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The Antipsychotic Effects of Omega-3 Fatty Acids in Rats
BASIC INVESTIGATION
The Antipsychotic Effects of Omega-3 Fatty Acids in Rats Mehmet Hanifi Kokacya, MD, Sema Inanir, MD, Umit Sertan Copoglu, MD, Recep Dokuyucu, MD and Oytun Erbas, MD
Abstract: Background: In humans, omega-3 fatty acids are necessary for cell membranes, brain function and nerve transmission continuation. When animals are exposed to a new environment—or as a result of an apomorphine application that creates an agonistic effect on D1 and D2 receptors—they display behavioral reactions like rearing and stereotypy. This study aims to reveal the possible antipsychotic and oxidative effects of omega-3 fatty acids by comparing with chlorpromazine, a conventional antipsychotic drug, through evaluating the novelty-induced rearing and apomorphine-induced stereotypic behaviors, as well as malondialdehyde and glutathione levels in rats. Methods: Twenty-eight, adult, male, Wistar rats were used in the study. Briefly, 4 groups of rats (n 5 7) were administered docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) (300 mg/kg; DHA: 120 mg/kg + EPA: 180 mg/kg intraperitoneally [IP]), DHA + EPA (150 mg/kg; DHA: 60 mg/kg + EPA: 90 mg/kg IP), chlorpromazine (1 mg/kg, IP) and isotonic saline (1 mL/kg, IP). One hour later, apomorphine (2 mg/kg, subcutaneously) was administered to each rat. After the apomorphine administration, rats were observed for stereotypic behavior. Results: This study shows that omega-3 fatty acids, “similar to antipsychotics,” reversed the psychotic like effects, increase of oxidants and decrease of antioxidants that are composed experimentally in rats. Conclusions: The application of omega-3 fatty acids has antipsychotic effects and causes an oxidative imbalance. This study adds new evidence to the current literature regarding the possible antipsychotic effects of omega-3 fatty acids. Key Indexing Terms: Omega-3 fatty acids; Antipsychotic effect; Oxidative stress. [Am J Med Sci 2015;350(3):212–217.]
A
growing amount of evidence suggests that the main problem in schizophrenia is dopaminergic hyperactivation. Studies suggest that dopamine is effective in therapeutic interventions by inhibiting postsynaptic dopamine receptors.1 Although the disease’s etiology is still unknown, abnormal neuronal maturation, neuronal and glial cell migration, dendritic and axonal branching and truncation, programmed cell death or other reasons, such as stress, trauma, infection and substance use, are thought to be involved in the disease onset.2–5 Oxidative stress results when excessive amounts of oxidants are produced and enzymatic and nonenzymatic antioxidant defense systems are insufficient. The failure of antioxidant mechanisms and membrane metabolism disorders is believed to play a role in neuronal destruction.6 Although oxidative stress is not the main cause of schizophrenia, it has a great impact on the pathophysiology of schizophrenia. Dopamine metabolism is one of the possible sources of oxidative stress forming reactive products.7 Oxidative stress From the Departments of Psychiatry (MHK, USC) and Physiology (RD), Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey; and Departments of Psychiatry (SI) and Physiology (OE), Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey. Submitted September 12, 2014; accepted in revised form June 4, 2015. The authors have no financial or other conflicts of interest to disclose. Correspondence: Recep Dokuyucu, MD, Department of Physiology, Mustafa Kemal University, Hatay 31100, Turkey (E-mail: drecepfatih@ gmail.com).
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resulting from elevated dopamine levels can cause late-onset and permanent central nervous system damage by increasing the striatal glutamatergic neurotransmission.7 It is believed that oxidative stress causes disease as a result of its toxic effects on carbohydrates, proteins, lipids and DNA metabolism.8 The effects of oxidative products can lead to malfunctioning enzymes, neurotransmitters and receptor proteins and may cause a disruption in membrane integrity by reducing the fluidity and permeability of the cell membrane.9 In accordance with the hypothesis of the membrane phospholipids in other psychiatric disorders and neurodegenerative diseases, structural abnormalities of neuronal membrane phospholipids can be an important factor for schizophrenia etiology.10 Studies on lipid peroxidation markers, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), showed that elevated levels of MDA were found in patients with negative symptoms, indicating greater oxidative damage in this patient group.7 Elevated MDA levels have also been found in peripheral tissues of patients with schizophrenia.11–13 Glutathione (GSH) depletion, a nonenzymatic antioxidant component, and GSH-related enzyme deficits are also extensively documented in schizophrenia.14–16 Ballesteros et al17 reported significantly lower GSH and higher glutathione disulfide (GSSG) levels in the blood of schizophrenia individuals. In mammals, omega-3 polyunsaturated fatty acids (n-3 PUFA) are required for cell membranes, brain function and transmission maintenance of nerve impulses. PUFAs are found in the brain and blood cells. Docosahexaenoic acid (DHA), a PUFA, regulates cell transport and synaptic functions.18 The main structural components of membrane phospholipids are PUFAs, which are not de novo—synthesized from fatty acids in the body.10 The presence of PUFAs is known to increase membrane fluidity. Consequently, in case of a PUFA deficiency, membranes become more rigid and result in changes in conformation and function of proteins, receptors and ion channels.19,20 While PUFAs are not antioxidants, they display antioxidant effects similar to classic antioxidants such as vitamin E.21 Also, it is well known that there is an oxidative imbalance in schizophrenia.22 Clarifying the mechanisms of PUFA deficits in schizophrenia may provide important insight into the underlying pathophysiology. In rats, the addition of n-3 to ketamineinduced schizophrenia models was shown to lower positive, negative and cognitive symptoms.22 In another study, addition of n-3 inhibited the startle reflex, reduced lipid damage in the hippocampus and striatum and reduced protein destruction in the prefrontal cortex. Additionally, n-3 may play a prophylactic role against symptoms associated with schizophrenia.23 One study showed that animals supplemented with trans fats, which contain n-3 fatty acids, from an early age presented stronger behavioral and biochemical amphetamine-induced responses.24 There are limited preclinical studies about the antipsychotic effect of n-3, and these have conflicting results. In the present study, it is hypothesized that n-3 will produce an antipsychotic effect in a rat model. To determine its efficacy in psychosis, MDA and GSH levels were compared with the effects
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of n-3 and chlorpromazine by evaluating the novelty-induced rearing and apomorphine-induced stereotypic behavior in rats.
