Protective effects of melatonin on long-term administration of fluoxetine in rats

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Protective effects of melatonin on long-term administration of fluoxetine in rats ⁎

Majid Khaksara,c, Ahmad Oryana, Mansour Sayyaria, Aysa Rezabakhshb,c,1, , ⁎⁎ Reza Rahbarghazic,d,1, a

Department of Pathobiology, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran c Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran b

A R T I C L E I N F O

A B S T R A C T

Keywords: Fluoxetine Melatonin Pathology Biochemical–hematological parameters

The degree and consequence of tissue injury are highly regarded during long-term exposure to selective antidepressant fluoxetine. Melatonin has been shown to palliate different lesions by scavenging free radicals, but its role in the reduction of the fluoxetine-induced injuries has been little known. Thirty-six mature male Wistar rats were randomly assigned into control and experimental groups. The experimental rats were included as following; 24 mg/kg/bw fluoxetine for 4 weeks; 1 mg/kg/bw melatonin for 4 weeks; fluoxetine + 1-week melatonin, fluoxetine + 2-week melatonin and fluoxetine + 4-week melatonin. In the current experiment, we investigated weight gain, hematological and biochemical parameters, pathological injuries and oxidative status. We noted the positive effect of melatonin in weight loss of fluoxetine-treated rats (p < 0.05). The significant reduction of superoxide dismutase, glutathione peroxidase, catalase activities in blood, liver, and kidneys and changes in serum total antioxidant capacity caused by fluoxetine were reversed by melatonin (p < 0.05). Melatonin reduced the increased lipid peroxidation and transaminase activity in rats received fluoxetine (p < 0.05). We also showed the potency of fluoxetine in inducing leukopenia, thrombocytopenia and hypochromic and macrocytic anemia which was blunted by melatonin. Both RBCs and platelets indices were also corrected. Rats received melatonin in combination with fluoxetine showed a reduction in the severity of degeneration and inflammatory changes in different tissues, brain, heart, liver, lungs, testes and kidneys as compared to the fluoxetine group. Therefore, melatonin fundamentally reversed the side effects of fluoxetine in the rat model which is comparable to human medicine.

1. Introduction Due to the widespread occurrence of depression, not limited exclusively to mental suffering individuals, different psychopharmacological and antidepressant agents have been prescribed extensively in human medicine (McHenry, 2006). In 1988, the first selective serotonin reuptake inhibitors (SSRIs), named fluoxetine (Prozac), were introduced to human medicine. This drug has potential to increase 5-brain HT level in the serotonergic synaptic space (Anderson, 1998; Fuller, 1995). In comparison to any other available antidepressant, fluoxetine

possesses superior side effects because of its selectivity for serotonin receptors, especially following repeated administrations (Ferguson, 2001). Noticeably, the reductions of both appetite and body weight are considerable among its therapeutic effects (Chojnacki et al., 2015). As a consequence, prolonged use of fluoxetine contributes to desensitization of presynaptic 5-HT1A and 5-HT1B auto-receptors with augmentation of extracellular 5-HT (Blier and De Montigny, 1994). Many longitudinal and retrospective investigations revealed the occurrence of tissue toxicity such as sexual dysfunction, hepatotoxicity, nephrotoxicity and etc. following fluoxetine therapy (Inkielewicz-Stępniak, 2011;

⁎

Corresponding author at:Faculty of Pharmacy, Tabriz University of Medical Sciences, Daneshgah St., Tabriz 51664-14766, Iran. Corresponding author at:Tabriz University of Medical Sciences, Imam Reza St., Daneshgah St., Tabriz 51666-14756, Iran. Tel.: +984133373879; fax: +984133363870. E-mail addresses: [email protected], [email protected] (M. Khaksar), [email protected] (A. Oryan), [email protected] (M. Sayyari), [email protected] (A. Rezabakhsh), [email protected] (R. Rahbarghazi). 1 These authors contributed equally to this work. ⁎⁎

http://dx.doi.org/10.1016/j.etp.2017.05.002 Received 6 October 2016; Received in revised form 5 May 2017; Accepted 5 May 2017 0940-2993/ © 2017 Elsevier GmbH. All rights reserved.

Please cite this article as: Khaksar, M., Experimental and Toxicologic Pathology (2017), http://dx.doi.org/10.1016/j.etp.2017.05.002

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2.2. Experimental procedure

Modabbernia et al., 2012). The administration of this drug for pregnant women has been shown to be associated with aberrant or insufficient intrauterine growth and impaired somatosensory and psychomotor maturation in the fetus (Berg et al., 2013). Up to now, different underlying mechanisms related to the fluoxetine toxicity have been described (Herbet et al., 2014; Inkielewicz-Stępniak, 2011). For example, an excessive production of free radicals along with the inability of the antioxidant response system and accelerated lipid and protein peroxidation have been reported in rats receiving fluoxetine, orally (Inkielewicz-Stępniak, 2011). In a work done by Herbet and co-workers, they acclaimed that a 14-day treatment with 10 mg/kg fluoxetine increased the parameters of oxidative stress in rats (Herbet et al., 2014). Unlike some harmful effects, Corbett et al. however, showed a 10-fold increase in the rate of brain neurogenesis in the rat model when the animals were treated with fluoxetine (Corbett et al., 2015). A few studies exist regarding the effects of fluoxetine on tissues other than the brain. With this background, it seems that further investigations are essential to elucidate different effects of serotonin reuptake inhibitors in smart in vitro and in vivo models. Melatonin is a multifunctional indolamine that is preferentially produced in the pineal gland, although many researchers discovered the broad-spectrum effective role of melatonin biosynthesis in other organs such as skin, retina, and gut (Gonzalez-Arto et al., 2016; J Reiter et al., 2013; Maldonado et al., 2010). It has been characterized that melatonin acts synergistically with serotonin in different physiological phenomena. For instance, melatonin could regulate carbohydrate and lipid metabolism, appetite, and possesses remarkable anti-depressant activity (Wolden-Hanson et al., 2000; Zanuto et al., 2013). Administration of melatonin with antidepressants has been suggested for treatment of depressive disorders, especially in postmenopausal women (Hall and Steiner, 2013; Targum et al., 2015; Toffol et al., 2014). Among the numerous benefits promised, melatonin is publicly known as the best anti-oxidants, since it responds easily to free radicals and has the characteristic of free radical scavenging activity (Manchester et al., 2015; Zhang and Zhang, 2014). Unlike to other antioxidants, melatonin does not enter redox cycling without any potent pro-oxidants capacity (Bizzarri et al., 2013). All the above-mentioned realities pinpoint the necessity of melatonin administration in the treatment of complex depression disorders. In the current study, we have explored the possibility of using melatonin in reducing fluoxetine side effects in the model of rat. Therefore, we aimed to evaluate the effects of the combined administration of fluoxetine and melatonin on biochemical and hematological parameters and oxidative status. Amelioration of the pathological changes induced by fluoxetine was monitored in different tissues. The results of the current experiment could shed lights to neutralize the exacerbating effect of fluoxetine by a natural hormone.

