Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata

Neurophysiological Studies on the Effect of Acetone on Pentylenetetrazole-Induced Seizure in Rats

Ahmed M. Shehata Department of Physiology- National Organization for Drug Control and Research

ABSTRACT

Recent interest in the anticonvulsant effects of acetone has stemmed from studies related to the ketogenic diet (KD). Despite knowledge of acetone's anticonvulsant properties, the neurochemical basis for this effect is not well known. The present study aimed to explore the neurochemical basis underlying the anticonvulsant effect of acetone in pentylenetetrazol (PTZ) - induced convulsions. This was achieved through determining the neurochemical changes of acetone in pentylenetetrazol (PTZ) – treated rats. Male adult rats received either saline, acetone (15 m mol/kg i.p.), PTZ (60 mg/kg, i.p.), or acetone 3 h before PTZ injection. Result showed that the maximum concentration of acetone reached about 3 h after acetone administration. Thus, the animals were administered pentylentetrazole three hours after acetone treatment. Pentylenetetrazole treated rats exhibited epilepsy, increased brain levels of excitatory amino acids (glutamate and aspartic acids) and decreased levels of inhibitory amino acid (γ-aminobutyric acid and glycine). In addition, pentylenetetrazole treatment decreased total antioxidant activity and reduced glutathione (GSH) in brain. Acetone pretreatment remarkably decreased seizure incidence rate and increased seizure latency. Moreover, acetone significantly minimized the disturbing effect of pentylenetetrazole on the redox status and the balance between excitatory and inhibitory amino acids. The study indicated that the epilepsy might be mediated, at least partially, through the disturbance in the redox status and imbalance between excitatory and inhibitory amino acids in the brain. Moreover, the study indicated that the antiepileptic effect of acetone might be due to its antioxidant effect and sustaining the balance between excitatory and inhibitory amino acids. Moreover, the study might recommend the concurrent intake of ketogenic diets with the conventional antiepileptic drugs. In addition, synthesizing new chemical entities yielding acetone during its metabolism in the body might provide new candidates as antiepileptic drugs.

INTRODUCTION awaiting safer drugs with improved antiepileptic effectiveness. Moreover, Epilepsy is considered one of the combinations of conventional most common neurological disorders antiepileptic drugs (AEDs) might fail (2) worldwide, with a prevalence of 0.5- to effectively control seizures . 1% in the general population(1). Consistently, about 30% of epileptic Epilepsy continues to be a disease patients do not respond to clinically

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Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata established AEDs(3). The the brain with a ketogenic diet neurobiological relationships between intake(16). The ketone bodies, b- epilepsy and affective disorders are hydroxybutylate, acetoacetate and receiving increased attention(4,5). acetone are significantly increased in Epilepsy is a neurological disorder the plasma of patients receiving a characterized by spontaneous, ketogenic diet(17). Among these three recurrent and paroxysmal cerebral ketone bodies, acetoacetate and discharge leading to persistent acetone easily pass through the blood alterations in function and brain barrier, and partly replace morphology of neurons(6). The burst glucose as fuel in the brain(18), leading firing associated with prolonged to the changes in energy metabolism epileptic discharges could lead to a in the brain(14,18). These changes might large number of changes and cascades be related to the anticonvulsive effect of events at the cellular level. These associated with a ketogenic diet in effects include activation of glutamate animal models of neurodegenerative receptors, changes in composition of diseases. Acetone is the principal glutamate and γ-aminobutyric acid ketone body elevated in the ketogenic (GABA) receptors, cytokine diet (KD), with demonstrated robust activation and oxidative stress, anticonvulsant properties across a modulation in neurogenesis and variety of seizure tests and models of neuroplasticity or activation of some epilepsy(19). In addition, the late cell death pathways(1,7,8). anticonvulsant effects of acetone have Consistently, N-methyl-D-aspartate been reported in various animal (NMDA) receptor antagonists were models of epilepsy (19, 20). shown to possess anticonvulsant The present study was conducted properties against several insults to explore the neurochemical basis including pentylenetetrazole (PTZ)- underlying the antiepileptic effect of induced seizures and to enhance the acetone in rats. This was achieved effects of AEDs (9,10). through determining the effect of Despite progress in current acetone on the brain content of the antiepileptic therapy, neither are excitatory (glutamate and aspartic) seizures adequately controlled nor and inhibitory (GABA and glycine) medications free of untoward side amino acids and the redox status (total effects(11). On the other hand, a antioxidant activity and reduced ketogenic diet has been used glutathione). successfully to treat patients with (12) intractable epilepsy . In addition, MATERILAS & METHODS many studies on the anticonvulsant effects of a ketogenic diet have been Experimental animals: male adult performed, however, the mechanism Sprague Dawley rats (150-200 g) (13,14) remains unknown . Several were kindly provided from our hypotheses have been proposed to breeding center at NODCAR and kept explain the anticonvulsant effects of a for a week for acclimatization under (15) ketogenic diet. , such as the normal conditions and constant alterations in energy metabolism in temperature (25±1C°) with ad libitum