Assessment of stereotypic behavior was performed by 2 observers blind to the study groups.
MATERIALS AND METHODS
Measurement of Brain Lipid Peroxidation Lipid peroxidation was determined in tissue samples by measuring MDA levels as thiobarbituric acid reactive substances (TBARS).32 Trichloroacetic acid and TBARS reagents were added to the tissue samples, then mixed and incubated at 100°C for 60 minutes. After cooling on ice, the samples were centrifuged at 3000 repetitions per minute for 20 minutes, and the absorbance of the supernatant was read at 535 nm. MDA levels were calculated from the standard calibration curve using tetraethoxypropane and expressed as nanomoles/gram of protein.
Animals The experimental protocol performed in the study was approved by the Institutional Animal Care and Ethics Committee of the Gaziosmanpasa University. Twenty-eight adult male Wistar rats (age, 7–8 weeks old; weight, 220–240 g) were used in the study. All animals were kept under standard 12-hour light/dark cycles in a temperature-controlled (22 6 2°C) environment with ad libitum access to rodent chow. All experimental procedures were performed during the light cycle. Chemicals All drugs were freshly prepared. Apomorphine hydrochloride (Sigma Chemical, Co, St Louis, MO) was dissolved in saline containing 0.1% ascorbic acid before experiments. DHA25 and eicosapentaenoic acid (EPA) (Marincap; Kocak, Turkey) was prepared. Saline (0.9% NaCl) was used as a control solution. All solutions were administered intraperitoneally (IP). Assessment of Novelty-Induced Rearing Behavior Novelty-induced rearing behavior is used to assess the central excitatory locomotor behavior in rodents.23 Four groups of rats (n 5 7) were administered DHA + EPA (300 mg/kg; DHA: 120 mg/kg + EPA: 180 mg/kg IP),26 DHA + EPA (150 mg/kg; DHA: 60 mg/kg + EPA: 90 mg/kg IP),27 chlorpromazine (1 mg/kg; IP) or isotonic NaCl (1 mL/kg, IP). One hour later, novelty-induced rearing behavior was assessed by placing the animals directly from their home cages into a transparent Plexiglas cage (45 3 25 3 25 cm). The rearing frequency (number of times the animal stood on its hind limbs, with its fore limbs against the walls of the observation box or free in the air) was recorded for 10 minutes. All rats were monitored individually by 2 observers who were blind to the study groups. The arena was cleaned with 5% alcohol to eliminate olfactory bias before beginning with a fresh animal.28 Apomorphine-Induced Stereotypic Behavior Test Mesolimbic and nigrostriatal dopaminergic pathways play crucial roles in the mediation of locomotor activity and stereotypic behavior. Apomorphine-induced stereotypy is because of the stimulation of dopamine receptors and has been used as a convenient method for in vivo screening of dopamine agonists or antagonists and assessment of dopaminergic activity.29,30 Four groups of rats (n 5 7) were administered DHA + EPA (300 mg/kg; DHA: 120 mg/kg + EPA: 180 mg/kg IP), DHA + EPA (150 mg/kg; DHA: 60 mg/kg + EPA: 90 mg/kg IP), chlorpromazine (1 mg/kg, IP) and isotonic saline (1 mL/kg, IP). One hour later, apomorphine (2 mg/kg, subcutaneously) was administered to each rat. First, rats were placed into cylindrical metal cages (18 3 19 cm) containing vertical (1 cm apart) and horizontal (4.5 cm apart) metal bars (2 mm) with upper lids for a 10-minute orientation period. After apomorphine administration, the rats were immediately placed back into the metal cages and observed for stereotypic behavior. Signs of stereotypy, which include sniffing and gnawing, were observed and scored as follows: absence of stereotypy (0), occasional sniffing (1), occasional sniffing with occasional gnawing (2), frequent gnawing (3), intense continuous gnawing (4) and intense gnawing on the same spot (5). The stereotypic behavior was rated after each minute, and a mean of 15-minute periods was calculated and recorded.31 Copyright © 2015 by the Southern Society for Clinical Investigation.