Six different protocols were used to perform the in vivo assay. Protocol I: control rats (given 1 ml of normal saline orally for a period of 4 weeks); Protocol II: the rats only received fluoxetine (24 mg/kg/ bw; Cat no: F132, Sigma) (Inkielewicz-Stępniak, 2011) for over a course of 4 weeks; Protocol III: the rats received 24 mg/kg/bw fluoxetine for 4 weeks and 1 mg/kg/bw melatonin (Kireev et al., 2008) (Cat. no.: M5250, Sigma) solution during the first week; Protocol IV: the rats received 24 mg/kg/bw fluoxetine for 4 weeks with 2-weeks administration of 1 mg/kg/bw melatonin solution; Protocol V: the rats received 24 mg/kg/bw fluoxetine for 4 weeks with 4-weeks administration of 1 mg/kg/bw melatonin solution and finally Protocol VI: the rat received only 1 mg/kg/bw melatonin for 4 weeks. Drugs of each group were mixed by sterile normal saline and administrated once daily by using a gastric tube. Each group was included six rats. 2.3. Weight change assay To explore the side effects of fluoxetine on weight gain/loss, the rats were weighed at the beginning and end to the experiment. Thereafter, differences in the weight change were calculated. 2.4. The procedure of sample preparation To analyze the possible effects of fluoxetine and melatonin administration in in vivo model of rats, blood, serum and plasma samples, as well as different tissues lysates, were provided in accordance with manufacturers’ instructions. For hematological examinations, blood samples were taken from the jugular vein and pooled in tubes containing EDTA. Total blood cell counts with the percent of cell component were determined by using an automated hematology analyzer (Sysmex KX-21N). To harvest serum from each rat, the blood samples were allowed to clot in sterile tubes and centrifuged at 3000 rpm for 15 min. The supernatant sera from each sample were then collected and stored at −20 °C until use. Different chromogenic assay for each sample was performed using Alcyon 300 auto-analyzer (Abbott Co.). To harvest the tissue samples, 2 g of either hepatic or renal tissues were weighed, homogenized in the presence of a solution of 1.5% KOH and then centrifuged at 3000 rpm for 10 min. The supernatant fluids were subjected to both hematological and biochemical analysis. 2.5. Measurement of the enzymatic oxidative status response in plasma, renal and hepatic niches To assess the enzymatic antioxidant response, the activities of enzymes within the antioxidant systems such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (Cat) were monitored either in blood, kidneys, and liver. Shortly, the total activity of these enzymes was estimated in the plasma samples and tissue lysates, using Randox kit according to the manufacturer's instruction (Cat. no.: SD125; RS504; Antrim Co.).

2. Material and methods 2.1. Animals and ethical issues

2.6. Measurement of lipid peroxidation by TBARS assay

Thirty-six 1-month male Wistar rats at the normal weight, 85 ± 10 g, were purchased from Razi Institute (Karaj, Iran). The rats were allowed to acclimate to the convenient housing condition with standard temperature (20–24 °C), 12 h light/dark cycle on a 12-h light period between 6:00 a.m. and 6:00 p.m. and free access to feed. The daily time point administration refers to melatonin and fluoxetine at 6:00 a.m. before the light was switched on. After finishing treatment, rats in each group were euthanized immediately with high doses of Ketamine and Xylazine combination. All implementations of the current experiment were conducted in accordance with the previously published NIH standards (Publication No. 85-23, revised 1996) and approved by the Animal Care Committee of Tabriz University of Medical Sciences.

The Lipid peroxidation end-product status was assessed based on the serum and tissue malondialdehyde (MDA) level and subsequent generation of MDA-2-thiobarbituric acid (TBA) reactive substances as previously described (Malekinejad et al., 2012). A panel serial dilution of the standard solution of MDA including 0.5, 1, 2, 4, 8 and 12 nmol/ ml of 1,1,3,3-tetraethoxypropane was prepared. Then, 500 μl of the serum from each group was mixed with 3 ml of 1% phosphoric acid, vortexed for 5 min and 1 ml of 0.67% thiobarbituric acid overlaid and incubated in boiling water for 45 min. After cooling, 3 ml of n-butanol solution was added into the samples, mixed and centrifuged at 3000 rpm for 10 min. Ultimately, the absorbance of MDA−TBA adduct 2

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ameliorate the side effects of fluoxetine on weight loss in late stages.

was determined at 570 nm. The concentration of MDA was obtained using a standard curve of the MDA standard solutions.