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Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata water and food until starting the euthanized by decapitation after experiment. seizures assessment; brains were Chemicals: All chemicals, unless removed and dissected bilaterally. specified other-wise, were purchased One brain half was homogenized in form Sigma-Aldrich Chemical Co. 75% (v/v) aqueous methanol (HPLC (St. Louis, MO). grade, Sigma-Aldrich, MO, USA), Drug: Pentylenetetrazole was homogenates were centrifuged at purchased from Sigma-Aldrich 4000×g, 20 min, 4 °C, and Chemical Co. (St. Louis, MO). supernatants were employed for the A total number of forty rats with determination of brain amino acids an average weight of 175g, were contents. The second brain half was administered an i.p. dose of acetone homogenized in ice cold saline and (15 mmol/kg) and blood samples were was used for the estimation of brain taken at time intervals 1, 2, 3, 5 and redox status (total antioxidant activity 10 h to asses the time corresponding and reduced glutathione). to the highest concentration of Methods: acetone. The rats were decapitated and Level of acetone in plasma was trunk blood was collected in 1 ml determined by HPLC method heparinized tubes. The heparinized according to Brega et al.(22). Free samples were centrifuged at 3,000 amino acids were determined using rpm for 20 min at 4°C. Plasma was precolumn derivatization HPLC-UV aspirated from the centrifuged method(23). Total antioxidant activity specimen using a transfer pipette. was determined using the Plasma samples were used to colourimetric method of Blois(24). determine acetone. Reduced glutathione was determined In addition, a total number of by HPLC method according to thirty two rats were divided into four Jayatilleke and Shaw (25). groups, group I received saline and Statistical Analysis. served as control. Group II received Data presented as means ± SE. single intraperitoneal injection of PTZ One-way ANOVA followed by LSD in convulsive dose of 60 mg/kg(21). test were used to evaluate significant Group III was injected differences from the control group. P< intraperitoneally with acetone (ACET, 0.05 was considered to be statistically 15 mmol/kg). Group IV was injected significant. Statistical processor intraperitoneally PTZ, 3h following system support (SPSS) for Windows acetone injection. After PTZ software, release 10.0 (SPSS, Inc, injection, rats were placed singly in Chicago, IL) was used. plexiglass cages and were observed for 30 min. Incidences and latency of RESULT clonic convulsive attacks, which lasts over 3 s with an accompanying loss of As shown in Figure 1. the righting reflex were recorded. Seizure maximum concentration of acetone latency for rats showing no convulsive attained after 3h of acetone attacks within the observation period administration to give 800.0 ± 95.5 was taken as 30 min(21). Rats were

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Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata mg/L and gradually declined to give decreased glutamate content. Acetone 100 .8 ± 35 mg/L after 10 h. pretreatment remarkably prevented Table 1 depicts that PTZ- treated the disturbing effect of PTZ on the rats exhibited clonic convulsions with free amino acid levels 3.5-min average seizure latency. (Table 2). Data in tables 3 shows Acetone pretreatment significantly that pentylenetetrazole treatment decreased the convulsions' incidence significantly decreased both total and prolonged seizure latency. antioxidant activity and GSH content Data in table 2 shows that PTZ in rat brain. Acetone treatment significantly (P <0.05) reduced brain significantly (P <0.05) increased both GABA and glycine contents and total antioxidant activity and GSH increased glutamate and aspartate content in rat brain. Acetone contents compared to control group pretreatment significantly minimized (Table 2). Acetone treatment the PTZ- adverse effect on the total significantly increased GABA and antioxidant activity and GSH content.

