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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 [1]. These findings provide support for the association of stress with the increased severity of inflammatory diseases [2], 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 [13].

Antidepressant drugs are widely employed for the treatment of stress and stress-related depression and anxiety [14]. Fluoxetine, a non-tricyclic antidepressant drug, effectively treats a wide spectrum of mood disorders [15] 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 [23]. 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals.  Nine-week-old naive male Swiss albino mice (Charles River, Barcelona, Spain) weighing 30–40 g were used. Prior to experimental testing, they were housed in standard cages containing a supply of food pellets (SDS Dietex, France) and water available ad libitum. They were kept on a 12-hr light: dark cycle (light on at 6:00 p.m.), temperature-controlled condition (22 ± 2°C) and a relative humidity of 55 ± 10%. All experimental procedures in this study were in compliance with National Institutes of Health Guide for Care and Use of Laboratory Animals, the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Animal Welfare Committee of the University of Santiago de Compostela. In addition, all efforts were made to minimize animal suffering and to reduce the number of animals used.

Procedure.  Mice were randomly divided into six experimental groups of five animals each according to the treatment to which they were to be submitted: group 1, control (i.e. no stress or placebo); group 2, non-stressed mice injected with placebo; group 3, non-stressed mice injected with fluoxetine; group 4, stressed mice with no treatment; group 5, stressed mice injected with placebo; and group 6, stressed mice injected with fluoxetine.

Immobilization stress in mice.  Animals were restrained for as long as 6 hr in ventilated polypropylene conical tubes 7.5 cm long and 3.5 cm diameter at its widest point, tapering to a blunt end of 1.5 cm. The immobilization did not produce signs of pain in the animals, as judged from the lack of vocalization. The animals, however, were emotionally aroused and showed struggling, urination and defecation [24]. All stressed mice were subjected to the same stress schedule. In the non-stressed group, the mice were handled without stress.

Treatment with drugs.  Fluoxetine HCl was obtained as commercially available 20-mg capsules (Prozac; Lilly Co., Madrid, Spain), prepared following the technique of Brandes et al. [25] and subcutaneously injected at dosages of 5 mg/kg of body-weight, in a volume of 1 ml/kg of H2O. The same volume of diluent was used as placebo. Drug was administered 30 min. before the animals were subjected to immobilization stress.

Measurement of reactive oxygen species levels.  Flow cytometry was used to separate the different immune cell populations according to size (forward light scatter) and relative granularity (side light scatter). Forward light scatter and side light scatter were used after excitation of the immune cells with a 488-nm argon laser beam [11,26]. The level of intracellular reactive oxygen species was measured in lymphocytes, granulocytes and monocytes by monitoring the emitted fluorescence of these cells.

After 6 hr of immobilization stress, five mice from each experimental group were anaesthetized with halothane and then killed. Blood was collected in heparinized tubes. Erythrolysis was carried out using 2 ml of lysis solution (BD Bioscience, Paris, France) in 100 μl blood from each mouse. The samples were then placed in the dark for 10 min. After centrifugation (447 × g; 5 min.; 4°C), the supernatant was removed. Then, 2 ml of cell wash solution (BD Bioscience) was added to the sediment containing the white cells and mixed. A second centrifugation was carried out in the same conditions. The supernatant was again completely removed. One ml of Hanks’ balanced salt solution buffer and 5 μl of 2′,7′-dichlorofluorescein diacetate (50 μM) were added to the sediment (white cells) obtained by centrifugation, mixed and incubated in the dark for 15 min. at 37°C [11]. As soon as the incubation was completed, the samples were placed on ice for 5 min. to stop the reaction. The levels of reactive oxygen species of each mouse were measured using flow cytometry. Oxidation of the intracellular oxidation-sensitive probe resulted in increased mean fluorescence intensity (per cent of positive cells multiplied by the mean channel of fluorescence intensity of the positive peak) [27].

Preparation of mice blood cells.  Blood was collected from the inferior vena cava of the anaesthetized mice of each experimental group. The separation of mononuclear from polymorphonuclear cells was performed using the discontinuous Percoll (Amersham Biosciences, Barcelona, Spain) gradient method. Cell preparations were adjusted to 106 cells/ml for the assay. The viability of cells was estimated by trypan blue dye exclusion and was <95%.

Antioxidant investigations.  Mice leucocytes were used for the estimation of the antioxidant enzymes superoxide dismutase, diaphorase and catalase as well as the non-enzymatic antioxidant, glutathione, according to the following methods:

Superoxide dismutase activity.  The reaction mixture was obtained by mixing 0.3 ml of 13 mM methionine, 0.1 ml of 75 μM nitroblue tetrazolium, 0.3 ml of 0.1 nM ethylenediaminetetraacetic acid and 0.3 ml of 2 μM riboflavin in Hanks’ balanced salt solution. The 106/ml cells were incubated with 0.1 ml of Hanks’ balanced salt solution for 2 hr and with 1.3 ml of the reaction mixture. The absorbance was determined at 560 nm. A unit of superoxide dismutase was defined as the quantity of enzyme required to produce a 50% inhibition of nitroblue tetrazolium reduction under the specified conditions indicated above. Riboflavin loses one electron in the presence of light and triggers the generation of inline image, which reduces nitroblue tetrazolium to blue formazan [28].