3.2. Melatonin prohibited the lipid peroxidation and stimulated the antioxidant status

2.7. Serum total antioxidant status assay

The oxidative status and lipid peroxidation levels in terms of MDA, Cat, GPx, SOD activities in different tissues and TAC levels in blood were measured in the rats received melatonin, fluoxetine alone or their combination (Fig. 1b and the Supplementary Table 1). According to our results, there was a statistically significant decrease (p < 0.05) in liver and kidney, but not blood, oxidative enzyme levels in the fluoxetinetreated rats over a period of 4 weeks (Fig. 1. and the Supplementary Table 1). We obtained no significant result in SOD hepatic levels between control and fluoxetine rats (p > 0.05). Compared to increased hepatic GPx levels, the amount of Cat and SOD enzymes was decreased as compared to non-treated rats (Fig. 2). The levels of renal Cat, GPx and SOD contents were decreased in rats given fluoxetine. Melatonin in combination with fluoxetine successfully returned the blood and tissues antioxidant enzyme activity time-dependently (p < 0.05). We observed slightly, but not significant, increase to the levels of SOD enzyme in the liver of rats given combined regime versus the fluoxetine-treated group (p > 0.05). A trend similar to antioxidant enzymes as was obtained in TAC index (p < 0.05). As outlined in Fig. 1B and the Supplementary Table 1, the TAC activity in the blood of the group under single fluoxetine treatment was significantly lower than the other groups (pFlu(1m)versusFlu(1m)+Mel(4w)andMel(4w) < 0.05). Although the TAC capacity decreased in the rats given fluoxetine, but it did not reach to significant levels as compared to the control rats. Fluoxetine application caused a striking increase in the level of MDA liver but not in blood and kidney levels (Fig. 1b and the Supplementary Table 1). The decline in blood, liver and kidney MDA levels in the rats received melatonin or combined regimes were statistically significant compared to those of the fluoxetine group. Therefore, we imagined that administration of melatonin possibly blunted the adverse effects of fluoxetine on oxidative status and lipid peroxidation levels in blood and tissues examined.

Serum total antioxidant status (TAS) was calculated based on the peroxidase activity with the generation of blue to a green color appearance in the presence of 2,2′-azinobis-3-ethylbenzo-thiazoline-6sulfonic acid (ABTS). In brief, the freshly prepared sera were mixed with reactive reagents in accordance with manufacturer's recommendations (Cat No.: NX232; Randox). Absorbance was finally read at 600 nm and the TAS was expressed in mmol/l by the following formulas:

Factor = concentration of standard/ΔA blank − ΔA standard ; mmol/ l = factor × (ΔA blank − ΔA sample)

2.8. Assessment of the serum level of transaminase activity, alkaline phosphatase, and gamma-glutamyl transpeptidase The most important enzymes of pertaining to the healthy state function of liver, including, alanine amino transferase (ALT; Pars Azmun Co.), aspartate amino transferase (AST; Pars Azmun Co.), alkaline phosphatase (ALP; Pars Azmun Co.) and gamma-glutamyl transpeptidase (GGT; Pars Azmun Co.) were also measured as has previously been described (Aslani et al., 2013). 2.9. Histopathological assessment To evaluate the probable pathological lesions, tissues samples were provided from the brain, heart, lungs, liver, kidneys and testes. The samples were fixed in 10% neutral formalin, dehydrated in gradient ethanol solution (from 60 to 100%) and cleared by xylol. Next, 2 μm sections of the renal tissue and 6 μm sections from other tissues were prepared from the paraffin-embedded slices and then stained with hematoxylin−eosin (H & E). The stained slides were then imaged, using an Olympus microscopy (Tokyo, Japan).

3.3. Melatonin prohibited the adverse effects of fluoxetine on the serum level of AST, ALT, ALP and GGTP enzymes

2.10. Statistical analysis The data are expressed as the mean ± standard deviation (mean ± SD) and were analyzed using SPSS software package version 16.0. The data analysis between the groups was also performed via the one-way analysis of variance (ANOVA) with Duncan post hoc test. Totally, two rats were given fluoxetine and three rats with the combined regimen of fluoxetine and melatonin died over a period of the experiment. To avoid sample attrition, the dead rats were replaced to fulfill an equal number of rats per each group. The results of various groups were compared based on mean differences which were significant at p < 0.05. In histograms, the statistical difference between the groups is presented by brackets with *p < 0.05, **p < 0.01 and ***p < 0.001. The result of pathological changes was analyzed using Kruskal−Wallis test with the post-hoc Mann−Whitney test. We considered statistical significance at p < 0.05.

We followed the changes in clinical enzyme concentrations of AST, ALT, GGTP and ALP indicative of organs injury. Compared to the control and melatonin-treated values in all the rats, fluoxetine with or without melatonin increased the mean plasma levels of AST, ALT, GGTP and ALP (p < 0.05) (Fig. 2). Deviation from the normal levels in the rats that received fluoxetine alone was prominently shown, indicating the occurrence of liver toxicity. A heavy induction in the ALT level of serum was evident 4 weeks after fluoxetine administration (Fig. 2). This induction was markedly blunted when melatonin was used, especially after 1 week of administration (pFlu(1m)versusFlu(1m)+Mel(1w) < 0.01) (Fig. 2). The AST values were also increased significantly post-fluoxetine treatment compared to the baseline values (pFlu(1m)versusControl < 0.001). Similar to ALT, the co-administration of melatonin decreased the AST levels significantly (pFlu(1m)versusFlu(1m)+Mel(1w) < 0.001; pFlu(1m)versusFlu(1m)+Mel(4w) < 0.05; pFlu(1m)versusMel(1m) < 0.001) but similarly, the levels in different treatment protocols were still higher than the control levels through different treatment protocols (Fig. 2). Although, no significant difference was observed in the GGTP activity between the fluoxetine-administered rats and the untreated controls (p > 0.05), but melatonin treatment showed a downward trend and reached to near-normal levels (pFlu(1m)versusMel(1m) < 0.05) (Fig. 2). Corroborating to our findings, similar results were obtained for the ALP levels; this enzyme that was increased by fluoxetine intake (pFlu(1m)versuscontrol < 0.01 also decreased significantly in all the rats received melatonin (pFlu(1m)versusFlu(1m)+Mel(1w), Mel(2w)andMel(4w) < 0.01; pFlu(1m)versusMel(1m) < 0.05) (Fig. 2). The data obtained from the current experiment suggests that long-time administra-

