Biol Trace Elem Res (2014) 160:409–417 DOI 10.1007/s12011-014-0067-8
Combination of Omega-3 Fatty Acids, Lithium, and Aripiprazole Reduces Oxidative Stress in Brain of Mice with Mania Pandiyan Arunagiri & Krishnamoorthy Rajeshwaran & Janakiraman Shanthakumar & Thangavel Tamilselvan & Elumalai Balamurugan
Received: 15 April 2014 / Accepted: 7 July 2014 / Published online: 18 July 2014 # Springer Science+Business Media New York 2014
Abstract Manic episode in bipolar disorder (BD) was evaluated in the present study with supplementation of omega-3 fatty acids in combination with aripiprazole and lithium on methylphenidate (MPD)-induced manic mice model. Administration of MPD 5 mg/kg bw intraperitoneally (i.p.) caused increase in oxidative stress in mice brain. To retract this effect, supplementation of omega-3 fatty acids 1.5 ml/kg (p.o.), aripiprazole 1.5 mg/kg bw (i.p.), and lithium 50 mg/kg bw (p.o) were given to mice. Omega-3 fatty acids alone and in combination with aripiprazole- and lithium-treated groups significantly reduced the levels of superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation products (thiobarbituric acid reactive substances) in the brain. MPD treatment significantly decreased the reduced glutathione (GSH) level and glutathione peroxidase (GPx) activity, and they were restored by supplementation of omega-3 fatty acids with aripiprazole and lithium. There is no remarkable difference in the effect of creatine kinase (CK) activity between MPDinduced manic model and the treatment groups. Therefore, our results demonstrate that oxidative stress imbalance and mild insignificant CK alterations induced by administration of MPD can be restored back to normal physiological levels through omega-3 fatty acids combined with lithium and aripiprazole that attributes to effective prevention against mania in adult male Swiss albino mice.
Keywords Oxidative stress . Creatine kinase . Mania . Omega-3 fatty acids . Aripiprazole
P. Arunagiri : K. Rajeshwaran : J. Shanthakumar : T. Tamilselvan : E. Balamurugan (*) Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu 608 002, India e-mail:
[email protected]
Introduction Bipolar disorder (BD) is a severe mental disorder, which is associated with elevated suicide rates [1]. Mania is the cardinal feature of BD and traditionally has been assessed using self- and observer-rating scales [2]. Many signs and symptoms related to BD can be replicated in animal models with dopaminergic stimulants, such as amphetamine and cocaine. For instance, amphetamine administration in rats induces hyperlocomotion, insomnia, and enhanced sexual drive [3], which are behavioral alterations that resemble manifestations of human bipolar mania [4]. In recent years, increased oxidative stress has been implicated in the pathogenesis of numerous diseases including cancer, atherosclerosis, schizophrenia, and BD [5, 6]. Martins and his colleagues have demonstrated that methylphenidate (MPD) induces imperative mood and increased oxidative stress in young rats [7]. Increased neuronal oxidative stress levels generate deleterious effects on signal transduction, structural plasticity, and cellular resilience, mostly by inducing lipid peroxidation in membranes, proteins, and genes. Such changes in diverse oxidative stress parameters have been reported in BD and schizophrenia [5, 6]. Oxidative stress caused by reactive oxygen species (ROS) occurs as a consequence of imbalance between the production and inactivation of these species. Important antioxidant enzymes include superoxide dismutase (SOD, EC.1.15.1.1), which catalyzes dismutation of superoxide anion to H2O2, which is then deactivated to H 2 O by catalase (CAT, EC.1.11.1.6) and glutathione peroxidase (GPx, EC.1.11.1.9). Thiobarbituric acid reactive substance (TBARS) levels are considered a direct index of cell lipid peroxidation and it was ameliorated by coordinated effects of primary antioxidant system including SOD, CAT, and GPx. GPx is a selenium (Se) ion-containing enzyme that is responsible for the reduction of hydro and organic peroxides in the presence of reduced glutathione (GSH) [8]. Lithium and aripiprazole has been well
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demonstrated for decreasing the oxidative stress involving various diverse mechanisms, as it is well documented with animal model and clinical studies in BD [9, 10]. Some side effects of antipsychotics limit their long-term use and are probably associated with oxidative stress and/or energy impairment [11]. Previous reports showed that lipid peroxidation was diminished by olanzapine and aripiprazole, but not haloperidol and clozapine administration in the prefrontal cortex. On the other hand, haloperidol and clozapine, but not olanzapine, ultimately cause oxidative stress [12]. Also, Eren and his colleagues reported the beneficial effects of lamotrigine, aripiprazole, and escitalopram and clearly reviewed antiepileptic drugs on oxidative stress molecular pathways [13, 14]. The brain and other high-energy tissues are more prone to stress in energy metabolism. In this context, neuropsychiatry disorders, such as schizophrenia and BD, have been related to dysfunction in brain metabolism. The metabolism dysfunction includes mitochondrial impairment [15], increase in ROS production, and expression of biochemical markers of