47.94 19.29

279.17 276.67 216.67 210.00

Day 21 ± ± ± ±

23.92 15.00 40.65 14.77

281.67 276.00 213.33 235.00

± ± ± ±

25.17 12.25 39.94 10.87

Values are mean ± S.D., n = 10.

2.5. Protein measurements Protein concentrations in all samples were measured spectrophotometrically by Lowry et al. assay [23] with bovine serum albumin as standard. 2.6. Statistical analysis The statistical analysis of the obtained data was performed by Sigma Stat (version 2.03) software for windows. The differences in MDA values and SOD activity among the different groups were analyzed via Kruskal–Wallis one-way analysis of variance on ranks and all pairwise multiple comparisons were performed by Dunn’s Method. The differences in GPx and CAT activities among the different groups were analyzed via oneway analysis of variance and all pairwise multiple comparisons were performed by Tukey Test. The calculated p-values by the Sigma Stat (version 2.03) statistical software program are given as p < 0.05 and p < 0.001. 3. Results and discussion No significant difference could be observed in the body weight change and daily food intake among different experimental rat groups (Tables 1 and 2, respectively). There was also no significant difference between liver and heart MDA levels among the different experimental groups (Fig. 1A and B, respectively). Kidney MDA levels measured in the RS and RS + LA groups were significantly greater (p < 0.05) than those detected in the LA and control group (Fig. 1C). Kidney MDA levels in the RS group were also significantly higher (p < 0.05) compared to the RS + LA group. The obtained data thus demonstrates that restraint stress leads to increased lipid peroxidation in the kidney and has no effect on liver and heart MDA levels. Although this observation is in partial agreement with data reported from similar studies on rats, it is important to note that there are discrepancies regarding lipid peroxidation following Table 2 Food intake values Group

Food intake (g/day/100 g body weight)

Control Restraint stress (RS) Lipoic acid (LA) RS + LA

8.19 8.00 7.82 7.96

Values are mean ± S.D., n = 10.

± ± ± ±

1.17 1.02 0.69 1.72

Fig. 1. Effect of restraint stress and alpha-lipoic acid on MDA levels. * p < 0.05 compared to control and LA. ** p < 0.05 compared to RS + LA. (A) Liver MDA values (mean ± S.E.M., n = 9), control = 0.39 ± 0.05 nmol/g protein; RS = 0.48 ± 0.03 nmol/g protein; LA = 0.47 ± 0.05 nmol/g protein and RS + LA = 0.57 ± 0.09 nmol/g protein. (B) Heart MDA values (mean ± S.E.M., n = 9), control = 1.5 ± 0.3 nmol/g protein; RS = 2.1 ± 0.32 nmol/g protein; LA = 1.71 ± 0.09 nmol/g protein and RS + LA = 1.98 ± 0.1 nmol/g protein. (C) Kidney MDA values (mean ± S.E.M., n = 9), control = 2.22 ± 0.04 nmol/g protein; RS = 8.68 ± 0.62 nmol/g protein; LA = 2.31 ± 0.2 nmol/g protein and RS + LA = 5.66 ± 0.24 nmol/g protein.