3. Results 3.1. Fluoxetine resulted in weight loss The rats showed a decline in body weight 4 weeks after fluoxetine treatment as compared with the control groups (p < 0.001) (Fig. 1a). Notably, the application of melatonin significantly reduced the negative effects of fluoxetine on the growth rate after 4-week administration of melatonin when compared with the fluoxetine-treated rats and others (Fig. 1a). Collectively, our findings showed that chronic fluoxetine treatment led to weight loss in rats and melatonin had potential to 3

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Fig. 1. Enhanced weight loss in rats after exposure to fluoxetine over a period of 4 weeks (A). The weight loss was in the rats receiving the combined regime of fluoxetine and melatonin. The effects of fluoxetine, melatonin and their combination on the SOD, GPx and Cat levels, TAC, and MDA generation in blood, liver and kidney tissues (B). One-way ANOVA with Tukey post-hoc test. Differences between the control and treated groups are significant at *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 6) (SOD = superoxide dismutase; GPx = glutathione peroxidase; Cat = catalase; TAC = total antioxidant capacity; MDA = malondialdehyde).

4

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Fig. 2. Changes in the serum levels of ALT, AST, GGTP, and ALP in the control and chronically-isolated male rats treated with 24 mg/kg/bw fluoxetine and 1 mg/kg/bw melatonin or their combination for 4 weeks. One-way ANOVA with Tukey post-hoc test. Differences between the control and treated groups are significant at *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 6) (ALT = alanine transaminase; AST = aspartate aminotransferase; GGTP = gamma-glutamyl transpeptidase; ALP = alkaline phosphatase).

tion of fluoxetine exerts an adverse effect on the integrity of liver function tests surveyed. Application of combined regimes potently attenuated the increase in ALT, AST, ALP, and GGPT levels, indicating the inhibitory effect of melatonin against the liver insult by fluoxetine.

effect of antidepressant on the hematological parameters. 3.4. Fluoxetine-induced pathological injuries were attenuated by melatonin On histopathological examination of the tissues slides, we found different changes presented to the brain, heart, lungs, kidneys, testes and liver (Figs. 3–5 and Table 3). In all tissues, there were significant results between non-treated control and rats with fluoxetine alone or in combination with melatonin (Table 3). In all tissues, but not the brain, significant statistical differences were evident between Flu rats with Flu + Mel groups. We found non-significant pathological scoring in rats from Flu + Mel (4W) in testicular and renal tissues (Table 3). Fourweek treatment of the rats with fluoxetine (24 mg/kg/bw/per day) caused mild to moderate hemorrhage and congestion while other lesions such as mild demyelination with concurrent mild encephalitis and vasculitis were also observed (Fig. 3A: a-f and Table 3). No significant results were observed in fluoxetine rats as compared to fluoxetine plus melatonin groups (p > 0.05; Table 3). Large foci of hemorrhages and muscle fiber degenerations together with edema, hyperemia and myocarditis were presented in the cardiac tissue (Fig. 3B: a−f). In addition, we found significant results between fluoxetine and fluoxetine + melatonin (4W) rats (p < 0.01). Our results also confirmed various lesions including hyperemia, hemorrhage, edema, mild portal hepatitis, diffused necrosis of hepatocytes in the treatment groups (Fig. 3C: a−f and Table 3). It was well-established that fluoxetine can lead not only to brain and heart injury but also the lungs injuries were documented by the presence of bronchial-associated lymphoid tissue (BALT) hyperplasia, fibrinous bronchopneumonia, accumulation of fibrin network in the alveoli in fluoxetine group, chronic interstitial pneumonia, peribronchiolitis, infiltration of mononuclear inflammatory cells, hyperemia, fibrin clot in some alveols and bronchioles in Flu+ mel (1w) rats. In rats with the regime of Flu+ mel (2w) vivid hyperemia, peribronchiolitis, BALT hyperplasia, hypertrophy of the muscle fibers and fibrous connective tissue surrounding the bronchioles, chronic interstitial pneumonia were observed. In Flu+ mel (4w) group, chronic peribronchiolitis, mild hyperemia, chronic interstitial pneumonia, BALT hyperplasia, hypertrophy of the muscle fibers and fibrous connective tissue surrounding the bronchioles (Fig. 4A: a-l, Table 3). Significant pathological changes were recorded between Flu and Flu+ Mel (4w) (p < 0.01; Table 3). Based on the pathologic