Measurement of Brain Protein Levels Total protein concentration in brain samples was determined according to Bradford’s33 method using bovine serum albumin as the standard. Measurement of Tissue Glutathione Levels GSH content in tissue samples was measured spectrophotometrically according to Ellman’s34 method. In this method, thiols interact with 5,5’-dithiobis (2-nitrobenzoic acid) and form a colored anion with a maximum peak at 412 nm. GSH levels were calculated from the standard calibration curve and expressed as micromoles/gram of protein. Statistical Analysis Statistical evaluation was performed by 1-way analysis of variance. A post hoc Bonferroni’s test was used to identify differences between the experimental groups. MDA and GSH levels were evaluated between and within the groups by the Kruskal-Wallis variance analysis and the Mann-Whitney’s U test where appropriate. Results are presented as mean 6 standard error of the mean. A value of P , 0.05 was considered to be significant.
RESULTS DHA + EPA (150 mg/kg), DHA + EPA (300 mg/kg) and chlorpromazine significantly decrease apomorphine-induced stereotypy scores compared to the control group (P , 0.001) (Figure 1). DHA + EPA (300 mg/kg) and chlorpromazine significantly decrease rearing behavior scores and brain MDA levels compared to the control group (P , 0.001) (Figures 2 and 3). DHA + EPA (150 mg/kg) significantly decrease rearing behavior scores and brain MDA levels compared to the control group (P , 0.01) (Figures 2 and 3). DHA + EPA (150 mg/kg) and chlorpromazine significantly increase brain GSH levels compared to the control group (P , 0.05) (Figure 4). DHA + EPA (300 mg/kg) significantly increase brain GSH levels compared to the control group (P , 0.001) (Figure 4).
DISCUSSION This study demonstrates the beneficial effects of n-3 on rearing behavior and stereotypy, which are accepted as indicators of antipsychotic effects. Theoretically, antipsychotic effects are formed by means of antidopaminergic activity in certain regions of the central nervous system. Antipsychotics have various side effects that are poorly tolerated. Moreover, two thirds of schizophrenics do not respond optimally to antipsychotic drugs.35 Therefore, clinical and nonclinical investigations focus on new drugs or supplements, which have fewer side effects.
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FIGURE 1. Apomorphineinduced stereotypy scores. Data are expressed as mean 6 standard error of the mean. Statistical analysis was performed by 1-way analysis of variance and Bonferroni’s post hoc test. *P , 0.001 (different from saline).
Hence, some molecules, which are not antipsychotic, have been investigated on rats in terms of possible antipsychotic effects.34,35 When the rats were exposed to a new environment, they exhibited a condition called novelty-induced behavior syndrome, which comprises behaviors such as rearing, grooming and gnawing.36 This behavioral response is regulated by multiple neurotransmitter systems, including Gamma-Amino Butyric acid A (GABAA), opioid and dopamine D2 neurotransmitter receptors.37 As a result of the apomorphine application, which acts as D1 and D2 receptor agonists, stereotyped behaviors are seen in animals.38 The anatomical structures, neurotransmitter systems and resulting stereotypic movements seem similar to that of psychotic disorders in humans, and improvement of these symptoms with antipsychotic drugs is also supportive evidence.39 In this study, rats exposed to a new environment and psychotic symptoms characterized by rearing and stereotypic behaviors were created through the application of apomorphine. Similar to chlorpromazine, DHA and EPA were shown to significantly decrease rearing behavior and stereotypy. Antipsychotic effects arise mainly with dopamine antagonism. The other underlying mechanisms of beneficial effects may be the incorporation of PUFAs into brain cell membranes.40–42 Chlorpromazine is the 1st antipsychotic drug exhibiting therapeutic effects in the treatment of schizophrenia. Therefore, it is classified as a classical or typical neuroleptic. Classical neuroleptics, like chlorpromazine, simply block dopamine D2 receptors in the central nervous system.43 Chlorpromazine blocks certain stereotypic behaviors in animals induced by dopamine agonists like apomorphine and
amphetamine, such as gnawing, circling, chewing and hyperactivity.44 These behaviors are thought to occur as a result of the activation of dopamine receptors located postsynaptically. In some animal studies that assess antipsychotic effects of a molecule, chlorpromazine was used as a reference antipsychotic.45,46 In this study, it was found that DHA + EPA (150 mg/kg), DHA + EPA (300 mg/kg) and chlorpromazine significantly decrease apomorphine-induced stereotypy scores compared to the control group. Omega-3 fatty acids are critical to normal brain function47 and have neuroprotective effects. It is a well-known fact that omega-3 fatty acids, which are major structural components of cell membranes, are decreased in patients with schizophrenia.48,49 There are reports of increased capacity for dopamine synthesis specific to the dorsal striatum, which is important for the etiology of schizophrenia, in individuals at a high risk for developing schizophrenia.50,51 In a study, Bondi et al52 showed that dietary n-3 PUFA deficiency influenced behavior and dopamine neurotransmission in both adult and adolescent rats. They found evidence of elevated dopamine availability specific to the dorsal regions of the striatum of adolescents, which is important for the etiology of schizophrenia. In a rat model of schizophrenia, Zugno et al53 showed that omega-3 fatty acids prevented the ketamine-induced increase in acetylcholinesterase activity, which is associated with the positive, negative and cognitive symptoms of schizophrenia. In clinical studies, EPA use often causes clinically significant beneficial effects in patients with schizophrenia and could be a novel therapeutic approach to schizophrenia.54 Mahadik et al25 have shown that when omega-3 fatty acids are
FIGURE 2. Rearing behavior scores. Data are expressed as mean 6 standard error of the mean. Statistical analysis was performed by 1-way analysis of variance and Bonferroni’s post hoc test. *P , 0.01 and**P , 0.001 (different from saline).