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restraint stress. In two studies, 8-week-old male Fischer rats [24] and male Wistar rats [25] were immobilized with stainless wire gauze for 6 h and blood samples were collected after cessation of stress. A significant increase in plasma lipid peroxidation was observed during and after stress [24,25]. In a separate study, male Wistar rats were exposed to 1 h of restraint stress twice daily for 14 days. This study showed that restraint stress had minimal effect on lipid peroxidation in liver, heart and kidney [26]. In another study, 7 week male Sprague-Dawley rats were immobilized by taping all four limbs for 8 h. Upon completion of immobilization, animals were sacrificed and plasma, liver, kidney samples were obtained. Immobilization stress-induced a significant increase in plasma and liver lipid peroxidation but had no effect on kidney MDA levels [27]. These differences in aforementioned data may be due to both the model and duration of restraint stress. Reactive oxygen species generated in the kidneys is closely linked to renal partial oxygen pressure [28]. Studies have shown that renal oxygen usage normally increases linearly with tubular sodium absorption above a basal level [29,30]. With reference to these studies, it is important to note that a significant amount of sodium and water retention occurs in male Wistar rats subjected to immobilization stress [31]. Thus, a likely increase in sodium absorption occurring in rats exposed to restraint stress can cause increased renal oxygen consumption and reactive oxygen species generation leading to enhanced lipid peroxidation in the kidneys. Previous studies have evaluated the protective role of LA treatment in diabetes- and gentamicin-induced lipid peroxidation [32,33], however this is the first study to assess the effect of LA on lipid peroxidation and antioxidant enzymes in restraint stress. As shown in Fig. 1C, restraint stress-induced lipid peroxidation in the kidney was significantly decreased via LA treatment. This finding is in agreement with previous studies which have shown that the administration of LA decreases the oxidation of lipids [32–34]. The protective effect of LA against lipid peroxidation was examined in microsomal fractions obtained from normal and Vitamin E-deficient animals and it was shown that DHLAmediated protection against lipid peroxidation was dependent on Vitamin E [35]. The rapid loss of Vitamin E during lipid peroxidation can be diminished by ascorbic acid (Vitamin C) which is known to recycle the Vitamin E radical. Knowing that LA supplementation can increase Vitamin C levels and metabolism [36], one may predict that the protective effect of LA against lipid peroxidation occurs through increased tissue levels of ascorbate. Liver GPx activity was found to be significantly higher (p < 0.001) in LA treated rats and in rats treated with RS + LA when compared to RS treated and control rats (Fig. 2A). Similarly, heart GPx activity detected in LA treated rats and in rats treated with RS + LA were significantly higher (p < 0.001) than those detected in rats treated with RS and controls (Fig. 2B). Alpha-LA treatment also caused a significant increase in kidney GPx activity, compared to RS and control groups (Fig. 2C) (Table 3). Previous studies have shown that intraperitoneal administration of LA for 11 days (5–16 mg/kg/day) leads to increased liver

Fig. 2. Effect of restraint stress and alpha-lipoic acid on GPx activity. * p < 0.001 compared to control and RS. (A) Liver GPx activity (mean ± S.D.; n = 9), control = 27.99 ± 2.04 U/g protein; RS = 25.26 ± 2.85 U/g protein; LA = 40.08 ± 3.87 U/g protein and RS + LA = 39.52 ± 5.12 U/g protein. (B) Heart GPx activity (mean ± S.D.; n = 9), control = 50.28 ± 5.77 U/g protein; RS = 53.02 ± 7.42 U/g protein; LA = 74.64 ± 2.51 U/g protein and RS + LA = 80.09 ± 7.47 U/g protein. (C) Kidney GPx activity (mean ± SD; n = 9), control = 17.1 ± 6.8 U/g protein; RS = 20.8 ± 2.7 U/g protein; LA = 58.3 ± 6.6 U/g protein and RS + LA = 65.0 ± 10.0 U/g protein.

and kidney glutathione (GSH) levels in rats [37]. The redox potential of GSSG/GSH (−0.24 V) and LA/DHLA (−0.32 V) allows the reduction of oxidized glutathione (GSSG) by dihydrolipoic acid [8], forming GSH. Alpha-LA/DHLA complex also reduces the active site of GPx in the presence of hydrogen peroxide [37], leading to increased enzyme activity as observed in the liver, heart and kidney (Fig. 2) of experimental rat models. Liver Cu/Zn SOD activity was found to be significantly lower (p < 0.05) in LA treated rats and in rats treated with RS + LA when compared to RS treated and control rats (Fig. 3A). Sim-

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Table 3 Effect of restraint stress and alpha-lipoic acid on CAT activity Measurement CAT (k/g protein)

Liver Heart Kidney

Enzyme activity Control (9)

RS (9)

LA (9)

RS + LA (9)

30.6 ± 8.8 9.7 ± 3.8 32.8 ± 7.1

36.9 ± 5.3 7.2 ± 2.7 30.4 ± 3.6

33.3 ± 10.2 12.9 ± 3.2 34.6 ± 6.5

34 ± 9.5 13.2 ± 3.7 36 ± 7

Values are mean ± S.D. n for each measurement is italicized in parentheses.