3.3.1. Changes in the serum levels of biochemical parameters We also assessed the serum biochemical parameters in the different groups. The results, outlined in Table 1, indicate that the mean blood cholesterol, triglyceride, urea, phosphorus and uric acid levels increased significantly (p < 0.05) in the fluoxetine-treated rats in comparison to other groups. The total serum protein and HDL showed significant decline following fluoxetine treatment (p < 0.05). In spite of marked changes in the mean levels of glucose, calcium, magnesium, albumin and creatinine in the rats receiving fluoxetine, no significant differences were observed in the fluoxetine-treated rats with others (p > 0.05). The levels of cholesterol, triglyceride, glucose, phosphorus, urea, creatinine and uric acid decreased and closed to near-normal values in the rats undergone the combination treatment (Table 1). Our results confirmed the efficiency of melatonin to return the levels of blood biochemical parameters to the normal values in the rats treated with fluoxetine, indicating the restoration of hepatic and renal functions. 3.3.2. Melatonin blunted detrimental effects of fluoxetine on the hematological parameters To ascertain the possible effect of fluoxetine on hematological parameters, numerous indices in blood were measured. Based on our data, it was shown that long-term administration of fluoxetine led to leukopenia, anemia, and thrombocytopenia (Table 2). Treatment of rats with fluoxetine, over a period of 4 weeks, remarkably diminished the total WBC, RBCs and platelet counts compared to the control and melatonin-treated rats (Table 2). Unlike the changes in neutrophil count, we found a prominent lymphocytosis in these rats. Regarding the data in the anemia panel with a decrease in RBCs number, Hb, Hct, MCH and MCHC indices and increase in MCV value, we hypothesize that a hypochromic and macrocytic anemia occurred when rats received fluoxetine. We also showed destructive thrombocytopenia with prominent reduction in the number of platelets and increase in the PDW and MPV levels. Noticeably, the combination of fluoxetine with melatonin from 1 to 4 weeks potently blunted the detrimental 5

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findings related to testicular parenchyma, it seems fluoxetine easily passed through the blood−testis barrier or affected its physical integrity and elicited testis injury during the chronic administration of fluoxetine on the model of rat. Different lesions, including a dramatic reduction of spermatogenesis in the seminiferous tubules, were seen in the treatment groups in testicular tissue (Fig. 4B: a−f and Table 3). The influence of fluoxetine supplementation contributed to histopathological changes characterizing by massive hemorrhage, tubular necrosis is seen in the treatment groups (Fig. 5a−l and Table 3). We notified that the addition of melatonin resulted in the reduction of pathological changes in rats received the combined regime in testis and kidney (p < 0.05; Table 3). These findings collectively support the idea that different regimes of melatonin remarkably diminished the extent and severity of the lesions in various tissues elicited by the fluoxetine. Melatonin therapy was more effective when it was administrated for 4 weeks so that this therapeutic modality returned the injured organs to their original healthy state (Fig. 6).

60.0 ± 16.2 34.0 ± 0.6#

55.2 ± 7.4

58.4 ± 7.7 53.3 ± 6.1### 30.6 ± 1.9

Corroborating the results of the current experiment, enhanced lipid peroxidation with reduced TAC index and SOD, GPx and Cat activity were obtained when the rats given 24 mg/kg/bw fluoxetine over a period of 4 weeks suggesting oxidative status and deleterious effects of this reagent (Zlatković et al., 2014) while the effects of fluoxetine were evidently blunted by the combination of melatonin and fluoxetine. Different underlying mechanisms have been proposed for fluoxetine side effects in various tissues and organs (De Long et al., 2014). For example, it has been elucidated that this drug nonspecifically decreased the mitochondrial electron transport chain enzyme activity, F1F0ATPase activity and also initiated mitochondrial membrane permeability (De Long et al., 2014; Lee et al., 2010). These changes, in turn, reduced the oxidative phosphorylation rate in rats brain (Curti et al., 1999). In line with the generation of reactive oxygen species, fluoxetine could also up-regulate the multiple the genes involved in apoptosis and related signaling pathways (Lee et al., 2010). This drug has potential to up-regulate Bcl-2 in several cancerous cell lines by inducing cytochrome C release, and caspase activation, leading to DNA fragmentation (Lee et al., 2010). Consistent with our study, Djordjevic et al. previously detected increased antioxidant status and apoptotic signaling in the rat hepatic tissue during 21 days of fluoxetine administration (Djordjevic et al., 2011). Conflicting results have been elucidated regarding the dosage and duration of drug administration in in vivo condition (Nahon et al., 2005). Nahon et al. acclaimed that fluoxetine potentially inhibited the opening of the mitochondrial permeability transition pore thereby prevented the release of mitochondrial cytochrome C in response to the staurosporine-induced apoptosis (Nahon et al., 2005). Noticeably, Oryan et al previously acclaimed that administration of another S2-receptor blocker agent, named as metrenperone, healed the acute and chronic tendon injuries in the rabbit model (Oryan et al., 2009). Significant increase in the serum enzyme levels of ALT, AST, ALP and GGTP in our experiment mainly suggests hepatotoxicity effects of this drug. It is well-established that an active intermediate metabolite of fluoxetine, named norfluoxetine, is produced via extensive hepatic biotransformation in in vivo condition (Hiemke and Härtte, 2000). It has been shown SSRIs induced hepatotoxicity via oxidative/nitrosative stress (Zlatković et al., 2014). Notably, the accumulation of norfluoxetine in the hepatic tissue targets energy metabolism (Bendele et al., 1992). Due to the high levels of hepatic transaminase activity and thiobarbituric acid reactive substances in the serum of fluoxetine-treated rats, one could hypothesize that the hepatocytes lose their membrane integrity followed by increase of serum leaking enzymes. Our results affirmed the previous data of Inkielewicz-Stepniak, who acclaimed that chronic high dose of fluoxetine, raised the levels of MDA and transaminase activity in the serum (Inkielewicz-Stępniak, 2011). It has been stated that the fluoxetine-

93.3 ± 11.3 Data are expressed as mean ± SD (n = 6). Analysis was performed, using one-way ANOVA followed by a post hoc Tukey test. # p < 0.05. ### p < 0.001 (between fluoxetine only with fluoxetine plus melatonin). *** p < 0.001 (between the control animals and treated rats).

