Effects of melatonin and metformin co-administration on testicular

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Aug 21, 2016 - administration on experimental testicular ischemia/ reperfusion (I/R) .... injection (300 mg/kg, i.p.) at the end of the reperfusion period. The left testis ... formaldehyde), post-fixed in 70% alcohol, and embedded in paraffin blocks.

Journal of Pediatric Urology (2016) 12, 410.e1e410.e7

Effects of melatonin and metformin co-administration on testicular ischemia/reperfusion injury in rats


Department of Clinical Science, Science and Research Branch, Islamic Azad University, Tehran, Iran


Science and Research Branch, Islamic Azad University, Tehran, Iran c Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, Iran

Correspondence to: A. Asghari, Department of Clinical Science, Science and Research Branch, Islamic Azad University, Tehran, Iran, Tel.: þ98 914 414 7924; fax: þ98 214 486 8536 [email protected] (A. Asghari) Keywords Testis; Ischemia/reperfusion; Melatonin; Metformin Received 26 January 2016 Accepted 27 June 2016 Available online 21 August 2016

Ahmad Asghari a, Ghasem Akbari a, Afshin Meghdadi b, Pejman Mortazavi c Summary Introduction Torsion of the spermatic cord is a common urologic emergency among infants and adolescents. It requires early diagnosis and surgical intervention to prevent subfertility and infertility. Objective The aim of this study was to investigate the effects of melatonin (MEL) and metformin (MET) coadministration on experimental testicular ischemia/ reperfusion (I/R) injury in rats. Material and methods Fifty male Wistar rats were randomly divided into five experimental groups (n Z 10), as follows. Group 1 was sham operated. In group 2, 1-hour ischemia was induced by the left testicular artery and vein clipping followed by 7 days of reperfusion. In groups 3 and 4, MEL (3 mg/kg) or MET (100 mg/kg) was administered orally for 7 days via oral gavage after ischemia, and in group 5 both agents were co-administered. At the end of trial, the left testis was removed for histological analysis and oxidative stress measurement. Histological findings in seminiferous tubule were evaluated according to Johnsen’s scoring system.


Results I/R reduced superoxide dismutase (SOD) activities and testicular Johnsen’s scores accompanied by an elevation in malondialdehyde (MDA) and myeloperoxidase (MPO) levels (p < 0.05). MEL and MET, and their combination restored SOD activity, tissue scores, MDA and MPO levels (p < 0.05). There was no significant difference among individual or combined treatment of these parameters (p > 0.05). Discussion In the present experiment, using a rat model it has been demonstrated that testicular I/R caused a significant increase in testicular injuries. This was in accordance with previous studies that have demonstrated the effect of I/R in testicular tissue. Treatment of MEL and MET had a benefit effect, but, there was no significant difference among individual or combined treatment. Conclusions The results of the present study suggest that MEL and MET may be useful for protecting the testes from the I/R injury. However, the combined use of these agents does not further increase the protection from this damage.

Histological scores in the experimental groups.


Histological score

Sham Ischemia/reperfusion Melatonin Metformin Melatonin þ metformin

9.95 2.99 8.50 8.30 8.67

* p < 0.05 compared with the other groups. http://dx.doi.org/10.1016/j.jpurol.2016.06.017 1477-5131/ª 2016 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.


