Significant outcomes
• The inhibitory effect of escitalopram on respiratory chain complexes was different from other selective serotonin reuptake inhibitors (SSRIs), which usually increase the activity of these complexes.
• Escitalopram presents a specific effect on respiratory chain complexes, since the enzymes of Krebs cycle were not affected.
Limitations
• A complementary study to examine different doses in order to verify a possible dose-dependent effect of escitalopram.
• The effect of escitalopram in an animal model of depression should also be studied.
Introduction
Depressive disorders are serious and disabling, including major depression. This sort of depression is the most prevalent mental disorder which is estimated to be prevalent in 13.5–21.2% of the cases (Reference Kessler and Walters1–Reference Kessler, Berglund, Demler, Jin, Kr and Walters3). The World Health Organisation estimates that major depression is the fourth most important cause of loss in disability-adjusted life years worldwide (Reference Rex, Schickert and Fink4–Reference Longone, Rupprecht, Manieri, Bernardi, Romeo and Pasini6). Interpersonal relationships are affected by this disability to a larger extent; spouses and children are the ones impacted by this aspect of impaired social functioning. It also has high economic costs, both in terms of direct treatment costs and working days lost through illness (Reference Greenberg, Stiglin, Finkelstein and Berndt7). Along with schizophrenia, depression is responsible for 60% of suicides worldwide and it is predicted to be the second main cause of disability in 2020 in all patients regardless of their age and gender (8). Although it is believed that depression has genetic antecedents, which also suggests a biological contribution to its origin, the exact pathophysiology of depression is not clearly understood (Reference Cannon and Keller9).
Current studies of pathophysiological mechanisms and the pharmacological treatment of major depressive disorder are emphasised mainly on the monoamine hypothesis (Reference Skolnick10). This theory suggests that major depression results from an unbalance of neurotransmission such as serotonin (5-HT), norepinephrine and dopamine, and the treatments are based on normalising the levels of these neurotransmitters (Reference Charney11).
The treatment of depression has focused on the neurotransmitter serotonin for the last decade. As tricyclic compounds have a less tolerable profile, SSRIs have been more widely prescribed for being safe and effective when compared with those older compounds (Reference Brunello, Mendlewicz and Kasper12). Escitalopram is functionally classified as an SSRI, which enhances serotonergic transmission by blocking the presynaptic active membrane transport mechanism for the reuptake of serotonin and consequently increases serotonergic activity at the postsynaptic receptor (Reference Johnson13,Reference Richelson14).
Microdialysis studies have shown that a single intraperitoneal (IP) injection of SSRIs increased the extracellular concentration of 5-HT in mice cortex (Reference David, Bourin, Jego, Przybylski, Jolliet and Gardier15). However, the data obtained from animal studies using antidepressants are contradictory and they still remain unclear whether these SSRIs show a true anxiogenic or anxiolytic effect or have no effect (Reference Borsini, Podhorna and Marazziti16).
Studies have shown that mitochondrial dysfunction has been implicated in the pathogenesis of a number of diseases affecting the brain such as dementia, cerebral ischaemia, Alzheimer's disease and Parkinson's disease (Reference Brennan, Bird and Aprille17–Reference Moreira, Santos, Seiça and Oliveira23). Tissues with high-energy demands, such as the brain, contain a large number of mitochondria, which makes them more susceptible to reduction of the aerobic metabolism. Mitochondria are intracellular organelles which optimise adenosine triphosphate (ATP) production (Reference Calabrese, Scapagnini, Giuffrida-Stella, Bates and Clark24). Oxidative phosphorylation is the process in which most cell energy production is obtained. This process requires the action of various respiratory enzyme complexes located in a special structure of the inner mitochondrial membrane, the mitochondrial respiratory chain (Reference Boekema and Braun25).
The Krebs cycle is the major, final, common pathway for oxidation of carbohydrates, lipids and some amino acids, which produces reducing equivalents in the form of nicotinamide adenine dinucleotide and flavin adenine dinucleotide that result in large production of ATP through oxidative phosphorylation (Reference Kelly, Gordon, Alpers and Strauss26). Some enzymes of Krebs cycle stand out with important roles. Citrate synthase (EC 4.1.3.7), an important regulatory step of Krebs cycle, is found within cells in the mitochondrial matrix and catalyses the condensation of oxaloacetate and the acetyl group of acetyl coenzyme-A (acetyl CoA) (Reference Shepherd and Garland27). Malate dehydrogenase (EC 1.1.1.37) is a key enzyme that plays an important metabolic function in aerobic energy-producing pathways and in the malate shuttle. It catalyses the dehydrogenation of l-malate to oxaloacetate at the end of Krebs cycle (Reference Kelly, Gordon, Alpers and Strauss26,Reference Labrou and Clonis28). Succinate dehydrogenase (SDH) (EC 1.3.99.1) is one of the most reliable indicator of the mitochondrial capability to supply an adequate amount of ATP, as it is part of both Krebs cycle and respiratory chain (complex II). SDH catalyses the oxidation of succinate to fumarate in the Krebs cycle and feed electrons to the respiratory chain ubiquinone (UQ) pool (Reference Tyler29,Reference Rustin, Munnich and Rötig30).