cellular degeneration [9]. Creatine kinase (CK, EC 2.7.3.2) catalyzes the reversible transfer of the phosphoryl group from phosphocreatine to adenosine diphosphate (ADP), regenerating adenosine triphosphate (ATP). It is also known that a decrease in CK activity is associated with neurodegenerative pathways that results in neurodegenerative diseases [16], BD, and other pathological states [17]. Omega-3 fatty acids are essential for the physiological function of neuronal cell membrane. Normal function of neuronal cell membrane requires appropriate composition of fatty acids in its structure. Decreased omega-3 fatty acid intake and increased oxidative stress could contribute to brain docosahexaenoic acid (DHA) depletion and low blood levels in Alzheimer’s disease (AD) patients [18]. Omega-3 fatty acid supplementation becomes especially important because the majority of diets contain a great quantity of omega-6 and insufficient omega-3 fatty acids. Several epidemiological studies show a protective effect associated with increased fish consumption on dementia and cognitive performance [19]. Recent reports from our laboratory have shown that omega-3 fatty acid supplementation with aripiprazole and lithium in MPD-induced manic model resulted in significant changes in the behavioral activities test evaluated by forced swim test (FST), actophotometer test, and open field test (OFT) [20]. However, as far as the reports are concerned, there are no data regarding the effect of omega-3 fatty acids after MPD administration related to the CK activity, antioxidant system, and lipid peroxidation in the brain. The current study was performed to determine the activity of antioxidant enzymes including SOD, CAT, GPx, and GSH to assess the scavenging effects on ROS and MDA level as an indicator of oxidative damage or lipid peroxidation in mice brain after acute treatment of MPD. There is also lack of data regarding the use of
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these drugs on brain energy metabolism and oxidative stress impairment in manic model. Taking all these into consideration, in the present study, we had evaluated the effects of omega-3 fatty acid combined with aripiprazole and lithium in MPD-induced manic model by analyzing CK activity, TBARS, SOD, CAT, GPx, and GSH levels in mice brain.
Materials and Methods Animals and Drug Administration The male adult Swiss albino mice (weighed 25–30±5 g) were housed in wire-topped plastic cages, as six animals per cage. Control and experimental mice received a standard diet of rodent chow (12–15 g/day) and water ad libitum. All mice were kept on an alternating 12-h light and 12-h dark cycle. All experiments were performed at the same time every day and in the light period (9:00–11:00 A.M.). All experimental procedures were approved, and all the animals were taken care according to the Institutional Animal Ethical Committee of Rajah Muthiah Medical College and Hospital, Annamalai Nagar, Tamil Nadu, India (Reg No. 160/1999/CPCSEA, Proposal number 933). After 7 days of acclimatization period, the mice were randomly assigned to eight groups consisting of six mice per group. The study group was administered 5 mg/kg/day of MPD intraperitoneally (i.p.), whereas the control group was administered distilled water. The dosage of MPD administration to mice is similar to that of Barbosa et al. [21]. It was procured from Ipca pharmaceutical company; 1.5 ml of 0.1 % fish oil (FO) with a homogenous 1 % Tween suspension [22] contained 120–180 mg eicosapentaennoic acid (EPA)/ docosahexaeonoic acid (DHA), and lithium carbonate (50 mg/kg bw) given orally [23]; aripiprazole (1.5 mg/kg bw) [24] was kindly provided by Sun Pharmaceutical, Karnataka, India, and dissolved in water and administrated intraperitoneally (i.p.). All other chemicals used in this study were of analytical grade obtained from HiMedia Laboratories, Mumbai, India. The dose of lithium, aripiprazole, and omega-3 fatty acids were chosen based on previous literature [21–24], and their combinatorial effects on behavior studies were confirmed [20]. The experimental design of the current study was as follows; each of the following groups consists of six animals. Group I: Control animals Group II: Control+lithium (50 mg/kg bw)+aripiprazole (1.5 mg/kg bw)+omega-3 fatty acids (1.5 ml/ kg bw) Group III: Mania animals (methylphenidate (5 mg/kg b.w)) Group IV: Mania+lithium (50 mg/kg bw)
Omega-3 Fatty Acid with Lithium and Aripiprazole Reduces Mania
Group V: Mania+aripiprazole (1.5 mg/kg bw) Group VI: Mania+omega-3 fatty acids (1.5 ml/kg bw) Group VII: Mania+lithium (50 mg/kg bw)+aripiprazole (1.5 mg/kg bw) Group VIII: Mania+lithium (50 mg/kg bw)+aripiprazole (1.5 mg/kg bw)+omega-3 fatty acids (1.5 ml/ kg bw)
Biochemical Assays Preparation of the Homogenate After the behavioral analysis, animals were sacrificed by decapitation immediately (OFT, actophotometer test, FST) [20]; mice brain tissues were stored at −80 °C until used for the biochemical analysis. The mice brain tissues were thawed, weighed, and then placed in chilled 0.1 mol/l Tris–HCl buffer, pH 7.4. The samples were homogenized using a Potter– Elvehjem homogenizer (Wheaton Science Products, Millville, NJ, USA) filled with Teflon pestle to produce 10 % homogenates and used for determining the biochemical parameters described below.
