Author for correspondence: Mustafa Nazıroǧlu, Department of Biophysics, Medical Faculty, Süleyman Demirel University, Postakutusu 68 Cunur, TR-32260 Isparta, Turkey (fax +90 246 2371165, e-mail firstname.lastname@example.org).
Abstract: Previous studies have shown that generation of free radicals is increased following pentylenetetrazol kindling, due to increased cytosolic Ca2+ concentrations. Topiramate, a voltage-gated calcium channel inhibitor, has an evident effect in the treatment of childhood epilepsy; however, topiramate may cause nephrotoxicity. We investigated the effects of topiramate and vitamin E administration on pentylenetetrazol-induced nephrotoxicity in rats by evaluation of lipid peroxidation, nitric oxide, glutathione peroxidase, catalase and superoxide dismutase values. Forty male Wistar rats were randomly divided into five equal groups. Group 1 was used as control and group II received a single dose of pentylenetetrazol. Fifty and 100 mg/kg topiramate daily were intragastrically administered to rats in groups III and IV for 7 days, respectively. Intragastric 100 mg topiramate (daily for 7 days) and intraperitoneal vitamin E (150 mg/kg, daily for 3 days) combination were given to animals in group V before a single-dose pentylenetetrazol administration. Serum and kidney samples were taken after 3 hr of pentylenetetrazol administration. Pentylenetetrazol resulted in a significant increase in nitric oxide levels of serum and kidney, and lipid peroxidation levels of kidney although superoxide dismutase and catalase activities in the kidney was reduced by pentylenetetrazol administration. The lipid peroxidation levels in serum and kidneys and the nitric oxide levels in kidneys of groups III, IV and V were decreased by topiramate although the superoxide dismutase and catalase activities in the kidneys were increased. Lipid peroxidation and nitric oxide levels were reduced by the topiramate and vitamin E combination compared to only topiramate. Glutathione peroxidase activity was not affect by pentylenetetrazol, topiramate and vitamin E administrations. In conclusion, topiramate and vitamin E have protective effects on pentylenetetrazol-induced nephrotoxicity by inhibition of free radicals and by support of the antioxidant redox system.
Reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide and singlet oxygen, act as subcellular messengers in such complex processes as mitogenic signal transduction, gene expression and regulation of cell proliferation when they are generated excessively or when enzymatic and non-enzymatic defence systems are impaired [1–4]. For several years, pharmacological investigations of exogenous compounds or therapeutic agents have focused on a possible interaction with ROS [4,5] in order to assess their capacity to prevent or minimize free radical damages to biological targets. There is also evidence that ROS play an important role in the pathogenesis of many diseases, particularly in kidney diseases due to the vulnerability of the kidneys to oxidative stress [3,5]. The major intracellular antioxidant enzymes, glutathione peroxidase (GPx) and catalase (CAT) detoxify hydrogen peroxide to water, although the major fat-soluble antioxidant vitamin E (α-tocopherol) has an inhibitor role on lipid hydroxyl radicals. Therefore, ROS can be indirectly evaluated by measurement of antioxidants such as GPx and CAT [1,5].
Epilepsy is one of the most common neurological disorders. There is emerging focus on the role of oxidative stress and mitochondrial dysfunction both a consequence and a cause of epileptic seizures . Oxidative stress has been shown in several rodent models of experimental epilepsy such as amygdale kindling model  and the pentylenetetrazol model . Administration of the chemical convulsant leads to a decrease in GABAergic function and to stimulation and modification of density or sensitivity of different glutamate receptor subtypes . Increased activity of glutamatergic systems induced by status epilepticus causes energy impairment and enhanced formation of ROS as described for kainite-evoked seizures.
Topiramate is a new antiepileptic drug that inhibits voltage-gated sodium and calcium channels, blocks glutamate amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainite receptors and increases the GABAA receptor-mediated chloride enhances . Increasing body of evidence indicates that topiramate possesses not only antiepileptic but also neuroprotective properties due to its multiple mechanisms of action. It has been reported, however, that it may cause adverse effects such as renal toxicity [11,12] although there is no information of the effect of oxidative stress on renal toxicity. Reports on the effects of topiramate in relation to oxidative stress and nitric oxide are present in some tissues and cells except kidney although they are also controversial. For examples, Cardile et al.  and Pavone et al.  reported that topiramate increased oxidative stress in astrocytes as measured malondialdehyde although Kubera et al.  have recently reported that topiramate plays an antioxidant role and attenuates lipid peroxidation levels in piriform cortex of rats.