Creatine kinase (EC 2.7.3.2) takes part in metabo- lism of high-energy-consuming tissues such as brain, where it functions as an effective buffering system of cellular ATP levels. The enzyme catalyses the reversible transfer of the phosphoryl group from phosphocreatine to adenosine diphosphate (ADP), regenerating ATP. It is also known that a reduction of creatine kinase activity might impair energy homeostasis, ending in cell death (Reference Tomimoto, Yamamoto, Homburger and Yanagihara31–Reference Aksenov, Aksenova, Butterfield and Markesbery35).
On the basis of the hypothesis that metabolism impairment might be involved in the pathophysiology of depression, in this study, we evaluated the activities of enzymes of Krebs cycle, mitochondrial respiratory chain complexes and creatine kinase in brain of rats subjected to acute administration of escitalopram.
Materials and methods
Animals
Adult and male Wistar rats (250–300 g) were obtained from Central Animal House of the Universidade do Extremo Sul Catarinense. They were caged in group of five with free access to food and water and were maintained on a 12-h light–dark cycle (lights on 7:00 h), at a temperature of 23 ± 1 °C. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior recommendations for animal care, with the approval of the Ethics Committee from Universidade do Extremo Sul Catarinense.
Drugs
Animals received daily IP injections of escitalopram (10 mg/kg), in 1.0 ml/kg volume for 14 days (n = 6 animals per group). All drugs were dissolved in saline solution (vehicle). Control animals received only vehicle (1.0 ml/kg). The selection of this regimen was based on previous studies showing important neurochemical and antidepressant effects for this drug (Reference Jayatissa, Bisgaard, Tingström, Papp and Wiborg36).
Tissue and homogenate preparation
Twelve hours after the last injection, the rats were killed by decapitation, the brain was removed and the prefrontal cortex, hippocampus, striatum, cerebellum and cerebral cortex were homogenised (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM ethylenediaminetetraacetic acid, 10 mM Trizma base, 50 IU/ml heparin). The homogenates were centrifuged at 800 × g for 10 min and the supernatants kept at −70 °C until used for enzymes' activity determination. The longest period between homogenate preparation and enzyme analysis was always less than 5 days. Protein content was determined by the method described by Lowry and colleagues (Reference Lowry, Rosebough, Farr and Randall37) using bovine serum albumin as standard. Biochemical preparation included mitochondria from different cells (neurons, astrocytes, microglia, etc.).
Activities of mitochondrial respiratory chain enzymes
NADH dehydrogenase (complex I) was evaluated by the method described by Cassina and Radi (Reference Cassina and Radi38) by the rate of NADH-dependent ferricyanide reduction at λ = 420 nm. The activities of succinate-2,6-dichloroindophenol (DCIP)-oxidoreductase (complex II) and succinate:cytochrome c oxidoreductase (complexes II–III) were determined by the method described by Fischer and colleagues (Reference Fischer, Ruitenbeek, Berden and Trijbels39). Complex II activity was measured by the monitoring of the decrease in absorbance due to the reduction of 2,6-DCIP at λ = 600 nm. The activity of complexes II–III was measured by cytochrome c reduction from succinate at λ = 550 nm. The activity of cytochrome c oxidase (complex IV) was assayed according to the method described by Rustin and colleagues(Reference Rustin, Chretien and Bourgeron40), measured by the monitoring of the decrease in absorbance due to the oxidation of previously reduced cytochrome c at λ = 550 nm. The activities of the mitochondrial respiratory chain complexes were calculated as nmol/min × mg protein.
Activities of enzymes of Krebs cycle
Citrate synthase activity. Citrate synthase activity was assayed according to the method described by Shepherd and Garland (Reference Shepherd and Garland27). The reaction mixture contained 100 mM Tris, pH 8.0, 100 mM acetyl CoA, 100 mM 5,5′-di-thiobis-(2-nitrobenzoic acid), 0.1% triton X-100 and 2–4 μg supernatant protein and was initiated with 100 μM oxaloacetate and monitored at 412 nm for 3 min at 25 °C.