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FIGURE 3. Brain MDA levels. Data are expressed as mean 6 standard error of the mean. Statistical analyses were performed by the Kruskal-Wallis variance analysis and the MannWhitney’s U test. *P , 0.001 and **P , 0.000 (different from saline).
added to diets in humans, there is an improvement in the symptoms of schizophrenia. A growing body of evidence suggests that the addition of EPA accelerates the treatment response and increases tolerability of antipsychotic medication.14 In a study considering dose-dependent effectivity of DHA and EPA in patients with schizophrenia treated with clozapine, DHA and EPA were found to be clinically important and have statistically significant effects. This effect was higher in 2 g/d dose than the 1 g/d and 4 g/d doses.16 In a meta-analysis, it was suggested that EPA addition has no prominent antipsychotic effect, yet addition of EPA and/or DHA may have beneficial effects in terms of extrapyramidal and metabolic side effects.15 Fenton et al55 treated patients with schizophrenia with 3 g/d of ethyl EPA and found improvement in residual symptoms, and cognitive impairment was no greater than that of patients with schizophrenia treated with a placebo. In this study, DHA and EPA were found to increase antioxidants and reduce oxidants. Similar to chlorpromazine and more effective than DHA + EPA 150 mg/kg, DHA + EPA 300 mg/kg decreased brain MDA levels and rearing behavior and also increased GSH levels more than both the 150-mg dose of DHA + EPA and chlorpromazine. Oxidative stress is a part of schizophrenia pathology and appears as a promising field for developing new therapeutic strategies. Studies show that the use of lower or adequate doses of omega-3 fatty acids exerts antioxidant effects, and higher doses may increase oxidative stress.56 Additionally, short-term n-3 PUFA additions slightly decreased lipid peroxidation in erythrocyte membranes; this effect was more evident in cases of medium and high doses of n-3 PUFA additions, whereas in the same study, n-3 PUFAs increased MDA production in
a dose-dependent manner.57 Low doses of DHA potentiate cellular antioxidant defense systems and reduce lipid peroxidation. This effect is lost in the medium term and is reversed in the long term, and the effect of DHA on cellular redox status was expressed as time dependent and dose dependent.58 When this study is considered in conjunction with the aforementioned studies, the therapeutic effect of omega-3 fatty acids and their positive effects on oxidative mechanisms can be considered dose dependent. In animal models of schizophrenia induced by ketamine, omega-3 inhibits oxidative stress in the prefrontal cortex, hippocampus59 and striatum (lipid and protein peroxidation measured by biochemical parameters).23 Addition of omega-3 essential fatty acids increases superoxide dismutase activity in the hypothalamus of rats and lowers the levels MDA and nitric oxide, thus affecting the antioxidant system and lipid peroxidation. In a meta-analysis focusing on the MDA levels in patients with schizophrenia, a strong increase in MDA levels was confirmed.60 These studies point to a growing body of evidence proving the importance of oxidative stress in schizophrenia, both in pathogenesis and treatment modalities. It is suggested that antioxidants might be useful in the treatment of schizophrenia. For instance, Dakhale et al indicated that a vitamin C and oral antipsychotic combination reduced brief psychiatric rating scale scores and MDA levels.41 This study has some limitations. First, chlorpromazine, a very effective antagonist of D2 dopamine receptors, exerts additional antiadrenergic, anticholinergic and antihistaminergic effects.29 Hence, the efficacy of chlorpromazine on rearing behavior may be associated with its sedative effect, which is mainly maintained by the drug’s anticholinergic and
FIGURE 4. Brain GSH levels. Data are expressed as mean 6 standard error of the mean. Statistical analyses were performed by the Kruskal-Wallis variance analysis and the MannWhitney’s U test. *P , 0.05 and **P , 0.001 (different from saline).
Copyright © 2015 by the Southern Society for Clinical Investigation.
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antihistaminergic properties. Second, the results are to be interpreted with caution because of the small sample size. When all these preclinical and clinical studies are considered, consistent with the results of this study, it can be concluded that omega-3 fatty acids could have therapeutic effects on schizophrenia by decreasing antioxidants and regulating oxidative metabolism through increasing antioxidants.
CONCLUSIONS
15. Fusar-Poli P, Berger G. Eicosapentaenoic acid interventions in schizophrenia: meta-analysis of randomized, placebo-controlled studies. J Clin Psychopharmacol 2012;32:179–85. 16. Peet M, Horrobin DF. A dose-ranging exploratory study of the effects of ethyl-eicosapentaenoate in patients with persistent schizophrenic symptoms. J Psychiatr Res 2002;36:7–18. 17. Ballesteros A, Jiang P, Summerfelt A, et al. No evidence of exogenous origin for the abnor-mal glutathione redox state in schizophrenia. Schizophr Res 2013;146:184–9.
The results of this study suggest that DHA and EPA omega-3 fatty acid applications may have antipsychotic effects similar to the chlorpromazine, as well as increase antioxidants and reduce oxidants, therefore displaying possible antipsychotic effects by exerting positive effects on oxidative metabolism. Because a vast number of studies show the improving effects of antioxidants as an adjunct therapy, this study proposes that omega-3 fatty acids might be beneficial because of the antioxidant effects in patients with schizophrenia. One of the limitations of this study is regarding usage of a camera (only 2 observers who were blind to the study groups) during assessment of novelty-induced rearing behavior test. There is a need for additional well-designed, placebo-controlled trials with supplementation therapy in schizophrenia.