There are no studies on topiramate changes on antioxidant enzyme activities, lipid oxidation and nitric oxide levels in the kidneys. Exposure of mitochondria to high cytosolic free Ca2+ was shown to increase formation of ROS . It has been reported that topiramate  in pentylenetetrazol-induced epileptic cells of mice and vitamin E in hippocampal slice cultures  modulated cytosolic Ca2+ levels by regulation of voltage-gated calcium channels (VGCC). Modulation of VGCC in kidneys due to treatment with topiramate or without vitamin E may cause a decrease in mitochondrial ROS and nitric oxide productions. Hence, we aimed at evaluating whether there is a protective effect of topiramate and vitamin E on oxidative stress, enzymatic antioxidants and nitric oxide status in pentylenetetrazol-induced kidney toxicity in rats.
Materials and Methods
Animals. Forty male Wistar albino rats, weighing 200 ± 20 g, were used for the experimental procedures. They were allowed 1 week to acclimate to the surroundings before beginning any experimentation. They were housed in individual plastic cages with bedding, and standard rat food and tap water were available ad libitum for the duration of the experiments unless otherwise noted. The temperature was 22 ± 2°. A 12-hr light:dark cycle was maintained, with lights on at 6 a.m., unless otherwise noted. The animals were handled in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Suleyman Demirel University.
Experimental design. Forty animals were randomly divided into five groups as fellows:
• Group I: placebos were given to the first group.
• Group II: pentylenetetrazol (60 mg/kg body weight) was administrated orally to rats for induction of epilepsy.
• Group III: topiramate (50 mg/kg/day) was administered orally (via gastric gavage) for 7 consecutive days before pentylenetetrazol administration.
• Group IV: topiramate (100 mg/kg/day) was administered (via gastric gavage) for 7 consecutive days before pentylenetetrazol administration.
• Group V: vitamin E + topiramate + pentylenetetrazol administration (n = 15). Topiramate (50 mg/kg/day) was orally administered (via gastric gavage) for 7 consecutive days before pentylenetetrazol administration. The group also received 150 mg/kg body weight (intraperitoneally) vitamin E (DL-α-tocopherol acetate) for three times and 150 mg/kg before pentylenetetrazol administration.
Epilepsy was induced in groups II, III, IV and V by administration of pentylenetetrazol (60 mg/kg). Induction of epilepsy was clinically monitored by presence of epilepsy symptoms and electroencephalography records. After 3 hr of pentylenetetrazol administration, all rats were killed and kidney samples were taken.
Anaesthesia and tissue and blood sampling. Rats were anaesthetized with a cocktail of ketamine (50 mg/kg) and xylazine (5 mg/kg) administered intraperitoneally before killing of each rats and removal of left kidney and blood samples. Blood (4–6 ml) was taken from the heart with a sterile injector and placed in tubes, protected against light. Serum samples were obtained from the blood by centrifugation (5 min. at 3000 ×g). The serum samples were used for immediate nitric oxide measurement.
Kidney tissues were washed twice with cold saline solution, placed in glass bottles, labeled and stored in a deep freeze (−30°) until processing (maximum 10 hr). After weighing, the kidneys were placed on ice, cut into small pieces with scissors, and homogenized (2 min. at 3000 ×g) in five volumes (1:5, w/v) of ice-cold Tris-HCl buffer (50 mM, pH 7.4), using a glass-Teflon homogenizer (Çalişkan Cam Teknik, Ankara, Turkey). All preparation procedures were performed at 4°. After addition of butylhydroxytoluol (4 µl per ml), the kidney homogenate samples were used for immediate nitric oxide, lipid peroxidation, GPx, CAT and superoxide dismutase (SOD) measurement.
Lipid peroxidation level determinations. Lipid peroxidation levels in the kidney homogenate were measured with the thiobarbituric acid reaction by the method of Draper and Hadley . The quantification of thiobarbituric acid-reactive substances was determined by comparing the absorption to the standard curve of malondialdehyde equivalents generated by acid-catalysed hydrolysis of 1,1,3,3-tetramethoxypropane. The values of lipid peroxidation in the kidneys were expressed as µmol/g protein. Although the method is not specific for lipid peroxidation, measurement of the thiobarbituric acid reaction is an easy and reliable method, which is used as an indicator of lipid peroxidation and ROS activity in biological samples.
