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Abstract: Emotional stress can be viewed as a cause of adverse circumstances that induces a wide range of biochemical and behavioural changes. Oxidative stress is a critical route of damage in various psychological stress–induced disorders such as depression. Antidepressants are widely prescribed to treat these conditions; however, no animal study has investigated the effect of selective serotonin reuptake inhibitors (SSRIs) on the levels of intracellular reactive oxygen species in peripheral blood leucocytes of stressed mice. In this study, mice were immobilized for a period of 6 hr. Fluoxetine (5 mg/kg of body-weight) was administered 30 min. before subjecting the animals to acute stress. The level of intracellular reactive oxygen species in leucocytes of the peripheral blood of stressed mice was investigated using a 2′,7′-dichlorofluorescein diacetate probe, and the antioxidant response of fluoxetine was evaluated by superoxide dismutase, diaphorase, catalase and reduced glutathione. Our results show that restraint stress significantly increases the generation of reactive oxygen species in the peripheral defence cells. Treatment with fluoxetine partially reverses the adverse effects of stress. The improvement in cellular oxidative status may be an important mechanism underlying the protective pharmacological effects of fluoxetine, which are clinically observed in the treatment of depressive disorders.
Psychological stress has a number of characteristic effects on the human cellular immune system. It has been shown that peripheral numbers and function of natural killer cells, as a part of the innate immune system, are strongly affected by stress . These findings provide support for the association of stress with the increased severity of inflammatory diseases , as well as the severity and duration of bacterial and fungal infections [3,4].
Oxidative stress has been implicated in the pathogenesis of a variety of diseases [5–7] with reactive oxygen species as a part of the intracellular effectors of damage. Aversive stimuli like stress [8–10] or anxiety [11,12] induce peripheral oxidative stress, which leads to an increase in the generation of reactive oxygen species in peripheral blood lymphocytes, granulocytes and monocytes. Restraint has been extensively used to study the impact of stress on the process of disease as well as the effects of drugs in stress-related pathology in animals .
Antidepressant drugs are widely employed for the treatment of stress and stress-related depression and anxiety . Fluoxetine, a non-tricyclic antidepressant drug, effectively treats a wide spectrum of mood disorders  and protects against the adverse effects of different types of stressors [16,17]. Furthermore, it attenuates some effects of stress on the immune system [18,19] and protects against oxidative damage [20–22]. Fluoxetine has emerged as the treatment of choice for depression because of its safer profile, fewer side effects and improved tolerability compared with the older tricyclic antidepressants . However, the underlying mechanisms of its therapeutic efficacy remain unclear, particularly those in reference to its antioxidant activity in the immune system. To further elucidate this relationship, we investigated the effects of fluoxetine on the intracellular redox status of peripheral blood cells obtained from mice exposed to restraint stress.
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- Materials and Methods
Emotional stressors like restraint stress can influence the immunological responses, and complex mechanisms have been proposed for these effects. In this study, a protective effect of fluoxetine on leucocytes from oxidative stress was observed. The present results showed that exposure to restraint stress induced peripheral oxidative stress, which is defined by an increase in the generation of reactive oxygen species in peripheral blood lymphocytes, granulocytes and monocytes. These adverse effects were partially reversed by fluoxetine. The restorative action of fluoxetine was also associated with protective augmentation of endogenous antioxidant defences (superoxide dismutase, diaphorase and catalase) and restoration of non-enzymatic components of the antioxidant cascade (glutathione). The present findings show that fluoxetine is capable of alleviating oxidative damage induced by psychological stress on the peripheral immune system.
Our findings are in agreement with other studies showing that the production of reactive oxygen species by immune cells might be influenced by psychological stress. However, the available results in reference to the influence of psychological stress on the production of reactive oxygen species are contradictory. Several studies have observed an increased production of reactive oxygen species under psychological stress [8–10,31], while others have shown a decreased reactive oxygen species production [32–34]. This discrepancy may have been the result of a number of research design problems, including: (i) age  and sex ; (ii) intensity and type of stressor ; (iii) plasma concentration of catecholamines ; and (iv) lack of adequate non-stressed controls, which are very important since a circadian rhythm in the generation of these compounds has been described .