18. Singh M. Essential fatty acids, DHA and human brain. Indian J Pediatr 2005;72:239–42.
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The Antipsychotic Effects of Omega-3 Fatty Acids in Rats Mehmet Hanifi Kokacya, MD, Sema Inanir, MD, Umit Sertan Copoglu, MD, Recep Dokuyucu, MD and Oytun Erbas, MD
Abstract: Background: In humans, omega-3 fatty acids are necessary for cell membranes, brain function and nerve transmission continuation. When animals are exposed to a new environment—or as a result of an apomorphine application that creates an agonistic effect on D1 and D2 receptors—they display behavioral reactions like rearing and stereotypy. This study aims to reveal the possible antipsychotic and oxidative effects of omega-3 fatty acids by comparing with chlorpromazine, a conventional antipsychotic drug, through evaluating the novelty-induced rearing and apomorphine-induced stereotypic behaviors, as well as malondialdehyde and glutathione levels in rats. Methods: Twenty-eight, adult, male, Wistar rats were used in the study. Briefly, 4 groups of rats (n 5 7) were administered docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) (300 mg/kg; DHA: 120 mg/kg + EPA: 180 mg/kg intraperitoneally [IP]), DHA + EPA (150 mg/kg; DHA: 60 mg/kg + EPA: 90 mg/kg IP), chlorpromazine (1 mg/kg, IP) and isotonic saline (1 mL/kg, IP). One hour later, apomorphine (2 mg/kg, subcutaneously) was administered to each rat. After the apomorphine administration, rats were observed for stereotypic behavior. Results: This study shows that omega-3 fatty acids, “similar to antipsychotics,” reversed the psychotic like effects, increase of oxidants and decrease of antioxidants that are composed experimentally in rats. Conclusions: The application of omega-3 fatty acids has antipsychotic effects and causes an oxidative imbalance. This study adds new evidence to the current literature regarding the possible antipsychotic effects of omega-3 fatty acids. Key Indexing Terms: Omega-3 fatty acids; Antipsychotic effect; Oxidative stress. [Am J Med Sci 2015;350(3):212–217.]
A
growing amount of evidence suggests that the main problem in schizophrenia is dopaminergic hyperactivation. Studies suggest that dopamine is effective in therapeutic interventions by inhibiting postsynaptic dopamine receptors.1 Although the disease’s etiology is still unknown, abnormal neuronal maturation, neuronal and glial cell migration, dendritic and axonal branching and truncation, programmed cell death or other reasons, such as stress, trauma, infection and substance use, are thought to be involved in the disease onset.2–5 Oxidative stress results when excessive amounts of oxidants are produced and enzymatic and nonenzymatic antioxidant defense systems are insufficient. The failure of antioxidant mechanisms and membrane metabolism disorders is believed to play a role in neuronal destruction.6 Although oxidative stress is not the main cause of schizophrenia, it has a great impact on the pathophysiology of schizophrenia. Dopamine metabolism is one of the possible sources of oxidative stress forming reactive products.7 Oxidative stress From the Departments of Psychiatry (MHK, USC) and Physiology (RD), Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey; and Departments of Psychiatry (SI) and Physiology (OE), Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey. Submitted September 12, 2014; accepted in revised form June 4, 2015. The authors have no financial or other conflicts of interest to disclose. Correspondence: Recep Dokuyucu, MD, Department of Physiology, Mustafa Kemal University, Hatay 31100, Turkey (E-mail: drecepfatih@ gmail.com).
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resulting from elevated dopamine levels can cause late-onset and permanent central nervous system damage by increasing the striatal glutamatergic neurotransmission.7 It is believed that oxidative stress causes disease as a result of its toxic effects on carbohydrates, proteins, lipids and DNA metabolism.8 The effects of oxidative products can lead to malfunctioning enzymes, neurotransmitters and receptor proteins and may cause a disruption in membrane integrity by reducing the fluidity and permeability of the cell membrane.9 In accordance with the hypothesis of the membrane phospholipids in other psychiatric disorders and neurodegenerative diseases, structural abnormalities of neuronal membrane phospholipids can be an important factor for schizophrenia etiology.10 Studies on lipid peroxidation markers, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), showed that elevated levels of MDA were found in patients with negative symptoms, indicating greater oxidative damage in this patient group.7 Elevated MDA levels have also been found in peripheral tissues of patients with schizophrenia.11–13 Glutathione (GSH) depletion, a nonenzymatic antioxidant component, and GSH-related enzyme deficits are also extensively documented in schizophrenia.14–16 Ballesteros et al17 reported significantly lower GSH and higher glutathione disulfide (GSSG) levels in the blood of schizophrenia individuals. In mammals, omega-3 polyunsaturated fatty acids (n-3 PUFA) are required for cell membranes, brain function and transmission maintenance of nerve impulses. PUFAs are found in the brain and blood cells. Docosahexaenoic acid (DHA), a PUFA, regulates cell transport and synaptic functions.18 The main structural components of membrane phospholipids are PUFAs, which are not de novo—synthesized from fatty acids in the body.10 The presence of PUFAs is known to increase membrane fluidity. Consequently, in case of a PUFA deficiency, membranes become more rigid and result in changes in conformation and function of proteins, receptors and ion channels.19,20 While PUFAs are not antioxidants, they display antioxidant effects similar to classic antioxidants such as vitamin E.21 Also, it is well known that there is an oxidative imbalance in schizophrenia.22 Clarifying the mechanisms of PUFA deficits in schizophrenia may provide important insight into the underlying pathophysiology. In rats, the addition of n-3 to ketamineinduced schizophrenia models was shown to lower positive, negative and cognitive symptoms.22 In another study, addition of n-3 inhibited the startle reflex, reduced lipid damage in the hippocampus and striatum and reduced protein destruction in the prefrontal cortex. Additionally, n-3 may play a prophylactic role against symptoms associated with schizophrenia.23 One study showed that animals supplemented with trans fats, which contain n-3 fatty acids, from an early age presented stronger behavioral and biochemical amphetamine-induced responses.24 There are limited preclinical studies about the antipsychotic effect of n-3, and these have conflicting results. In the present study, it is hypothesized that n-3 will produce an antipsychotic effect in a rat model. To determine its efficacy in psychosis, MDA and GSH levels were compared with the effects
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of n-3 and chlorpromazine by evaluating the novelty-induced rearing and apomorphine-induced stereotypic behavior in rats.