0.16 0.12* 0.52 0.52 0.53

Testicular ischemia/reperfusion injury in rats

Introduction Testicular torsion is a common urologic syndrome mainly caused by torsion of the spermatic cord that affects newborns, children, and adolescents. The primary pathophysiologic event in testicular torsion is ischemia followed by reperfusion; thus, testicular torsionedetorsion is an ischemia/reperfusion (I/R) injury to the testis. This syndrome often leads to male infertility. In the course of testicular I/R, overgeneration of reactive oxygen species (ROS) is a major initiating component of the testicular injury [1]. ROS, such as superoxide anions, hydrogen peroxide, hydroxyl radicals, and peroxynitrite anion, can oxidize cell membrane lipids, proteins, and DNA, leading to cellular dysfunction or death [2]. The neutrophil is one of important sources of ROS generation [3]. Enzymatic antioxidant defense systems such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) protect tissues from ROS and oxidative damage [1,4]. In recent years, many anti-inflammatory, antioxidants, and free-radical scavengers have been used for the treatment of testicular I/R-induced male infertility. Furthermore, treatment of antioxidants and ROS scavengers such as SOD, catalase, curcumin, allopurinol, N-acetylcysteine, resveratrol, and pentoxifylline have been proved by some research to prevent I/R injury in testes [5e8]. Melatonin (MEL, N-acetyl-5-methoxytryptamine) is a hormone produced by the pineal gland in a circadian rhythm and it is the most powerful endogenous antioxidant known [9]. Antioxidant effects of MEL can occur by either a direct or an indirect mechanism. MEL itself exerts direct antioxidant effects via scavenging the hydroxyl radical, peroxyl radical, singlet oxygen, peroxynitrite anion, and superoxide anion [9]. Additionally, it acts as an indirect antioxidant by stimulating several antioxidative enzymes, including glucose-6phosphate dehydrogenase, GPx, and SOD [10]. Metformin (MET) is a molecule of the biguanide family, and has the ability to decrease ROS [11,12]. At the cytoplasmic level, MET is able to lower the activity of mitochondrial complex I, which results in less ROS. Indeed, Zhou et al. [13] have described the activation of the AMP-activated protein kinase (AMPK) by MET. AMPK is a key regulator of cellular energy balance, and activated AMPK switches cells from an anabolic to a catabolic state. MET also has a beneficial effect on the cardiovascular system. The cardioprotective effect of MET has previously been investigated by Charlon et al. [14], who reported that MET treatment in rats for 5 days reduced infarct size by approximately 22%. Wang et al. [15] showed that low doses of MET may attenuate renal I/R injury by increasing the energy supply to the ischemic tissue and reducing the expression of inflammatory cytokines. In the present study, we aimed to evaluate the effects of MEL and MET co-administration on testicular damage in a rat testicular I/R injury model by assessing histological and biochemical parameters.

Methods Animals Fifty healthy adult male Wistar rats, (weighing 250e300 g) were purchased from the Pasteur Institute. They were

410.e2 maintained under constant room temperature of 22  1  C, relative humidity of 40  2%, on a 12-hour light/ dark cycle with commercial food and filtered tap water ad libitum. This study was conducted according to the guidelines of the animal care review board of the Islamic Azad University, Faculty of Veterinary Medicine, adhering to the guide for care and use of laboratory animals; the study was approved by the ethics committee (no. 41-0211/09).

Experimental groups The subjects were randomly divided into five experimental groups, each with 10 rats: group 1 (sham-operated group) were subjected to all operative procedures, except vessels occlusion. group 2 (I/R group) were subjected to I/ R. The animals in groups 1 and 2 received physiologic saline orally for 7 days via oral gavage. Group 3 (I/R þ MEL group) were subjected to I/R. A solution of 3 mg/kg MEL [16] in 0.9% saline solution was administered orally for 7 days via oral gavage after ischemia. Group 4 (I/R þ MET group) were subjected to I/R then were received oral administration of 100 mg/kg MET [17] in 0.9% saline solution for 7 days via oral gavage after ischemia. In group 5, a combination of MEL and MET (as above) was given in the same fashion.

Experimental protocol All surgical procedures were performed under anesthesia by intraperitoneal (i.p.) injection of 60 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride. After clipping, disinfecting with antiseptic povidoneeiodine solution, and draping, an abdominal incision was made; then the testicular artery and vein of the left testis were occluded with a mini vascular clamp for 1 h; after this process, the clamp was removed and the organ was allowed to reperfusion for 7 days. Sham operations were performed in a similar fashion, except the vessels were not clamped. The rats were euthanized by overdose of pentobarbital injection (300 mg/kg, i.p.) at the end of the reperfusion period. The left testis was harvested, cleared of adhering connective tissue. First, the testicle was divided into two by a sagittal section and one half was fixed in Bouin’s solution. The second half of the testicle tissue was stored at 80  C for the biochemical analysis.