Determination of diaphorase activity.  The 106/ml cells were incubated in the presence of 10 μl of potassium phosphate buffer with 40 μl of 7 μmol/ml nicotinamide adenine dinucleotide in 20 mM Tris–HCl buffer (pH 7.5), 0.2 ml of 0.2 M NaCl in Tris–HCl buffer and 1.650 ml of Tris–HCl buffer. The diaphorase activity of the leucocytes was assayed spectrophotometrically at 340 nm by following nicotinamide adenine dinucleotide oxidation [29].

Catalase activity.  The catalase activity of 106 leucocytes (mononuclear leucocytes and polymorphonuclear leucocytes) in the presence of 0.8 ml of potassium phosphate buffer and 0.4 ml of hydrogen peroxide solution was measured by monitoring the disappearance of hydrogen peroxide at 240 nm. The stock hydrogen peroxide solution employed was prepared with 85 μl of 30% (v/v) in potassium phosphate buffer, pH 7.0. One unit of catalase was defined as the amount of enzyme able to decompose 90% of the hydrogen peroxide [29].

Assay of glutathione.  The leucocyte glutathione level was determined colorimetrically on the basis of the absorbance of the reaction product of glutathione and 5,5 ′-dithiobis-2-nitrobenzoic acid [30]. The 106/ml leucocytes were incubated with 20 μl of glutathione reductase (6 U/ml), 50 μl of NADPH (4 mg/ml) and 20 μl of 5,5 ′-dithiobis-2-nitrobenzoic acid 1.5 mg/ml at 37°. The absorbance was determined at 415 nm. Glutathione levels were expressed per milligram of protein (μM/mg).

Statistical analyses.  Results are presented as mean ± S.E.M. Data were analysed by Student’s t-test. Statistical analysis was performed with SPSS 15.0.1. Mean differences with p < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Determination of reactive oxygen species by 2′,7′-dichlorofluorescein diacetate probe.

Differences in the intracellular redox status of the blood leucocytes between non-stressed and stressed mice were found (table 1). Results obtained using the Student’s t-test revealed that reactive oxygen species levels were significantly increased in peripheral blood lymphocytes (p < 0.001), monocytes (p < 0.01) and granulocytes (p < 0.001) of stressed mice compared with non-stressed animals. No difference was observed in intracellular levels of reactive oxygen species in each of the circulating leucocyte populations (lymphocytes, monocytes and granulocytes) among any of the non-stressed animal groups (p > 0.05). In contrast, treatment with fluoxetine partially reversed the adverse effects of stress on the oxidative status of mouse peripheral blood leucocytes. Fluorescence intensity appeared significantly decreased (p < 0.01) in all the studied cell populations of stressed mice injected with fluoxetine, compared with those of stressed mice injected with placebo, but still remained significantly (p < 0.05) elevated compared with non-stressed controls (fig. 1). This decrease is because of the effect of fluoxetine on reactive oxygen species level, which is directly correlated with fluorescence intensity.

Table 1.    Effects of stress on reactive oxygen species level in blood lymphocytes, monocytes and granulocytes oxidative status.
 LymphocytesMonocytesGranulocytes
  1. Mean fluorescent intensity (arbitrary units) of lymphocytes, monocytes and granulocytes is expressed as mean ± S.E.M. (n = 5). Fluorescence intensity emitted by immune cells is as a result of oxidation of intracellular 2′,7′-dichlorofluorescein diacetate by reactive oxygen species. Differences between non-stressed and stressed mice with no treatment were significant (p < 0.005; t = 11.119 to lymphocytes; t = 3.434 to monocytes; and t = 12.754 to granulocytes). Differences between non-stressed and stressed mice injected with placebo were significant (p < 0.01; t = 11.61 to lymphocytes; t = 3.27 to monocytes; and t = 6.868 to granulocytes). Differences between stressed mice with no treatment and those injected with fluoxetine (p < 0.05; t = 8.012 to lymphocytes; t = 2.126 to monocytes; and t = 9.157 to granulocytes) and between stressed mice injected with placebo or fluoxetine were significant (p < 0.01; t = 6.05 to lymphocytes; t = 3.107 to monocytes; and t = 5.56 to granulocytes). Differences between non-stressed mice with no treatment and those injected with placebo and between non-stressed mice with no treatment or injected with fluoxetine were not significant (p > 0.05).