Fig. 1. Level of Plsam Acetone Following Single Administration of Acetone (15 mmol/kg, i.p) in Rat

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mg/L 400

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0 123610 Hours

Table 1. Effect of Acetone (ACET, 15 mmol/kg, p.i.) on Pentylenetetrazole (PTZ- induced Clonic Seizures in Rats Groups Clonic Seizures Incidence (%) Clonic Seizure Latency (min.) PTZ 100 3.51 ± 0.55 ACET + PTZ 70 15.12 ± 1.38 * Data are expressed as means ±SEM; *P<0.05 significant different from control (n=8)

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Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata

Table 2. Effect of Acetone (ACET, 15 mmol/kg, i.p.), Pentylenetetrazole (PTZ, 60 mg/kg, p.i.) alone or in combination on Levels of Amino Acids Levels (μmol/g tissue) in Rat Brain Brain Amino Acids Contents (μmol/g Tissue) Groups Aspartic acid Glutamic acid GABA Glycine CONTOL 5.52 ± 0.37 6.95 ± 0.24 3.40 ± 0.14 2.17 ± 0.08 PTZ 6.37 ± 0.14 * 8.20 ± 0.16 * 2.20 ± 0.10 * 1.83 ± 0.04 * ACET 4.95 ± 0.28 + 6.25 ± 0.32 *, + 3.88 ± 0.10 *, + 2.33 ± 0.09 + ACET + PTZ 5.32 ± 0.15 + 7.35 ± 0.16 + 3.10 ± 0.09 + 2.28 ± 0.05 + Data are expressed as means ±SEM; P<0.05 significant different, (n=8) * Significant different from control + significant different from PTZ group

Table 3. Effect of Acetone (ACET, 15 mmol/kg, i.p.), Pentylenetetrazole (PTZ, 60 mg/kg, p.i.) alone or in combination on Total Antioxidant Activity and Content of Reduced Glutathione in Rat Brain Group Total antioxidant Reduced glutathione Control 57.39 ± 1.04 1.65 ± 0.07 PTZ 32.85 ± 1.88 * 1.29 ± 0.04 * ACET 54.78 ± 2.45 + 1.63 ± 0.04 + ACET+ PTZ 59.71 ± 2.74 + 1.59 ± 0.10 + Data are expressed as means ±SEM; *P< 0.05 significant different, (n=8) * Significant different from control + Significant different from PTZ group

DISCUSSION In addition, the convulsive effect of PTZ might be due to the activation The present data showed that of the excitatory neurotransmission acetone required from 2-3 h to reach through the excitatory amino acids the maximum concentration in the (aspartic and glutamic) and/ or by blood. It is important to note that the suppressing the inhibitory time required for the drug to reach the neurotransmission by inhibitory maximum level in the blood is amino acids (GABA and glycine). In dependent on the route of the present study, the increase in administration and the metabolic glutamate and the decrease in GABA status which determines the rate of contents might be due to the inhibition drug's biotransformation and of the enzymatic activity of GABA clearance. Consistently, a previous synthesizing enzyme glutamic acid study indicated that acetone takes decarboxylase (GAD) and/or the about 2 hours to reach the maximum decrease in GAD protein levels. level after intravenous Consistently, a recent study reported administration(26). that antibodies to GAD are found at