Preparation of testicular tissue homogenates The sample of testicular tissues was washed three times in cold normal saline solution (0.9%). Then, the tissues were homogenized in ice-cold TriseHCl buffer solution within a homogenizer for 2 min at 11,200g. The homogenate was centrifuged at 3500g (4  C) for 60 min, and supernatant was obtained. The levels of myeloperoxidase (MPO) were determined in the supernatant, and malondialdehyde (MDA) levels were studied in the homogenate. For a further extraction procedure, the supernatant was extracted in ethanol/chloroform mixture (5/3 v/v). After a second centrifugation at 3500g for 20 min, the clear upper layer was taken and used for SOD activity determination [18].


Biochemical analysis Testicular injury was quantified by measuring testicular MPO activity, using a protocol described from the previous report [19]. The MPO activity was determined after adding O-dianisidine dihydrochloride and hydrogen peroxide. The rate at which a colored product formed during the MPOdependent reaction of O-dianisidine (0.167 mg/mL; Sigma Chemical Co., Gillingham, UK) and hydrogen peroxide (0.0005%; Sigma Chemical Co.) was measured at 460 nm for 3 min in a PerkineElmer (Waltham, MA, USA) spectrophotometer (Model 552A UV/VlS). One unit of MPO activity is defined as that which degrades 1 mmol of peroxideimin at 25  C. The tissue MDA level was determined by a method based on the reaction with thiobarbituric acid (TBA) [20]. In the TBA test reaction, MDA or MDA-like substances and TBA react with the production of a pink pigment with a maximum absorption at 532 nm. The reaction was performed at pH 2e3 at 90  C for 15 min. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid for protein precipitation. The precipitate was pelleted by centrifugation, and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm. The results were expressed as nmol/g tissue. Tissue SOD activity was measured according to the method of Paoletti and Mocali [21]. In brief, the superoxide anions were generated from manganese (II) chloride and mercaptoethanol in the presence of acidethylenediaminetetraacetic acid. The level of SOD was measured on the basis of its ability to inhibit nicotinamide adenine dinucleotide oxidation in reaction mixture after the addition of tissue homogenate. Nicotinamide adenine dinucleotide oxidation was measured by monitoring the decrease in absorbance at 340 nm during the reaction. The SOD activity was expressed as U/mg tissue.

Histologic evaluation The testes were fixed in Bouin’s solution (7.5 mL saturated picric acid, 2.65 mL glacial acetic acid, and 2.5 mL 7% formaldehyde), post-fixed in 70% alcohol, and embedded in paraffin blocks. Sections (5 mm) were obtained, deparaffinized, and stained with hematoxylineeosin. The testicular tissue was evaluated in random order with standard light microscopy by an observer who was unaware as to which group the rat had belonged. Histological findings in seminiferous tubule were evaluated according to Johnsen’s scoring system [22]. The Johnsen score is based on the premise that with testicular damage there is successive disappearance of the most mature cell type, with progressive degeneration of germinal epithelium, with the disappearance of spermatozoa and spermatids, then spermatocytes, and finally Sertoli cells, in that order. A score of 1e10 was given to each tubule according to the maturity of the germ cells: a score of 1 indicated no seminiferous epithelial cells and tubular sclerosis. A score of 2 indicated no germ cells, only Sertoli cells. A score of 3 indicated spermatogonia only. A score of 4 indicated no spermatids,

A. Asghari et al. few spermatocytes, and arrest of spermatogenesis at the primary spermatocyte stage. A score of 5 indicated no spermatids and many spermatocytes. A score of 6 indicated no late spermatids, few early spermatids, arrest of spermatogenesis at the spermatid stage, and disturbance of spermatid differentiation. A score of 7 indicated no late spermatids and many early spermatids. A score of 8 indicated few late spermatids. A score of 9 indicated many late spermatids and disorganized tubular epithelium. A score of 10 indicated full spermatogenesis.