3.9 ± 0.6 0.5 ± 0.1 2.2 ± 0.3

60.7 ± 5.3

3.6 ± 0.7 0.5 ± 0.1 2.1 ± 0.2###

63.4 ± 6.4###

4. Discussion

9.8 ± 0.3 3.4 ± 0.1 7.9 ± 0.3###

3.3 ± 0.2

3.5 ± 0.1 7.1 ± 0.5***

3.6 ± 0.4###

10.3 ± 0.7

106.7 ± 17.0

67.0 ± 10.0 59.5 ± 7.5### 0.5 ± 0.1 2.0 ± 0.8###

62.0 ± 8.0###

3.4 ± 0.1

7.2 ± 0.8***,

#

3.5 ± 0.1

3.3 ± 0.1###

9.4 ± 0.6

119.5 ± 1.5

29.2 ± 2.2

Control Flu (1m) Flu (1m)+ Mel (1w) Flu (1m)+ Mel (2w) Flu (1m)+ Mel (4w) Mel (1m) 56.2 ± 3.7 70.3 ± 9.2# 55.0 ± 2.0# 56.5 ± 9.0 149.0 ± 8.3### 47.0 ± 12.0### 28.7 ± 3.5 28.1 ± 5.1# 29.2 ± 3.0 104.5 ± 24.5 121.3 ± 30.8 106.5 ± 7.5 10.1 ± 1.0 9.5 ± 0.7 9.0 ± 1.2 3.8 ± 0.4 5.7 ± 1.1### 3.5 ± 1.0### 3.5 ± 0.1 3.6 ± 0.2 3.4 ± 0.1 8.1 ± 0.8*** 6.2 ± 0.1 #, 7.0 ± 0.3*** 0.5 ± 0.4 0.6 ± 0.1 0.4 ± 0.1 2.1 ± 0.4 4.2 ± 0.1### 2.4 ± 0.2###

68.7 ± 3.7 81.0 ± 2.7### 53.5 ± 9.5###

3.8 ± 0.1 3.2 ± 0.2 3.5 ± 0.2

###

Magnesium (mg/dl) Total serum protein (g/dl) Albumin (g/dl) Urea (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl)

Blood biochemical parameters

Table 1 Effect of melatonin and fluoxetine on blood biochemical parameters.

Phosphorus (mg/ dl)

Calcium (mg/ dl)

Glucose (mg/dl)

Cholesterol (mg/dl) Triglyceride (mg/dl) HDL (mg/dl)

Groups

M. Khaksar et al.

6

18.8 ± 1.5

###

16.6 ± 1.1

###

13.4 ± 0.1 *** ### ,

***, ###

8.0 ± 0.9

***, ###

8.8 ± 0.1 *** ### ,

###

19.2 ± 2.5

8.0 ± 0.6

7.4 ± 0.3 *** ### ,

32.3 ± 0.3 ###

###

###

30.9 ± 1.9

##

30.3 ± 1.3

29.7 ± 0.8

#

27.7 ± 1.0 ** # ## ### , , ,

30.5 ± 0.5**

MCHC (g/dl)

882.2 ± 32.8

###

813.2 ± 45.4

###

736.1 ± 48.4

659.0 ± 51.3 *** ### ,

496.3 ± 43.0 *** ### ,

800.3 ± 73.6***

PLT (×103/μl)

17.4 ± 0.1

17.4 ± 0.4

17.2 ± 0.3

17.0 ± 0.2

57.1 ± 1.1, ### 57.0 ± 2.1 ### 56.9 ± 3.1 ### 53.9 ± 0.7 ###

###

###

##

##

###

43.1 ± 0.9 ###

###

32.9 ± 3.4 ***,

###

32.1 ± 3.5 ***,

###

31.2 ± 3.6 ***,

###

20.7 ± 3.5 ***,

19.1 ± 1.1 *, ##, 69.1 ± ### 3.7 ***,

Hct (%)

41.9 ± 1.6 ***

*

MCV (fL)

58.5 ± 3.9 ***

17.8 ± 1.0

MCH (pg/cell)

###

5.7 ± 0.8

###

5.4 ± 0.7

###

3.0 ± 0.4

***

13.9 ± 0.2 ###

8.0 ± 0.1

###

###

,

**

,

***

,

,

**

***

7.2 ± 0.6 *,

RBC (×106/μl)

10.4 ± 5.9 ± 0.8 *, 1.5 **, ###

9.9 ± 1.4 **, ###

9.4 ± 1.3 ***, ###

5.8 ± 1.1 ***, ###

12.8 ± 0.4 **, ***

Hb (g/dl)

#

68.2 ± 3.1

69.4 ± 10.0

66.0 ± 6.3

62.7 ± 2.5

#

,

**

**

78.3 ± 12.5

62.1 ± 5.6

Lymphocyte (%)

#

26.8 ± 3.1

25.6 ± 10.0

29.0 ± 6.3

32.3 ± 2.5

16.7 ± 12.5 ** # ,

**

32.9 ± 5.6

Neutrophil (%)

9.8 ± 1.0

,

***

###

##

***

***

***

8.8 ± 0.8 *,

6.2 ± 1.4

6.7 ± 2.0

6.1 ± 0.5 ## ### ,

10.9 ± 0.7 *,

WBC (×103/μl)

Mel (1m)

Flu (1m) + Mel (4w)

Flu (1m) + Mel (2w)

Flu (1m) + Mel (1w)

Flu (1m)

Control

Groups

Data are expressed as mean ± SD (n = 6). Analysis was performed, using one-way ANOVA followed by a post hoc Tukey test. # p < 0.05. ## p < 0.01. ### p < 0.001 (between fluoxetine only with fluoxetine plus melatonin). * p < 0.05. ** p < 0.01. *** p < 0.001 (between the control animals and treated rats). WBC = white blood cell; RBC = red blood cell; Hb = hemoglobin; Hct = hematocrit; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; PLT = platelet; RDW = red cell distribution width; PDW = platelet distribution width; MPV = mean platelet volume; P-LCR = platelet larger cell ratio.