Malate dehydrogenase activity. Malate dehydrogenase was measured as described by Kitto (Reference Kitto41). Aliquots (20 mg protein) were transferred into a medium containing 10 mM rotenone, 0.2% Triton X-100, 0.15 mM NADH and 100 mM potassium phosphate buffer, pH 7.4, at 37 °C. The reaction was started by the addition of 0.33 mM oxaloacetate. Absorbance was monitored as described above.
SDH activity. SDH activity was determined according to the method of Fischer and colleagues (Reference Fischer, Ruitenbeek, Berden and Trijbels39), measured by monitoring the decrease in absorbance due to the reduction of 2,6-di-chloro-indophenol (2,6-DCIP) at 600 nm with 700 nm as reference wavelength (ε = 19.1 mM−1 cm−1) in the presence of phenazine methasulphate (PMS). The reaction mixture consisting of 40 mM potassium phosphate, pH 7.4, 16 mM succinate and 8 μM 2,6-DCIP was preincubated with 40–80 μg homogenate protein at 30 °C for 20 min. Subsequently, 4 mM sodium azide, 7 μM rotenone and 40 μM 2,6-DCIP were added and the reaction was initiated by the addition of 1 mM PMS and was monitored for 5 min.
Activity of creatine kinase
Creatine kinase activity was measured in brain homogenates pretreated with 0.625 mM lauryl maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 μg protein in a final volume of 100 μl. After 15 min of pre-incubation at 37 °C, the reaction was started by the addition of 3.2 mmol of ADP plus 0.8 mmol 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 (Reference Hughes42). The colour was developed by the addition of 100 μl 2% α-naphtol 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/min × mg protein.
Statistical analysis
Data were analysed by Student's t-test and were expressed as mean ± standard deviation. All analyses were performed using the Statistical Package for the Social Science (SPSS) software, IBM Corporation, New York, USA.
Results
This work investigated the activities of respiratory chain complex in the presence of escitalopram in homogenates from prefrontal cortex, hippocampus, striatum, cerebellum and posterior cortex from rat brain. Our results showed that rats treated with this antidepressant presented a significant decrease in complex I activity (Fig. 1a) in cerebellum, hippocampus and striatum, whereas prefrontal and posterior cortexes were not affected. Similar findings occurred with complex II–III activity (Figure 1c), where the same structures were affected plus posterior cortex. Furthermore, complex II activity (Fig. 1b) was decreased only in striatum without affecting prefrontal cortex, hippocampus, cerebellum and posterior cortex. On the other hand, chronic administration of escitalopram did not affect complex IV activity (Fig. 1d) as well as all other important analysed enzymes in the Krebs cycle (Figs 2–4) and creatine kinase (Fig. 5).
Fig. 1. Complex I activity (a), complex II activity (b), complex II–III activity (c) and complex IV activity (d) activity after chronic administration of escitalopram in the prefrontal cortex, cerebellum, hippocampus, striatum and cerebral cortex of rats. Data were analysed by the Student's t-test. Values are expressed as nmol/min × mg protein, mean ± SD (n = 6). *Different from control; p < 0.05.
Fig. 2. Citrate synthase activity after chronic administration of escitalopram in the prefrontal cortex, cerebellum, hippocampus, striatum and posterior cortex of rats. Data were analysed by the Student's t-test. Values are expressed as nmol/min × mg protein, mean ± SD (n = 6). *Different from control; p < 0.05.
Fig. 3. Malate dehydrogenase activity after chronic administration of escitalopram in the prefrontal cortex, cerebellum, hippocampus, striatum and posterior cortex of rats. Data were analysed by the Student's t-test. Values are expressed as nmol/min × mg protein, mean ± SD (n = 6). *Different from control; p < 0.05.
Fig. 4. SDH activity after chronic administration of escitalopram in the prefrontal cortex, cerebellum, hippocampus, striatum and posterior cortex of rats. Data were analysed by the Student's t-test. Values are expressed as nmol/min × mg protein, mean ± SD (n = 6). *Different from control; p < 0.05.
Fig. 5. Creatine kinase activity after chronic administration of escitalopram in the prefrontal cortex, cerebellum, hippocampus, striatum and cerebral cortex of rats. Data were analysed by the Student's t-test. Values are expressed as nmol/min × mg protein, mean ± SD (n = 6). *Different from control; p < 0.05.
Discussion
Most recent findings suggest the possibility that a mitochondrial deficit is enough to trigger one or more psychiatric disorders (Reference Mattson, Gleichmann and Cheng43). Moreover, several works also support the hypothesis that metabolism impairment is involved in the pathophysiology of depression (Reference Retter, Mayer-Takacs and Adam-Vizi44–Reference Stanyer, Jorgensen, Hori, Clark and Heales47). Madrigal and colleagues (Reference Madrigal, Olivenza and Moro48) showed that stress (immobilisation for 6 h during 21 days) inhibited the activities of complexes of the mitochondrial respiratory chain. An abnormal cellular energy state can lead to alteration in neuronal function, plasticity and brain circuit, and thereby to the cognition (Reference Barrett, Kelly, Bell and King49,Reference Quraishi, Walshe and Mcdonald50). We have recently showed that creatine kinase is inhibited in animal models of neuropsychiatry disorders such as bipolar disorder (Reference Streck, Amboni and Scaini51) and after electroconvulsive shock (Reference Burigo, Roza and Bassani52).