Non-stressed
 Control144 ± 1541 ± 3398 ± 58
 Placebo129 ± 1848 ± 6412 ± 61
 Fluoxetine150 ± 1737 ± 4406 ± 48
Stressed
 Control212 ± 2162 ± 9476 ± 64
 Placebo200 ± 2868 ± 4454 ± 73
 Fluoxetine163 ± 1749 ± 7420 ± 55
image

Figure 1.  Intracellular levels of reactive oxygen species in circulating leucocytes of mice measured by flow cytometry. The oxidative status of lymphocytes, monocytes and granulocytes was quantified by monitoring the emitted fluorescence intensity resulting from oxidation of intracellular 2′,7′-dichlorofluorescein diacetate by reactive oxygen species. (A) Representative flow cytometric side light scatter versus forward light scatter dot plot of peripheral blood leucocytes of control Swiss albino mice. Region 1 (R1), Region 2 (R2) and Region 3 (R3) were defined to contain cells having typical forward scatter and side-scatter properties of lymphocytes, monocytes and granulocytes, respectively. (B) Representative flow cytometry histograms of peripheral blood leucocytes of: (i) control non-stressed animals; (ii) stressed mice injected with placebo; and (iii) stressed mice injected with fluoxetine. The fluorescence intensity was expressed as decimal logarithm versus the cell number.

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Effect of fluoxetine on restraint stress–induced decline in antioxidant enzyme activities.

Fig. 2 illustrates the effects of fluoxetine on leucocyte superoxide dismutase, diaphorase and catalase. No significant differences in antioxidant enzymes were observed from controls when fluoxetine was administered to non-stressed animals (data not shown). Results indicate that exposure to restraint stress significantly decreased all antioxidant enzyme activities (polymorphonuclear leucocytes – superoxide dismutase: p < 0.01; diaphorase: p < 0.01; catalase: p < 0.001; mononuclear leucocytes – superoxide dismutase: p < 0.01; diaphorase: p < 0.05; catalase: p < 0.001), as compared to non-stressed untreated controls, while antidepressant drug treatment to restraint-stressed animals significantly recovered enzyme activities (polymorphonuclear leucocytes – superoxide dismutase: p < 0.05; diaphorase: p < 0.01; catalase: p < 0.005; mononuclear leucocytes – superoxide dismutase: p < 0.01; diaphorase: p < 0.005; catalase: p < 0.001).

image

Figure 2.  Effect of fluoxetine administration on antioxidant enzyme activities in leucocytes. Results are given as mean ± S.E.M (n = 5). A, B and C: p < 0.05; p < 0.01; and p < 0.001, respectively, show significant differences between non-stressed control and stressed placebo groups; a,b,c and d: p < 0.05; p < 0.01; p < 0.005; and p < 0.001, respectively, show significant differences between stressed groups injected with placebo and fluoxetine. CAT, catalase; MNLs: mononuclear leucocytes; OD, optical density; PMNs: polymorphonuclear leucocytes; and SOD, superoxide dismutase.

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Effect of fluoxetine on restraint stress–induced decline in non-enzymatic antioxidant levels.

Restraint stress alone produced a significant decrease in leucocyte glutathione levels (p < 0.05), whereas no statistically significant differences were found relative to controls upon administering antidepressant drug to non-stressed animals. Administration of fluoxetine resulted in a significant recovery of glutathione (polymorphonuclear leucocytes: p < 0.001; mononuclear leucocytes: p < 0.01) in comparison with stress-induced decline in these levels (fig. 3).

image

Figure 3.  Effect of fluoxetine administration on glutathione reductase activity in leucocytes. Results are given as mean ± S.E.M (n = 5). A and B: p < 0.05 and p < 0.01, respectively, show significant differences between non-stressed control and stressed placebo groups; a and b: p < 0.01 and p < 0.001, respectively, show significant differences between stressed groups injected with placebo and fluoxetine. MNLs: mononuclear leucocytes; PMNs: polymorphonuclear leucocytes; and GSH: glutathione.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 [31] and sex [35]; (ii) intensity and type of stressor [36]; (iii) plasma concentration of catecholamines [36]; 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 [37].

Overproduction of reactive oxygen species intermediates may cause instability in important macromolecules and represents the molecular basis of many diseases including cancer [6], neurodegenerative diseases [5] and infections [7]. The antioxidant/oxidant balance is an important determinant of immune cell function [38]. 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 [41]. Therefore, H2O2 induces apoptosis in neutrophils [42] and sodium arsenite in eosinophils [43], 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 [39], our results support that stressed mice are predisposed to recurrent infection [44] and chronic inflammation [45], besides other pathologies [40].

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 [22]. 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 [50]. Secondly, increased glutaminergic transmission is characteristic of depression [51]. 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 [52]. 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) [53] and prostaglandin E2 [54] that are involved in enhancing reactive oxygen species [22]. Its inhibitory effects have been suggested to be mediated, in part, by the protein kinase A [53]. Additionally, the reduction in neutrophil counts by fluoxetine [55] limits the production of hypochlorus acid, which by reacting with reduced glutathione, decreases the amount of its form [56].

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Isabel Tarrío for her technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References