6.3 ± 0.2 ***, ### 7.6 ± 0.9 ### 6.6 ± 0.3 ***, ### 6.9 ± ### 1.9 6.9 ± 0.7.2 ± 4 ***, ### 2.9 ### 7.2 ± 0.1 ## 7.6 ± ### 0.7

###

###

26.0 ± 2.1 *** ### ,

17.5 ± 0.1*-

**

11.0 ± 0.5*-

**

7.6 ± 0.3***

10.4 ± 0.6

RDW (%)

PDW (fL)

MPV (fL)

7.8 ± 0.12.9 ± 1 ##, ### *** 0.8 ,

8.0 ± 2.2***

P-LCR (%)

Hematological parameters

Table 2 Effects of melatonin on fluoxetine-induced hematological parameters.

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has a cytotoxic effect when cells expose to pharmacologic concentrations. Therefore, one could hypothesize that melatonin favors different effects in a dose-dependent manner (Blask et al., 2002). We also detected that long-term application of fluoxetine resulted in increased levels of cholesterol, triglyceride, glucose, phosphorus, magnesium, urea, creatinine and uric acid; although there was a reduction in the total amount of serum protein, albumin, and HDL concentration. These data evidently showed the kidneys and liver damage in the rats undergone fluoxetine therapy. Despite the fluoxetine-derived hepatotoxicity effect on the serum lipid profile, Karimi et al. showed the decrease of tetraiodothyronine (T4) hormone in the female guinea pigs treated (Karimi et al., 2012). Fluoxetine disrupts the mitochondrial electron transport chain in beta cells and causes insufficient ATP production thereby decreasing insulin release in response to high levels of blood glucose (De Long et al., 2014). We here reported the increase in the amount of urea and creatinine with a declined albumin level, in addition to the reduced hepatic biosynthesis, could be due to induced nephropathy. Higher levels of uric acid could also be related to elevated ALT activity during a hepatic injury (Afzali et al., 2010). Calling attention, chronic injection of fluoxetine in the model of rat attributed to an inappropriate secretion of antidiuretic hormone, resulting in hyponatremia via affecting inner medullary collecting ducts with simultaneous up-regulation of Aquaporin 2 (Moyses et al., 2008). In agreement with the previous study, Rouch et al. also showed a blocked water transport in the medullary collecting ducts by alpha-2 adrenoceptors during fluoxetine therapy (Rouch and Kudo, 2006). In this study, we also affirmed the potency of fluoxetine in inducing leukopenia (apparently due to neutropenia), anemia and thrombocytopenia. The high value of P-LCR could be associated with hypercholesterolemia and/or hypertriglyceridemia (Grotto and Noronha, 2004). Again, melatonin reversed these adverse effects. Pathological monitoring revealed the injury of brain, heart, lungs, kidneys, testis and liver. It is noteworthy that rapid contribution and high concentrations of the active metabolite of fluoxetine are responsible for clinical signs in different organs, especially in liver, lungs, and brain (Souza et al., 1994). Previously, different pathological aspects of SSRIs have been shown on testes. In parallel with the current study, a 60-day administration of fluoxetine in rats resulted in decreased testicular, epididymal, and prostate volumes with a reduced the number of sperms (Erdemir et al., 2014). Therefore, these data possibly imply that long-term application of fluoxetine makes the rats prone to infertility. The administration of fluoxetine up to 60 mg/day for 7 weeks, in human, results in 6% decrease in the heartbeat with 2 and 7% increase in systolic pressure and ejection fraction, respectively (Roose et al., 1998). In contrary, treatment of the elderly depressed people with 20 mg/day for 6 weeks did not have any detrimental effects on electrocardiographic and echocardiographic parameters (Strik et al., 1998). Regarding the toxic effects of fluoxetine on lungs, it has previously been shown that maternal use of SSRIs during the late pregnancy caused persistent pulmonary hypertension in neonates (Chambers et al., 2006). We here pinpointed that the pathological effects of fluoxetine on different tissues were significantly reduced in the rats received the combined regimen of melatonin and fluoxetine in comparison with the fluoxetine group. A great body of documents supports the notion that during the inflammatory and degenerative disease of various tissues melatonin acts as a non-enzymatic scavenger and enhancer of SOD, Cat, and GPx (Costa et al., 1995). Because of high grade of lipophilicity and easy entrance to intracellular space, melatonin protects lipid membranes against lipid peroxidation and subcellular compartments (Costa et al., 1995). A large number of extra-pineal tissues, however, possess the capacity to synthesize considerable levels of melatonin in which a 400-fold production has been recorded in the gastrointestinal tract (Huether et al., 1992). Modulations of PGE2, cyclooxygenase and nitric oxide synthase by melatonin in vascular endothelium are key points in the acceleration of tissues healing via effective refinement of free radicals and neutralization (Takeuchi et al., 1994).