Assessment of stereotypic behavior was performed by 2 observers blind to the study groups.
MATERIALS AND METHODS
Measurement of Brain Lipid Peroxidation Lipid peroxidation was determined in tissue samples by measuring MDA levels as thiobarbituric acid reactive substances (TBARS).32 Trichloroacetic acid and TBARS reagents were added to the tissue samples, then mixed and incubated at 100°C for 60 minutes. After cooling on ice, the samples were centrifuged at 3000 repetitions per minute for 20 minutes, and the absorbance of the supernatant was read at 535 nm. MDA levels were calculated from the standard calibration curve using tetraethoxypropane and expressed as nanomoles/gram of protein.
Animals The experimental protocol performed in the study was approved by the Institutional Animal Care and Ethics Committee of the Gaziosmanpasa University. Twenty-eight adult male Wistar rats (age, 7–8 weeks old; weight, 220–240 g) were used in the study. All animals were kept under standard 12-hour light/dark cycles in a temperature-controlled (22 6 2°C) environment with ad libitum access to rodent chow. All experimental procedures were performed during the light cycle. Chemicals All drugs were freshly prepared. Apomorphine hydrochloride (Sigma Chemical, Co, St Louis, MO) was dissolved in saline containing 0.1% ascorbic acid before experiments. DHA25 and eicosapentaenoic acid (EPA) (Marincap; Kocak, Turkey) was prepared. Saline (0.9% NaCl) was used as a control solution. All solutions were administered intraperitoneally (IP). Assessment of Novelty-Induced Rearing Behavior Novelty-induced rearing behavior is used to assess the central excitatory locomotor behavior in rodents.23 Four groups of rats (n 5 7) were administered DHA + EPA (300 mg/kg; DHA: 120 mg/kg + EPA: 180 mg/kg IP),26 DHA + EPA (150 mg/kg; DHA: 60 mg/kg + EPA: 90 mg/kg IP),27 chlorpromazine (1 mg/kg; IP) or isotonic NaCl (1 mL/kg, IP). One hour later, novelty-induced rearing behavior was assessed by placing the animals directly from their home cages into a transparent Plexiglas cage (45 3 25 3 25 cm). The rearing frequency (number of times the animal stood on its hind limbs, with its fore limbs against the walls of the observation box or free in the air) was recorded for 10 minutes. All rats were monitored individually by 2 observers who were blind to the study groups. The arena was cleaned with 5% alcohol to eliminate olfactory bias before beginning with a fresh animal.28 Apomorphine-Induced Stereotypic Behavior Test Mesolimbic and nigrostriatal dopaminergic pathways play crucial roles in the mediation of locomotor activity and stereotypic behavior. Apomorphine-induced stereotypy is because of the stimulation of dopamine receptors and has been used as a convenient method for in vivo screening of dopamine agonists or antagonists and assessment of dopaminergic activity.29,30 Four groups of rats (n 5 7) were administered DHA + EPA (300 mg/kg; DHA: 120 mg/kg + EPA: 180 mg/kg IP), DHA + EPA (150 mg/kg; DHA: 60 mg/kg + EPA: 90 mg/kg IP), chlorpromazine (1 mg/kg, IP) and isotonic saline (1 mL/kg, IP). One hour later, apomorphine (2 mg/kg, subcutaneously) was administered to each rat. First, rats were placed into cylindrical metal cages (18 3 19 cm) containing vertical (1 cm apart) and horizontal (4.5 cm apart) metal bars (2 mm) with upper lids for a 10-minute orientation period. After apomorphine administration, the rats were immediately placed back into the metal cages and observed for stereotypic behavior. Signs of stereotypy, which include sniffing and gnawing, were observed and scored as follows: absence of stereotypy (0), occasional sniffing (1), occasional sniffing with occasional gnawing (2), frequent gnawing (3), intense continuous gnawing (4) and intense gnawing on the same spot (5). The stereotypic behavior was rated after each minute, and a mean of 15-minute periods was calculated and recorded.31 Copyright © 2015 by the Southern Society for Clinical Investigation.
Measurement of Brain Protein Levels Total protein concentration in brain samples was determined according to Bradford’s33 method using bovine serum albumin as the standard. Measurement of Tissue Glutathione Levels GSH content in tissue samples was measured spectrophotometrically according to Ellman’s34 method. In this method, thiols interact with 5,5’-dithiobis (2-nitrobenzoic acid) and form a colored anion with a maximum peak at 412 nm. GSH levels were calculated from the standard calibration curve and expressed as micromoles/gram of protein. Statistical Analysis Statistical evaluation was performed by 1-way analysis of variance. A post hoc Bonferroni’s test was used to identify differences between the experimental groups. MDA and GSH levels were evaluated between and within the groups by the Kruskal-Wallis variance analysis and the Mann-Whitney’s U test where appropriate. Results are presented as mean 6 standard error of the mean. A value of P , 0.05 was considered to be significant.