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Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata high levels with low GABA fluid GABA content and increased concentration in a subgroup of brain synaptosomal GABA patients with chronic epilepsy(27), content(16,32). A recent study of 5- indicating a role for immune-mediated month-old rats of the GAERS (genetic enzyme destruction and GABAergic absence epilepsy rats of Strasbourg) dysfunction. strain showed that ketogenic diet In accordance to this decreased cortical glutamate levels interpretation, a previous study using 13C nuclear magnetic resonance indicated that both excitatory and spectroscopy(33). Moreover, ketogenic inhibitory amino acids are involved in diet has been reported to reduce epilepsy and that glutamatergic aspartate level in brain regions, activation is responsible for the induces alterations in the metabolism persistent down-regulation of of excitatory amino acids, with greater postsynaptic GABA (A) receptors and effects on aspartate than on erosion of synaptic inhibition(28). glutamate(13,16). In the present study, Furthermore, GABAergic neurons the observation that acetone exposed to glutamate in vitro showed pretreatment restored the normal a reduced dendrite growth and altered levels and the balance between the glutamic acid decarboxylase (GAD) excitatory and inhibitory 65- and 67-kDa isoform protein neurotransmission in PTZ treated rats, expression from mouse cortical whereas acetone alone depressed the GABAergic neurons (29,30). Moreover, levels of excitatory amino acids in intraventricular microinjection of normal rats might indicate a NMDA induced behavioral seizures depressive effect of acetone. and motor disorders(31). It is worthy to Moreover, it seems that the note that glutamate and GABA are the antiepileptic effect of acetone is not most abundant neurotransmitters in dependent on its metabolites, because the central nervous system, especially acetone freely crosses blood–brain in the cerebral cortex, the site where barrier, and its concentrations in blood thinking occurs and different and cerebrospinal fluid are similar in sensations are interpreted and rats(34), whereas its metabolites integrated. seemingly can not cross biological Notably, seizure disorders are all membranes (14). related to low GABA activity which In addition, oxidative stress is one regulates the transmission of nerve of possible mechanisms in the impulses from one neuron to another. pathogenesis of epilepsy. It is Thus, with the inhibited GABAergic interpreted that oxidative stress neurotransmission, nerve cells fire too resulting from mitochondrial often and too easily. Hence, it is likely dysfunction gradually disrupts the that the antiepileptic effect of acetone intracellular calcium homeostasis, probably is due to the depressive which modulates neuronal excitability effect of acetone on the excitatory and synaptic transmission making neurotransmission. In accordance, neurons more vulnerable to additional previous studies indicated that stress, and leads to neuronal loss in ketogenic diet increased cerebrospinal epilepsy(6). In addition, the high

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Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata oxidative status is associated with the gm/kg) induced oxidative stress severity and recurrence of epileptic leading to disturbance of the seizure. Hence, treatment with biochemical and physiological antioxidants is critically important in functions(41), which might indicate that epileptic patients through scavenging the beneficial effect of acetone is a the excessive free radicals to protect dose dependent. against the neuronal loss(6,35). The The study concluded that epilepsy observation that acetone pretreatment might be due to the imbalance significantly attenuated the pro- between excitatory and inhibitory oxidant effect of PTZ treatment might neurotransmission and oxidative be attributed to acetone's antioxidant stress. Acetone offered an effect. antiepileptic effect probably due to its In accordance to our result, a restorative effect of the balance previous study indicated that acetone between excitatory and inhibitory was found to be active in animal neurotransmission and its antioxidant models of tonic-clonic seizures, effect. In addition, the study might typical absence seizures, complex encourage the concurrent intake of partial seizures, and atypical absence ketogenic diets with the conventional seizures associated with Lennox- antiepileptic drugs. In addition, the Gastaut syndrome where the study recommends the synthesis of therapeutic indices are either new chemical entities yielding acetone comparable or better than that of during its metabolism in the body to valproate(17). Mitochondrial be antiepileptic drugs candidates. dysfunction and oxidative stress has been suggested to play a role in the REFERENCES acute consequences of injuries that are known to provoke chronic epilepsy 1. Chuang, Y. (2010): and their involvement in the chronic Mitochondrial dysfunction and (1,35) stages of acquired epilepsy . In oxidative stress in seizure- accordance, ketogenic diet has caused induced neuronal cell death. Acta an elevation in glutathione peroxidase Neurol. Taiwan.,19:3-15 (36) activity in rat hippocampus , and 2. Kamiński, R.M., Mazurek, M., increased mitochondrial uncoupling Turski, W.A., Kleinrok, Z. and protein levels and activity – which Czuczwar, S.J. (2001): decreased reactive oxygen species Amlodipine enhances the activity (37) (ROS) production . In addition, of antiepileptic drugs against ketone bodies provided a protective pentylenetetrazole-induced effect against oxidative stress in seizures. Pharmacol. Biochem. neocortical neurons by decreasing Behav., 68 (4):661-668 (38) mitochondrial ROS production , and 3. Theodore, W.H. and Fisher, R. prevented oxidative stress cytotoxicity (2007): Brain stimulation for induced by H2O2 formed by epilepsy. Acta Neurochir. Suppl. (39,40) glucose/glucose oxidase . On the 97, 261–272 other hand, a recent study indicated 4. Dudra-Jastrzêbska, M., that a high dose of acetone (7.0 Andres-Mach, M.M., £uszczki,