Nitric oxide determination. The nitric oxide content of the serum and kidneys was measured in ELISA (ELx808 Absorbance Microplate Reader, Bio-Tek Instrument Inc., Winooski, VT, USA) by using commercial kits (nitrate/nitrite colorimetric assay kit, Cayman Chemical Inc., cat. no. 780001, Ann Arbor, MI, USA).
Superoxide dismutase activity assay. Total SOD activity was determined in kidney homogenate according to Woolliams et al. . The measurement of SOD was based on the principle in which xanthine reacts with xanthine oxidase to generate superoxide radicals that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazon dye. The SOD activity is then measured by the degree of inhibition of this reaction. SOD activity was expressed as units per mg protein.
Catalase activity assay. Catalase activity was assayed in kidney homogenate by the Aebi method . The principle of the assay is based on the determination of the rate constant (s−1, k) for H2O2 decomposition at 240 nm by the spectrophotometer. Results were expressed as unit per mg protein.
Assay of glutathione peroxidase activity. Glutathione peroxidase activity was measured in the kidney homogenate by the method of Paglia and Valentine . The principle of the method is as follows: GPx catalyses the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance of NADPH was measured by a spectrophotometer at 340 nm. The activity was expressed as units per mg protein.
Protein assay. The protein content in the kidney was measured by method of Lowry et al.  with bovine serum albumin as the standard.
Statistical analysis. Statistical analysis was performed by using SPSS statistical package (version 9.0, SPSS Inc., Chicago, IL, USA) for Windows. Mann–Whitney U test was used to determine the differences between the groups. The level of statistical difference at P < 0.05 was considered as significant.
The mean nitric oxide and pentylenetetrazol values in the kidneys in the five groups are shown in fig. 1 and serum nitric oxide levels are shown in table 1. The results show that the kidney nitric oxide, lipid proxidation (P < 0.05) and serum nitric oxide levels (P < 0.01) in the pentylenetetrazol group were significantly higher than in the control group. Both doses of topiramate decreased the nitric oxide and lipid peroxidation levels of the kidneys and the nitric oxide levels of serum (P < 0.05–P < 0.01) compared to the control group. Fifty milligrams topiramate plus vitamin E caused more reduction in nitric oxide and pentylenetetrazol levels than in 100 mg topiramate because the statistical difference (P < 0.001) in 50 mg topiramate plus vitamin E group in kidney lipid peroxidation levels was more significant than in the sole 50 mg topiramate (P < 0.01). One hundred milligrams topiramate did not cause more reduction in nitric oxide and lipid peroxidation levels compared to 50 mg topiramate. Hence, there was no statistical difference on nitric oxide and lipid peroxidation levels between 50 and 100 mg topiramate.
Table 1. The effects of topiramate (TPM) and vitamin E on serum nitric oxide levels and kidney superoxide dismutase (SOD), glutathione peroxidaseidase (GPx) and catalase (CAT) activities in pentylentetrazol (PTZ)-induced nephrotoxicity in rats (mean ± S.D., n = 8).
The mean SOD, CAT and GPx activities in the kidneys in the five groups are shown in table 1. The results show that SOD (P < 0.05) and CAT (P < 0.01) activities in the kidneys in the pentylenetetrazol group were significantly lower than in the control group. However, administration of topiramate caused an increase of kidney SOD and CAT enzyme activities in pentylenetetrazol plus 100 and 50 mg topiramate plus vitamin E (P < 0.05–P < 0.001). One hundred milligrams topiramate did not cause more increase in SOD and CAT activities than in the 50 mg topiramate group. Hence, there was no statistical difference on SOD and CAT values between 50 and 100 mg topiramate. However, the statistical difference (P < 0.01) in the 50 mg topiramate plus vitamin E group in kidney SOD activities was more significant than in only 50 mg topiramate (P < 0.05). There was no statistical significance in the GPx activities between the control and pentylenetetrazol-administered groups.