Fig. 3. Histopathological changes in the rat brain and heart (H & E). Melatonin reduced the degenerative changes caused by chronic administration of fluoxetine. In brain tissue, we observed a diffuse pattern of central chromatolysis (arrow head), hemorrhagia (arrow) mild demyelination as well as mild encephalitis (asterisk). Panel B; Myocarditis: arrows and muscle fiber degeneration: arrow heads.

induced liver injury is associated with the depletion of the GSH content as a result of fluoxetine oxidation during the neutralization of hydrogen peroxide and lipid peroxides (Gupta et al., 2005). On the other hand, there is a reduction in the level of mitochondrial MnSOD and its activity decreased in the liver of the chronically-isolated rats with fluoxetine (Filipović et al., 2010). Solubilization of fluoxetine metabolites in the inner membrane of mitochondria is touted as other cytopathic effects of this reagent (Souza et al., 1994). Melatonin could counteract with oxidant effects of numerous drugs and toxic agents (Guven et al., 2016; Moreira et al., 2015). Short-term exposure of mononuclear cells in the peripheral blood to melatonin prohibits the overwhelming effects of H2O2 on MnSOD down-regulation while intensifying positive effects of free radicals on catalase mRNA expression (Emamgholipour et al., 2016). The hepatoprotective advantage of melatonin is not only depended on direct scavenging of the free radicals and up-regulation of the antioxidant enzymes, but it also correlates with other mechanisms (Guo et al., 2014). It has been elucidated that melatonin exerts its mitochondrial protective effects by up-regulating SIRT1/PGC-1 alpha pathway in HepG2 cell line (Guo et al., 2014). Melatonin represents different behavior in pharmacological and physiological concentrations (Blask et al., 2002). For instance, in physiologic concentrations melatonin only inhibits cancer cell multiplication via action in the cell cycle, while it 8

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Fig. 4. Histopathological changes in the rat liver (H & E). Melatonin attenuated the degenerative changes due to chronic administration of fluoxetine. Diffuse necrosis of hepatocytes: arrows, mild portal hepatitis: arrow heads.

Fig. 5. Representative images of pathological injuries in the lungs and testes (H & E). Our data revealed the potency of melatonin in reducing the adverse effects of fluoxetine in the rat lungs and testes. In testes, a dramatic reduction in the rate of spermatogenesis was confirmed in fluoxetine-treated rats. Panel A; arrows: BALT, black arrow heads: fibrinous bronchopneumonia, asterisk: hypertrophy of muscle fibers and chronic interstitial pneumonia: white arrow heads. Panel B; arrow: reduction of spermatogenesis.

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Table 3 Pathological findings scores recorded in each group. Tissues

Control Mel Median (min-max score)

Flu

Brain Heart Lung Liver Kidney Testis

1 0 1 0 0 0

4 4 4 4 4 4

(0−1) (0−1) (0−1) (0−1) (0−1) (0−1)

1 0 1 1 0 0

(0−1) (0−1) (0−1) (0−1) (0−1) (0−1)

Flu + Mel (1W)

(2−4)*** (3−4)*** (3−4)*** (2−4)*** (2−4)*** (3−4)***

4 3 3 2 3 3

(2−4)*** (3−4)*** (3−4)*** (2−3)***,# (2−3)***,# (4−2)***,#

Flu + Mel (2W)

3 3 4 3 2 2

(3−4)*** (3−4)*** (2−4)*** (3−2)*** (2−3)***,## (2−3)***,##

Flu + Mel (4W)

3 2 2 2 1 1

(3−4)*** (1−3)**,## (1−2)**,## (1−3)**,## (0−2)*** (0−1)***

The pathological changes were scored as follows; score 0: no pathological changes; score 1: mild changes; score 2: moderate changes; score 3: severe changes; score 4: very severe changes. Statistical differences between control and different groups: ### p < 0.001. ** p < 0.01. *** p < 0.001. Statistical differences between Flu + Mel (1W), Flu + Mel (2W) and Flu + Mel (4W) versus Flu group: # p < 0.05. ## p < 0.01.

Fig. 6. Photomicrographs of the rat kidneys at the different experimental protocol. Similar to other tissues melatonin potently neutralized nephrotoxicity of fluoxetine by reducing the rate of inflammation and injury. Arrows: tubular necrosis; arrow heads: massive hemorrhagia.

Conflict of interest

5. Conclusions

The authors declare no conflict of interest exists.

Collectively, the current study has shown that chronic administration of fluoxetine has the potency to reinforce liver, lung, brain, heart, kidneys and testes toxicity as shown by the high levels of serum transaminase activity, the presence of histopathological injuries and the modulation of biochemical parameters. In agreement with our data, melatonin fundamentally reversed the side effects of fluoxetine in the rat model which are comparable to human medicine. Although, there is no definite human medicine role of melatonin, thus, our results must be interpreted with caution for human counterpart. There are also some limitations associated with the current experiment. For example, we did not perform any morphological studies in blood cells. Since fluoxetine's primary action is on the CNS, future biochemical studies should have dealt with this issue either in molecular level on the topics of transcription and translation. Toward a better understanding of the cycle of glutathione, it is better to measure the levels and activity of GRd, GSSH, and GSSG.

Acknowledgment Tabriz University of Medical Sciences supported this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://(http://dx.doi.org/10.1016/j.etp.2017.05. 002) References Afzali, A., Weiss, N.S., Boyko, E.J., et al., 2010. Association between serum uric acid level and chronic liver disease in the United States. Hepatology 52, 578–589. Anderson, I., 1998. SSRIs versus tricyclic antidepressants in depressed inpatients: a metaanalysis of efficacy and tolerability. Depress Anxiety 7, 11–17. Aslani, M.R., Mohri, M., Movassaghi, A.R., 2013. Serum troponin I as an indicator of myocarditis in lambs affected with foot and mouth disease. Vet. Res. Forum 4, 59–62. Bendele, R.A., Adams, E.R., Hoffman, W.P., et al., 1992. Carcinogenicity studies of fluoxetine hydrochloride in rats and mice. Cancer Res. 52, 6931–6935. Berg, C., Backström, T., Winberg, S., et al., 2013. Developmental exposure to fluoxetine modulates the serotonin system in hypothalamus. PLoS One 8, e55053. Bizzarri, M., Proietti, S., Cucina, A., et al., 2013. Molecular mechanisms of the proapoptotic actions of melatonin in cancer: a review. Expert Opin. Ther. Targets 17, 1483–1496.

Funding This work was partially supported by Tabriz University of Medical Sciences.

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