Statistical analysis Data were expressed as mean  standard deviation (SD). The analytical results were evaluated using the Statistical Package for the Social Sciences (SPSS) version 18.0 software program. Control variables were compared among the groups by a one-way analysis of variance (ANOVA) using Tukey’s honestly significant difference test. The KruskaleWallis test was used to compare group medians for histopathological scores. Statistical significance was accepted as p < 0.05.

Results The experimental procedures were well tolerated and no animal died during the experiment. The values of the tissue biochemical measurements for the different groups are shown in Table 1. As seen in Table 1, I/R reduced SOD activities accompanied by an elevation in MDA and MPO levels when compared with the sham group (p < 0.05). MEL, MET, and combined treatment significantly enhanced the SOD activity (p < 0.05). Although the highest SOD activity was found in MEL þ MET group, there were no significant differences in SOD activity between MEL and MET alone and (MEL þ MET) combination groups. In the MEL þ MET group, the MDA levels were lower than those in groups MEL and MET, but there were no statistically significant differences between the MDA levels of these groups. The MPO levels were significantly lower in groups MEL, MET, and MEL þ MET when compared with I/R group. The MPO levels were lower in group MEL þ MET than in groups MEL and MET, and there were no significant differences between these groups. The testicular Johnsen’s score of all groups are shown in Fig. 1. In the sham group, normal histologic structures were observed in ultrastructural level in rat testis cross-sections (Fig. 2A). The testes in the I/R group were found to markedly increase disorganization, sloughing and loss of maturation of germ cells when compared with the sham group (Fig. 2B). The scores of the treated groups were significantly higher than that of the I/R group. There was no difference between MEL, MET, and MEL þ MET groups according to testicular Johnsen’s score (Fig. 2CeE).

Discussion Testicular torsionedetorsion is an I/R process of the testis. In the course of testicular I/R, ROS are overproduced. ROS are difficult to quantify directly in tissue because of their high reactivity and short half-life. MDA, a stable end

Testicular ischemia/reperfusion injury in rats Table 1


Tissue oxidative stress activities of the groups.


SOD (U/mg tissue)

Sham Ischemia/reperfusion Melatonin Metformin Melatonin þ metformin

3.68 2.65 3.27 3.19 3.45


0.18 0.21* 0.31 0.20 0.19

MDA (nmol/g tissue) 127.54 170.81 143.22 146.24 139.37


14.42 19.13* 13.70 14.15 13.81

MPO (U/g tissue) 4.22 5.37 4.50 4.61 4.34


0.23 0.82* 0.52 0.55 0.85

MDA Z malondialdehyde; MPO Z myeloperoxidase; SOD Z superoxide dismutase. * p < 0.05 compared with the other groups.

Figure 1

Histogram of histological score measurements in experimental groups. * p < 0.05.

product of lipid peroxidation generated by ROS, usually is used as an indirect indicator of ROS [1]. In our study, an elevated level of MDA in testes of the I/R group indicates increased oxidative stress. During I/R, increased production of ROS inflicts significant injury on ischemic tissue through oxidation of cell membrane lipids, proteins, and DNA. ROS stimulate the release and the formation of various inflammatory mediators with powerful chemotactic potential [1]. These mediators lead to leukocyte activation. Higher MPO activity was detected in testes in the I/R group. Increase in MPO activity in testicular tissue may reflect activation of neutrophils in the I/R condition. Increased oxidative stress during I/R, as demonstrated by greater levels of lipid peroxidation, was accompanied by a decreased level of antioxidant enzymes, such as SOD [3]. In this study, significant decreases occurred in the SOD activities in the I/R group compared with the sham group, revealing the decompensating antioxidant power of testicular tissue. Recent investigations on oxidative stress and ROS generation after testicular I/R suggest that treatment with