RESULTS DHA + EPA (150 mg/kg), DHA + EPA (300 mg/kg) and chlorpromazine significantly decrease apomorphine-induced stereotypy scores compared to the control group (P , 0.001) (Figure 1). DHA + EPA (300 mg/kg) and chlorpromazine significantly decrease rearing behavior scores and brain MDA levels compared to the control group (P , 0.001) (Figures 2 and 3). DHA + EPA (150 mg/kg) significantly decrease rearing behavior scores and brain MDA levels compared to the control group (P , 0.01) (Figures 2 and 3). DHA + EPA (150 mg/kg) and chlorpromazine significantly increase brain GSH levels compared to the control group (P , 0.05) (Figure 4). DHA + EPA (300 mg/kg) significantly increase brain GSH levels compared to the control group (P , 0.001) (Figure 4).
DISCUSSION This study demonstrates the beneficial effects of n-3 on rearing behavior and stereotypy, which are accepted as indicators of antipsychotic effects. Theoretically, antipsychotic effects are formed by means of antidopaminergic activity in certain regions of the central nervous system. Antipsychotics have various side effects that are poorly tolerated. Moreover, two thirds of schizophrenics do not respond optimally to antipsychotic drugs.35 Therefore, clinical and nonclinical investigations focus on new drugs or supplements, which have fewer side effects.
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FIGURE 1. Apomorphineinduced stereotypy scores. Data are expressed as mean 6 standard error of the mean. Statistical analysis was performed by 1-way analysis of variance and Bonferroni’s post hoc test. *P , 0.001 (different from saline).
Hence, some molecules, which are not antipsychotic, have been investigated on rats in terms of possible antipsychotic effects.34,35 When the rats were exposed to a new environment, they exhibited a condition called novelty-induced behavior syndrome, which comprises behaviors such as rearing, grooming and gnawing.36 This behavioral response is regulated by multiple neurotransmitter systems, including Gamma-Amino Butyric acid A (GABAA), opioid and dopamine D2 neurotransmitter receptors.37 As a result of the apomorphine application, which acts as D1 and D2 receptor agonists, stereotyped behaviors are seen in animals.38 The anatomical structures, neurotransmitter systems and resulting stereotypic movements seem similar to that of psychotic disorders in humans, and improvement of these symptoms with antipsychotic drugs is also supportive evidence.39 In this study, rats exposed to a new environment and psychotic symptoms characterized by rearing and stereotypic behaviors were created through the application of apomorphine. Similar to chlorpromazine, DHA and EPA were shown to significantly decrease rearing behavior and stereotypy. Antipsychotic effects arise mainly with dopamine antagonism. The other underlying mechanisms of beneficial effects may be the incorporation of PUFAs into brain cell membranes.40–42 Chlorpromazine is the 1st antipsychotic drug exhibiting therapeutic effects in the treatment of schizophrenia. Therefore, it is classified as a classical or typical neuroleptic. Classical neuroleptics, like chlorpromazine, simply block dopamine D2 receptors in the central nervous system.43 Chlorpromazine blocks certain stereotypic behaviors in animals induced by dopamine agonists like apomorphine and
amphetamine, such as gnawing, circling, chewing and hyperactivity.44 These behaviors are thought to occur as a result of the activation of dopamine receptors located postsynaptically. In some animal studies that assess antipsychotic effects of a molecule, chlorpromazine was used as a reference antipsychotic.45,46 In this study, it was found that DHA + EPA (150 mg/kg), DHA + EPA (300 mg/kg) and chlorpromazine significantly decrease apomorphine-induced stereotypy scores compared to the control group. Omega-3 fatty acids are critical to normal brain function47 and have neuroprotective effects. It is a well-known fact that omega-3 fatty acids, which are major structural components of cell membranes, are decreased in patients with schizophrenia.48,49 There are reports of increased capacity for dopamine synthesis specific to the dorsal striatum, which is important for the etiology of schizophrenia, in individuals at a high risk for developing schizophrenia.50,51 In a study, Bondi et al52 showed that dietary n-3 PUFA deficiency influenced behavior and dopamine neurotransmission in both adult and adolescent rats. They found evidence of elevated dopamine availability specific to the dorsal regions of the striatum of adolescents, which is important for the etiology of schizophrenia. In a rat model of schizophrenia, Zugno et al53 showed that omega-3 fatty acids prevented the ketamine-induced increase in acetylcholinesterase activity, which is associated with the positive, negative and cognitive symptoms of schizophrenia. In clinical studies, EPA use often causes clinically significant beneficial effects in patients with schizophrenia and could be a novel therapeutic approach to schizophrenia.54 Mahadik et al25 have shown that when omega-3 fatty acids are
FIGURE 2. Rearing behavior scores. Data are expressed as mean 6 standard error of the mean. Statistical analysis was performed by 1-way analysis of variance and Bonferroni’s post hoc test. *P , 0.01 and**P , 0.001 (different from saline).