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دراسات فسيولوجية عصبية عن تأثير األسيتون في الصرع المستحدث بمادة بنتيلينتترازول في الجرذان

أحمد محمد شحاته قسم الفسيولوجي - الھيئة القومية للرقابة و البحوث الدوائية

نشأ حديثا االھتمام بتأثير االسيتون المضاد للصرع و ذلك من خالل الدراسات الخاصة بالنظام الغذائي الكيتوني. على الرغم من معرفة خصائص األسيتون المضادة للصرع معروفة جيدا اال أن األلية الكيميائية العصبية لھذا التأثيرغير معلومة بصورة كاملة. تھدف الدراسة الى اكتشاف التغيرات الكيميائية العصبية الكامنة وراء تأثير االسيتون المضاد للصرع الناتج من عقار بنتيلينتترازول في ذكور الجرذان البالغة. تم حقن الجرذان بعقار بنتيلينتترازول بتركيز ٦٠ مجم/كجم في التجويف البريتوني. و حقن االسيتون بتركيز ١٥ مللي مول/كجم. أظھرت النتائج أن أقصى تركيز لالسيتون في الدم يكون بعد ثالث ساعات من الحقن. تم ظھور أعراض الصرع في الجرذان بعد ثالث دقائق من حقن عقار بنتيلينتترازول .وتسبب حقن العقارفي زيادة مستوى االحماض االمينية المثيرة Glutamic and) (aspartic acids و نقص في محتوى االحماض االمينية المثبطة (GABA and glycine) و نقص النشاط الكلي المضاد لالجھاد التأكسدي و كذلك نقص محتوي الجلوتاثيون المختزل. أدى الحقن المسبق باالسيتون الي تأخير حدوث نوبات الصرع وتقليل معدل حدوثھا. كما منع االسيتون تأثير عقار بنتيلينتترازول علي مستوى االحماض االمينية المثيرة و المثبطة و منع حدوث االحھاد التأكسدي. تشيرالدراسة الى أن حدوث الصرع قد يكون من خالل نخفاض النشاط المضاد لألكسدة في المخ و انعدام التوازن بين األحماض األمينية المثيرة و المثبطة في المخ. كما تشير الدراسة الى ان تأثير االسيتون المضاد للصرع قد يكون بسبب تأثيره المضادة لألكسدة وتأثير الحافظ للتوازن بين األحماض األمينية المثيرة و المثبطة. وباإلضافة إلى ذلك ، تؤيد الدراسة تناول الوجبات الغذائية الكيتونية مع العقاقير التقليدية المضادة للصرع. وباإلضافة إلى ذلك توصي بتخليق عقاقير تنتج األسيتون من خالل عملية التمثيل الغذائي في الجسم لتكون أدوية مضادة للصرع.

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