Topiramate (50 mg/kg body weight/day) in human beings has been commonly used for treatment of epilepsy although the 100 mg topiramate group has been also been used. Hence, we wanted to investigate the effect of both doses of topiramate. We found that serum nitric oxide levels, and kidney nitric oxide and lipid peroxidation levels were increased in the kidneys after pentylenetetrazol administration although SOD and CAT activities in kidney decreased. Hence, pentylenetetrazol administration to rats was characterized by increased lipid peroxidation and nitric oxide levels and decreased enzymatic antioxidant activities. Administration of topiramate and vitamin E caused a decrease in serum nitric oxide values and kidney nitric oxide and lipid peroxidation levels although SOD and CAT activities increased. A limited number of in vivo or in vitro studies in tissues except kidneys have been reported regarding the effects of topiramate on antioxidant enzymatic system, lipid peroxidation and nitric oxide levels [15–18]. To the best of our knowledge, the current study is the first to compare the two doses of topiramate and vitamin E with particular reference to its effects on oxidative stress and antioxidant redox system using levels of lipid peroxidation, nitric oxide, SOD and CAT values in pentylenetetrazol-induced nephrotoxicity in rats.
The current study indicates that pentylenetetrazol administration at a convulsive dose of 60 mg/kg produced a significant increase in lipid peroxidation levels of serum and nitric oxide levels of kidney although CAT and SOD activities decreased. Our results are in accordance with the previous reports of pentylenetetrazol and nitric oxide increment in brain, erythrocytes and liver during epileptic seizures [8,15,24]. On the other hand, the current study is the first report regarding the increased lipid peroxidation and nitric oxide in epileptic rat kidneys. We found that pentylenetetrazol-induced epilepsy caused a significant decrease in SOD and CAT activities in the kidneys. However, Erakovic et al.  reported that exposure to a single pentylenetetrazol injection did not change brain SOD activity. They explained the unchanged SOD activity as enzyme degradation-caused oxidative stress and then simultaneous up-regulation of SOD. Similar to our results, Akbas et al.  reported that a single pentylenetetrazol treatment in a convulsive dose of 50 mg/kg reduced the erythrocyte and liver SOD and CAT activities of rats although the lipid peroxidation levels increased. They concluded that increased oxidative stress in the liver of epileptic rats might be due to the activation of the recently found glutamate receptors (mGlu5) in the liver.
Catalase and SOD are the main enzymes of the enzymatic antioxidant defence system, responsible for protection against the increase in ROS production . Hydrogen peroxide formed by the catalytic reaction of SOD is both a reactive form of oxygen and a normal cellular metabolite, and it is further detoxified by GPx and catalase . CAT and SOD activities in kidney were decreased in the pentylenetetrazol group, although their activities in kidney were increased in the topiramate and vitamin E treatment groups. The increased activities of SOD and CAT could be due to their depletion or inhibition as a result of the increased production of free radicals. The increase in kidney CAT and SOD values in animals during topiramate treatments has been attributed to the inhibition of free radicals and lipid peroxidation [15,17].
A large number of studies linked seizure-induced cell damage to excitotoxic mechanisms . Convulsions can results in augmented glutamate release, leading to Ca2+ uptake through N-methyl D-aspartate and VGCC. In fact, during convulsions induced by different means and in different models, extracellular Ca2+ decreases while cytosolic Ca2+ concentration increases . The mitochondria were reported to accumulate Ca2+ provided cytosolic Ca2+ rises exceed 400 nm, or provided mitochondrial uptake dominates mitochondrial Ca2+ extrusion , thereby leading to depolarization of mitochondrial membranes . Uptake of Ca2+ into mitochondria stimulates the tricarboxylate cycle resulting in augmented reduction of pyridine nucleotides, which may be one of the mechanisms of the coupling of neuronal and metabolic activity . On the other hand, exposure of mitochondria to high cytosolic free Ca2+ was shown to increase formation of ROS . It has been reported that topiramate  in pentylenetetrazol-induced epileptic cells of mice and vitamin E in hippocampal slice cultures  modulated cytosolic Ca2+ levels by regulation of VGCC. In the current study, lipid peroxidation and nitric oxide levels in the kidneys were lower in the topiramate groups than in the pentylenetetrazol group. Modulation of VGCC in kidneys by means of topiramate with or without vitamin E might be caused by the decrease in mitochondrial ROS and nitric oxide productions.
In conclusion, our kidney results in the pentylenetetrazol group are consistent with a generalized antioxidant abnormality in different tissues of epileptic animals and human beings. However, 50 and 100 mg/day topiramate with vitamin E supplementation shows protective effect on oxidative stress and antioxidant redox system in kidneys. The beneficial effect of topiramate and vitamin E on enzymatic antioxidant systems was regulated by CAT, SOD, nitric oxide and lipid peroxidation levels in the kidneys. Our results may help physicians when treating oxidative stress-dependent epileptic nephrotoxicity with topiramate and vitamin E.