It has been also reported that brain Na+, K+-ATPase and activities of respiratory chain complexes I, III and IV are inhibited after chronic variate stress in rats (Reference Gamaro, Streck, Matté, Prediger, Wyse and Dalmaz53,Reference Rezin, Cardoso and Gonçalves54) and that complexes I–III and II–III of mitochondrial respiratory chain are inhibited in rat brain after chronic stress (Reference Quraishi, Walshe and Mcdonald50).
In brain structure samples analysed in this study, the most affected area was the striatum, with abnormal activity in complexes I, II, II–III. The striatum is a dopaminergic area involved in memory (Reference Ferretti, Roullet and Sargolini55) and mood disorders (Reference Marais, Stein and Daniels56). Additionally, striatum also has relation to the start, stop and direction of motor movement (Reference Gokce, Runne, Kuhn and Luthi-Carter57); in other areas investigated, hippocampus, cerebellum and posterior cortex, there was an alteration in the activity of complexes I and II–III. The hippocampus is one of several limbic structures that have been related to mood disorders. In addition, the hippocampus has connections with the prefrontal cortex, an area that is more directly involved in emotion and cognition and, thereby, contributes to other major symptoms of mood disorders (Reference Duman and Monteggia58–Reference Hassel, Almeida and Kerr60).
In this study, we verified an unexpected decrease in some respiratory chain complexes from rat brain after chronic administration of escitalopram, especially in the activities of complexes I and II–III in most brain structures like cerebellum, hippocampus, striatum and posterior cortex. A recent study showed that citalopram, the isomer form of escitalopram, decreased the activity of respiratory chain complexes I and IV (Reference Hroudova and Fisar61). This is a relevant concept for medications to be considered, although enantiomers differ only in how they rotate plane-polarised light, they may have very different biological properties (Reference Burke and Cj62). Various pre-clinical and clinical studies with escitalopram for the treatment of major depression have been conducted and supply further evidence of the potency and efficacy of escitalopram (Reference Mitchell and Hogg63–Reference Mitchell and Hogg65) between its peculiarities is more rapid onset of action when compared with citalopram. We verified that escitalopram, like citalopram, showed inhibitory effect on mitochondrial respiratory chain.
It is known that complex I is a key element in the control of oxidative phosphorylation and its abnormal activity can lead to defects in energy metabolism and changes in neuronal activity (Reference Pathak and Davey66). In addition, complex I is the primary source of reactive oxygen species (ROS) in a variety of pathological processes (Reference Barja and Herrero67,Reference Sherer, Betarbet, Jh and Greenamyre68). This tendency of xenobiotics to inhibit complex I (mitochondrial NADH:UQ oxidoreductase; EC 1.6.5.3) may depend on the structure of this enzymatic complex, which consists of at least 40 different polypeptides in the inner mitochondrial membrane. This unique feature explains the mitochondrion's great vulnerability to lipophilic molecules (Reference Degli69–Reference Don and Hogg71). Inhibition of complex III usually results in the generation of ROS as a consequence of the intrinsic characteristics of the electron-transfer process to this complex from reduced UQ (Reference Scatena, Bottoni, Botta, Martorana and Giardina72).
Our study also showed that chronic treatment with escitalopram does not alter citrate synthase activity such as other enzymes of Krebs cycle, succinate dehidrogenase and malate dehydrogenase. Together, they play an important role in brain energy metabolism. It is important to consider that we have some limitations such as the time of administration of the drug in this study. Moreover, we observed only the effect of the drug per se and a different effect may be found if we test the drug in an animal model of variant chronic stress.
On the basis of the hypothesis that metabolism impairment might be involved in the pathophysiology of depression, our findings of mitochondrial changes induced by escitalopram support the idea that mitochondrial dysfunction could be a primary event. Escitalopram treatment affects the metabolic parameters of brain. Our findings suggest that therapeutic or side-effects of escitalopram may be due to its differential effects on the specific biochemical metabolic parameters of both cortical and sub-cortical brain regions.
Acknowledgements
This research was supported by grants from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), Fundação de Apoio à Pesquisa Científica e Tecnológica do Estado de Santa Catarina (FAPESC) and Universidade do Extremo Sul Catarinense (UNESC).