Fig. 3. Effect of restraint stress and alpha-lipoic acid on SOD activity. * p < 0.05 compared to control and RS. (A) Liver SOD activity (mean ± S.E.M.; n = 9), control = 11.35 ± 1.5 U/mg protein; RS = 13.08 ± 1.86 U/mg protein; LA = 7.69 ± 1.37 U/mg protein and RS + LA = 7.05 ± 1.15 U/mg protein. (B) Heart SOD activity (mean ± S.E.M.; n = 9), control = 22.10 ± 3.73 U/mg protein; RS = 21.18 ± 1.58 U/mg protein; LA = 3.08 ± 0.44 U/mg protein and RS + LA = 3.56 ± 0.31 U/mg protein. (C) Kidney SOD activity (mean ± S.E.M.; n = 9), control = 27.90 ± 2.32 U/mg protein; RS = 23.93 ± 0.44 U/mg protein; LA = 2.9 ± 0.3 U/mg protein and RS + LA = 5.62 ± 0.4 U/mg protein.

ilarly, heart Cu/Zn SOD levels detected in LA treated rats and in rats treated with RS + LA were significantly lower (p < 0.05) than those detected in rats treated with RS and controls (Fig. 3B). Alpha-LA treatment also caused a significant decrease in kidney Cu/Zn SOD levels compared to RS and control groups (Fig. 3C). In addition to their radical scavenging properties, LA and DHLA can chelate a number of metal ions including Cu2+ [38]. In fact, chelation of Cu2+ by LA is reported to inhibit Cu2+ catalyzed oxidations [39] and reduce copper-induced neurotoxicity in cell cultures [40]. The observed decrease in Cu/Zn SOD activity (Fig. 3) in rat tissues following LA treatment may be the result of a similar complex formation between LA and the active Cu2+ metal in the enzyme. In view of the observed decrease in Cu/Zn SOD activity, it is also important to mention that DHLA at concentrations of 0.01–0.1 mM has been reported to increase superoxide anion levels in rat liver mitochondrial particles [8]. Restraint stress did not alter tissue GPx, Cu/Zn SOD and CAT activities (Figs. 2 and 3 and Table 1, respectively). This observation is in accordance with a previous study which has shown minimal effect of restraint stress on antioxidant enzyme activities in heart, liver and kidney [26]. In the reported study, male Wistar rats were exposed to 1 h of restraint stress twice daily for 14 days. In a similar study, 6-week-old male Wistar rats were immobilized with stainless wire gauze for 6 h and mRNA expression of antioxidant enzymes were evaluated immediately after, then 24 and 48 h after cessation of immobilization. Expression of Cu/Zn SOD, GPx and CAT mRNA were examined in the liver, heart and kidney after immobilization stress. Immediately after 6 h of immobilization, mRNA levels of antioxidant enzymes were significantly decreased in the liver compared to baseline levels. The mRNA expression of liver antioxidant enzymes recovered to baseline levels 24 h after cessation of stress. Restraint stress did not alter mRNA levels of antioxidant enzymes in the heart, and kidney [41]. This study is novel for the reason that it examines the effect of LA on lipid peroxidation and antioxidant enzymes in restraint stress. Restraint stress is a model that is developed for biomedical studies primarily in connection with psychogenic stress, wherein a stress response is initiated in the brain in response to the psychological distress of being unable to move freely. Data herein shows that LA is protective against restraint stressinduced lipid peroxidation in the kidney. However LA-induced changes in antioxidant enzyme activities, such as increased GPx and decreased Cu/Zn SOD, in rat peripheral tissues may contribute to their versatile effects observed in different organ systems. That is, the beneficial or prooxidant effect of LA administration depends not only on the model conditions associated

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with oxidative stress but also on the tissue that is targeted for protection. [20]

Acknowledgement [21]

This study was supported by a grant from Akdeniz University Research Foundation, Turkey (2002.01.0122.005). [22]

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