anti-inflammatory and antioxidant drugs can protect the testis against reactive oxygen species insult [23]. MEL is known to be a free radical scavenger and inhibits the peroxidation of membrane lipids [24]. Its lipophilicity ensures that MEL rapidly enters cells and may accumulate in the nucleus. Thus, MEL protects DNA against oxidative damage [24]. Kotler et al. [25] have shown that MEL increases the mRNA levels for GPx and SOD in the rat brain cortex. In addition, MEL modulates the androgen milieu and controls the immune response [26]. Gorman and Yellon [27] have shown that MEL stimulates testis development as well. The protective effect of MEL on lipid peroxidation has previously been shown in many investigations related to I/R injury in other tissues and this protective effect was reported to arise from its action as a direct free radical scavenger [10]. Because of its wellknown antioxidant characteristics, we investigated its effects in combination with MET on the present experimental model of I/R. MET is a biguanide drug that improves sensitivity to insulin, increases the insulin-stimulated uptake and


A. Asghari et al.

Figure 2 (A) Light microscopy of testicular tissue in sham-operated group. Normal testicular architecture was seen (seminiferous tubule, arrow, and interstitial cells, arrowheads). (B) The testicular tissue in ischemia-reperfusion group showed seminiferous tubular degeneration, reduction of germ cell layer number, and spermatogenous arrest (seminiferous tubule, arrow). (C) The testicular tissue in the melatonin group displayed normality of most seminiferous tubules (arrows). (D) The testicular tissue in the metformin treated group displayed normality of most seminiferous tubules (arrows). (E) The testicular tissue in the melatonin þ metformin treated group displayed normality of most seminiferous tubules, and abnormality of a few tubules (arrows). Hematoxylin and eosin staining; bar, 100 mm.

utilization of glucose, reduces basal hepatic glucose production, causes weight reduction, and decreases hunger [28]. Recent studies have recommended the use of this drug for kidney protection. These studies have suggested that MET has antioxidant activities too [29]. The apoptosis, induced by oxidative stress, in endothelial cells was reduced and the vascular dysfunction was prevented following MET therapy [28]. Recently, the ameliorative effect of metformin was demonstrated against I/Rinduced injury in rats [30]. Indeed, there is evidence that, when MET is used alone, the beneficial effect of MET might be due to its mild inhibition of the mitochondrial respiratory chain. It is also suggested that MET treatment may attenuate the increase in MDA and total ROS generation and restore the decrease in both enzymatic and nonenzymatic antioxidants [30,31].

We determined that MEL and MET, and their combination restored SOD activity, MDA and MPO levels, but there was no significant difference among individual or combined treatment of these parameters. In this study, we evaluated testicular injury by observing changes in tubular architecture and also by applying Johnsen’s scoring system. Using a rat model it has been demonstrated that testicular I/R caused a significant decrease in testicular score [32]. In our experiment, we observed morphologic changes in testes following testicular I/R. The Johnsen’s criteria decreased significantly in I/R groups compared with the sham animals. Treatment of MEL and MET had a significant benefit to the tubular architecture or Johnsen’s score after I/R. But, there was no significant difference among individual or combined treatment of Johnsen’s score.

Testicular ischemia/reperfusion injury in rats


Conclusion In conclusion, although various drugs and chemicals have been used to protect testes against I/R injury to date, there has been no research on the role of MEL and MET coadministration within the testicular I/R. The results of this study confirmed the protective effects of MEL and MET in the I/R insult. These effects may be, at least in part, due to the inhibition of ROS production. To the best our knowledge, the effects of these two substances were compared for the first time on testicular damage in this study. However, no additional effect was observed when these two agents were used together.





Conflict of interest None.





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