Overproduction of reactive oxygen species intermediates may cause instability in important macromolecules and represents the molecular basis of many diseases including cancer , neurodegenerative diseases  and infections . The antioxidant/oxidant balance is an important determinant of immune cell function . Decreased antioxidant status favours the accumulation of free radicals, which conducts to oxidative stress and significant damage to immune cells, leading to their dysfunction [39,40]. Reactive oxygen species accumulation can cause apoptosis in many different cell systems . Therefore, H2O2 induces apoptosis in neutrophils  and sodium arsenite in eosinophils , which can be prevented by catalase and glutathione or N-acetyl-cysteine, respectively. Taking into account the crucial role of these immune cells in the protection of the organism , our results support that stressed mice are predisposed to recurrent infection  and chronic inflammation , besides other pathologies .
The present investigation showed that fluoxetine significantly reversed restraint stress–induced oxidative damage. Furthermore, we observed that antidepressant administration per se did not alter the optimal antioxidant status of unstressed mice relative to controls, meaning that fluoxetine does not influence redox status in the absence of oxidative stress conditions. This finding suggests that this SSRI has an antioxidative effect against psychological stress–induced oxidative damage in rodents, in agreement with other authors [20,21]. However, it should be noted that there are major and important differences among these studies related to: (i) duration of stressor; (ii) research animal; (iii) studied tissue; and (iv) evaluated parameters. Zafir’s research group [20,21] used a chronic immobilization stress (over 21 days) in rats, whereas we employed an acute stress model in mice. Furthermore, Zafir et al. [20,21] measured enzymatic activities in brain and liver homogenates and in serum, while the present study investigated the intracellular level of reactive oxygen species and antioxidative molecules and enzymes within immune cells, either gated in blood or isolated in two fractions, i.e. polymorphonuclear and mononuclear. It is well known that exposure to stress induces a variety of autonomic, visceral, immunological and neurobehavioural responses and activation of the hypothalamo–pituitary–adrenal axis [46,47]. Many of these effects are thought to be mediated by stress-induced neurochemical and hormonal abnormalities that are often associated with oxidative stress [48,49]. Three main pathways of reactive oxygen species generation in the course of depression have been described: (i) deficiency of monoamines or increased metabolism of monoamines; (ii) increased glutaminergic transmission; and (iii) activation of immune and inflammatory response systems . Taking into account the latest available evidence, we believe that the potentially favourable antioxidant effect of the fluoxetine could be mediated by the three previously commented mechanisms. First, monoamines inhibit lipid peroxidation, eliminate free radicals and chelate iron ions, which are important elements of free radical reactions. It has been noted that fluoxetine restores not only normal metabolism of monoamines but also their physiological levels in synaptic clefts. Considering the reactive oxygen species–scavenging potential of monoamines, this effect of fluoxetine imposes a limitation on free radical reactions and concentration of their products . Secondly, increased glutaminergic transmission is characteristic of depression . Pathologically high levels of glutamate can cause excitotoxicity by allowing high levels of calcium ions to enter the cell, which, if present in excess, stimulate the production of reactive oxygen species. Fluoxetine has a cytoprotective effect involving limitation of overproduction of calcium ions . Thirdly, fluoxetine is capable of reducing the immune and inflammatory components [53–55] that favour the generation of reactive oxygen species [22,56]. This antidepressant drug has been shown to inhibit the expression of pro-inflammatory cytokines (e.g. tumour necrosis factor-alpha)  and prostaglandin E2  that are involved in enhancing reactive oxygen species . Its inhibitory effects have been suggested to be mediated, in part, by the protein kinase A . Additionally, the reduction in neutrophil counts by fluoxetine  limits the production of hypochlorus acid, which by reacting with reduced glutathione, decreases the amount of its form .
Our present data show that fluoxetine is effective to counteract the adverse effects of stress. Stressed mice might be more predisposed to diseases such as infections and chronic inflammation than non-stressed mice, as the oxidative stress is present in their peripheral defence cells. As treatment with fluoxetine ameliorates stress-induced oxidative damage, this study demonstrates that improvement in cellular oxidative status may be an important mechanism underlying the protective pharmacological effects of fluoxetine, which are clinically observed in the treatment of depressive disorders.