Estimation of Lipid Peroxidation The level of lipid peroxidation (TBARS) was determined by analyzing TBA-reactive substance according to the method of Niehaus and Samuelsson [25]. The pink-colored chromogen formed by the reaction of 2-TBA with breakdown products of lipid peroxidation was measured spectrophotometrically at 532 nm. The values were expressed as nanomoles per milligram of protein.
Determination of SOD and CAT Activities Superoxide dismutase activity was measured using the method of Kakkar et al. [26] and calculated using the percentage of inhibition of formazan formation. One unit of the enzyme is defined as the amount of enzyme required for 50 % inhibition of NBT reduction per minute per milligram protein. CAT was assayed using the method of Sinha [27]. The reaction mixture contained 0.1 ml of tissue homogenate, 1 ml of phosphate buffer (0.01 mol, pH 7.0), and 0.2 mol H2O2. The reaction was arrested by the addition of a dichromate acetic acid reagent, and the chromic acetate formed was determined spectrophotometrically at 590 nm. The values are expressed as micromoles of H2O2 utilized per minute milligram protein.
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Determination of the Levels/Activities of Glutathione and Glutathione Peroxidase Estimation of reduced glutathione (GSH) in the brain tissue was performed by the method of Ellman [28]. This method is based on the development of a yellow color, when dithionitrobenzoic acid is added to compounds containing sulfhydryl groups. The color developed was read at 412 nm. Glutathione peroxidase (GPx) activity in the heart tissue was assayed by the method of Rotruck et al. [29]. A known amount of enzyme preparation was allowed to react with hydrogen peroxide and GSH for a specified time period. The GSH content remaining after the reaction was measured by Ellman’s reaction. The Activity of Creatine Kinase in Brain Homogenates CK activity was measured in brain homogenates pretreated with 0.625 mmol lauryl maltoside. The reaction mixture consisted of 60 mmol Tris–HCl, pH 7.5, containing 7 mmol phosphocreatine, 9 mmol MgSO4, and approximately 0.4– 1.2 μg protein in a final volume of 100 μl. After 15 min of preincubation at 37 °C, the reaction was started by the addition of 0.3 μM of ADP plus 0.08 μmol of reduced glutathione. The reaction was stopped after 10 min by the addition of 1 μmol of p-hydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes [30]. The color was developed by the addition of 100 μl 2 % αnaphthol and 100 μl 0.05 % diacetyl in a final volume of 1 ml and read spectrophotometrically after 20 min at 540 nm. Results were expressed as units per minute × milligram protein. Protein Determination The protein content of the brain tissue homogenate was estimated using the method of Lowry et al. [31]. About 0.5 ml of brain tissue homogenate was precipitated with 0.5 ml of 10 % TCA and centrifuged for 10 min, and the precipitate was dissolved in 1.0 ml of 0.1 N NaOH. About 0.1 ml of aliquot was taken and made up to 1.0 ml with distilled water. Then, 4.5 ml of alkaline copper reagent was added and allowed to stand at room temperature for 10 min. After incubation, 0.5 ml of Folin–Ciocalteu reagent was added, and the blue color developed was read at 620 nm after 20 min; bovine albumin was used as standard. Statistical Analysis All the values were expressed as mean±SD of six determinations. Statistical analysis of the data was carried out by oneway ANOVA on Statistical Package for Social Sciences (SPSS), and the group mean compared by Duncan’s multiple
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range test (DMRT). A value of P