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FIGURE 3. Brain MDA levels. Data are expressed as mean 6 standard error of the mean. Statistical analyses were performed by the Kruskal-Wallis variance analysis and the MannWhitney’s U test. *P , 0.001 and **P , 0.000 (different from saline).
added to diets in humans, there is an improvement in the symptoms of schizophrenia. A growing body of evidence suggests that the addition of EPA accelerates the treatment response and increases tolerability of antipsychotic medication.14 In a study considering dose-dependent effectivity of DHA and EPA in patients with schizophrenia treated with clozapine, DHA and EPA were found to be clinically important and have statistically significant effects. This effect was higher in 2 g/d dose than the 1 g/d and 4 g/d doses.16 In a meta-analysis, it was suggested that EPA addition has no prominent antipsychotic effect, yet addition of EPA and/or DHA may have beneficial effects in terms of extrapyramidal and metabolic side effects.15 Fenton et al55 treated patients with schizophrenia with 3 g/d of ethyl EPA and found improvement in residual symptoms, and cognitive impairment was no greater than that of patients with schizophrenia treated with a placebo. In this study, DHA and EPA were found to increase antioxidants and reduce oxidants. Similar to chlorpromazine and more effective than DHA + EPA 150 mg/kg, DHA + EPA 300 mg/kg decreased brain MDA levels and rearing behavior and also increased GSH levels more than both the 150-mg dose of DHA + EPA and chlorpromazine. Oxidative stress is a part of schizophrenia pathology and appears as a promising field for developing new therapeutic strategies. Studies show that the use of lower or adequate doses of omega-3 fatty acids exerts antioxidant effects, and higher doses may increase oxidative stress.56 Additionally, short-term n-3 PUFA additions slightly decreased lipid peroxidation in erythrocyte membranes; this effect was more evident in cases of medium and high doses of n-3 PUFA additions, whereas in the same study, n-3 PUFAs increased MDA production in
a dose-dependent manner.57 Low doses of DHA potentiate cellular antioxidant defense systems and reduce lipid peroxidation. This effect is lost in the medium term and is reversed in the long term, and the effect of DHA on cellular redox status was expressed as time dependent and dose dependent.58 When this study is considered in conjunction with the aforementioned studies, the therapeutic effect of omega-3 fatty acids and their positive effects on oxidative mechanisms can be considered dose dependent. In animal models of schizophrenia induced by ketamine, omega-3 inhibits oxidative stress in the prefrontal cortex, hippocampus59 and striatum (lipid and protein peroxidation measured by biochemical parameters).23 Addition of omega-3 essential fatty acids increases superoxide dismutase activity in the hypothalamus of rats and lowers the levels MDA and nitric oxide, thus affecting the antioxidant system and lipid peroxidation. In a meta-analysis focusing on the MDA levels in patients with schizophrenia, a strong increase in MDA levels was confirmed.60 These studies point to a growing body of evidence proving the importance of oxidative stress in schizophrenia, both in pathogenesis and treatment modalities. It is suggested that antioxidants might be useful in the treatment of schizophrenia. For instance, Dakhale et al indicated that a vitamin C and oral antipsychotic combination reduced brief psychiatric rating scale scores and MDA levels.41 This study has some limitations. First, chlorpromazine, a very effective antagonist of D2 dopamine receptors, exerts additional antiadrenergic, anticholinergic and antihistaminergic effects.29 Hence, the efficacy of chlorpromazine on rearing behavior may be associated with its sedative effect, which is mainly maintained by the drug’s anticholinergic and
FIGURE 4. Brain GSH levels. Data are expressed as mean 6 standard error of the mean. Statistical analyses were performed by the Kruskal-Wallis variance analysis and the MannWhitney’s U test. *P , 0.05 and **P , 0.001 (different from saline).
Copyright © 2015 by the Southern Society for Clinical Investigation.
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antihistaminergic properties. Second, the results are to be interpreted with caution because of the small sample size. When all these preclinical and clinical studies are considered, consistent with the results of this study, it can be concluded that omega-3 fatty acids could have therapeutic effects on schizophrenia by decreasing antioxidants and regulating oxidative metabolism through increasing antioxidants.
CONCLUSIONS
15. Fusar-Poli P, Berger G. Eicosapentaenoic acid interventions in schizophrenia: meta-analysis of randomized, placebo-controlled studies. J Clin Psychopharmacol 2012;32:179–85. 16. Peet M, Horrobin DF. A dose-ranging exploratory study of the effects of ethyl-eicosapentaenoate in patients with persistent schizophrenic symptoms. J Psychiatr Res 2002;36:7–18. 17. Ballesteros A, Jiang P, Summerfelt A, et al. No evidence of exogenous origin for the abnor-mal glutathione redox state in schizophrenia. Schizophr Res 2013;146:184–9.
The results of this study suggest that DHA and EPA omega-3 fatty acid applications may have antipsychotic effects similar to the chlorpromazine, as well as increase antioxidants and reduce oxidants, therefore displaying possible antipsychotic effects by exerting positive effects on oxidative metabolism. Because a vast number of studies show the improving effects of antioxidants as an adjunct therapy, this study proposes that omega-3 fatty acids might be beneficial because of the antioxidant effects in patients with schizophrenia. One of the limitations of this study is regarding usage of a camera (only 2 observers who were blind to the study groups) during assessment of novelty-induced rearing behavior test. There is a need for additional well-designed, placebo-controlled trials with supplementation therapy in schizophrenia.
18. Singh M. Essential fatty acids, DHA and human brain. Indian J Pediatr 2005;72:239–42.
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