induced kidney and testis injury

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Kaldir M, Cosar-Alas R, Cermik TF, Yurut-Caloglu V, Saynak. M, Altaner S, Caloglu M, Kocak Z, Tokatli F, Türe M,. Parlar S, Uzal C (2008) Amifostine use in ...
ORIGINAL ARTICLE

Beneficial effects of aminoguanidine on radiotherapyinduced kidney and testis injury K. Ekici1, O. Temelli1, H. Parlakpinar2, E. Samdanci3, A. Polat4, A. Beytur5, K. Tanbek4, C. Ekici6 & I. H. Dursun7 1 2 3 4 5 6 7

Department Department Department Department Department Department Department

of of of of of of of

Radiation Oncology, Medical Faculty, Inonu University, Malatya, Turkey; Pharmacology, Medical Faculty, Inonu University, Malatya, Turkey; Pathology, Medical Faculty, Inonu University, Malatya, Turkey; Physiology, Medical Faculty, Inonu University, Malatya, Turkey; Urology, Medical Faculty, Inonu University, Malatya, Turkey; Medical Biology and Genetics, Medical Faculty, Inonu University, Malatya, Turkey; Medical Oncology, Medical Faculty, Inonu University, Malatya, Turkey

Keywords aminoguanidine—kidney—oxidative stress— radiotherapy—testis Correspondence Kemal Ekici, MD, Department of Radiation Oncology, Faculty of Medicine, Inonu University, 44280 Malatya, Turkey. Tel.: +90 422 341 0660-5603; Fax: +90 422 341 0728; E-mail: [email protected] Accepted: September 22, 2015 doi: 10.1111/and.12500

Summary This experimental study was designed to investigate both protective and therapeutic effects of aminoguanidine (AG), on radiotherapy (RT)-induced oxidative stress in kidney and testis. Forty rats were divided into five groups equally as follows: (i) control, (ii) RT, (iii) AG, (iv) AG+RT and (v) RT+AG group. Histopathological findings and biochemical evaluations, including tissue malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione (GSH), total oxidant status (TOS), total antioxidant capacity, oxidative stress index (OSI), blood urea nitrogen (BUN), serum creatinine (Cr) and testosterone levels, were determined. MDA, TOS and OSI were significantly higher in RT-treated groups, whereas SOD, CAT, GPX and GSH were significantly lower in these groups when compared with the control rats in the kidney and testis tissue. AG treatment significantly decreased MDA, TOS and OSI levels and increased SOD, CAT, GPX and GSH levels, when compared to the RT-treated groups in both kidney and testis tissue. BUN and Cr levels did not change among the groups, whereas testosterone levels were found as reduced in the RT-treated rats. AG treatment significantly augmented these hazardous effects of RT on testis tissue. According to our results, AG has beneficial effects against RT-induced kidney and testis injury.

Introduction Radiotherapy (RT) is considered to be one of the most important modalities for the cure of cancers and is a crucial component of the applications for many genitourinary cancers (Perez & Halperin, 2008). Ionising radiation is a well-established carcinogen, which occurs via the oxidative stress, and the mostly reported damaged part of the cells is DNA. The most of these DNA damages produced by ionising radiation are caused by hydroxyl radicals (OH) formed from radiolysis of water (Ward, 1988; Eroglu et al., 2008). Kidneys are one of the most radiosensitive abdominal organs. The renal tolerance dose is 20 Gray (Gy) in bilateral irradiation and renal function completely becomes lost in doses of 25–30 Gy (Kucuktulu, 2012). Radiation nephropathy includes perfusion © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 683–692

disturbance, increased vascular permeability, inflammatory reactions and fibrosis (Cassady, 1995; Cohen & Robbins, 2003; Kaldir et al., 2008). Also, testis tissue, another radiosensitive organ, has a variety of cells that differ in their degree of radiosensitivity. The spermatogonia are very radiosensitive and kill at doses less than 3 Gy in differentiation period (van Beek et al., 1986). The infertility following irradiation is caused due to apoptosis of spermatogonia instead of becoming differences (Kanter et al., 2010). Therefore, it seems to be related that the renal and testis tissue damages induced by RT treatment mostly occur via oxidative stress happened due to the overproduction of reactive oxygen/nitrogen species (ROS/ RNS) and a decrease in the antioxidant level. Aminoguanidine (AG) is an inhibitor of nitric oxide synthase (NOS) and has a high selectivity for the inducible 683

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isoform of this molecule (iNOS). Recent studies have indicated that nitric oxide (NO) is a mediator of radiationinduced acute tissue damage (Courderot-Masuyer et al., 1999). Also, AG has a cytoprotective effect via its antioxidant properties in vitro and in vivo. Many studies have reported that AG acts as an antioxidant and prevents the loss of antioxidant enzyme activities and cellular damage in rats (Huang et al., 2009; Abo-Salem, 2012). For instance, Parlakpinar et al. (2004) reported that AG has a protective effect on nephrotoxicity induced by amikacin and may improve the therapeutic index of amikacin. Also, Sahna et al. (2006) showed that AG has renoprotective effects against renal ischaemia–reperfusion injury at the biochemical level. More recently, Oguz et al. (2013) declared that AG treatment reversed 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced testicular damage in rats. The protective effect of AG has been thought to be related to its effects on reduction in free-radical production and amelioration in antioxidant contents in the renal and testis tissue. All of these studies mentioned above encouraged us to plan this experimental design for the first time. The aim of this study was to investigate both the protective and therapeutic effects of AG on 8 Gy single fraction RTinduced oxidative stress in rat kidney and testis in the light of the histopathological and biochemical evaluations. Materials and methods Animals and experimental protocol In this study, 40 male Wistar Albino rats of 10–12 weeks of age and weighing 250–300 g were obtained from Inonu University Laboratory Animals Research Center and placed in a temperature (21  2 °C) and humidity (60  5%) controlled room in which a 12 : 12 h light: dark cycle was maintained. This study was approved by Animal Ethics Committee (Reference Number 2014/A-24) and was conducted in accordance with ‘Animal Welfare Act and the Guide for the Care and Use of Laboratory animals (NIH publication No. 5377-3, 1996)’. The rats were randomly allocated into five groups with 8 rats in each as follows: control group (C), received only saline solution for 5 days intraperitoneally (i.p.); RT group, 800 cGy RT was applied to abdominopelvic region (APR) as a single fraction; AG group, AG (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) was given in a dose of 100 mg kg1 per i.p. for five consecutive days; AG+RT group, AG was given (100 mg kg1 per i.p.) for 5 days prior to RT; and RT+AG group, 800 cGy RT was given to APR as a single fraction according to our previous study (Cagin et al., 2015), and AG was given (100 mg kg1 per i.p.) for five days after 10 days follow684

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ing RT. In literature, there are different time intervals after the RT-induced injury in the experimental studies. This interval time varies from 3 days to 1 month (Seal et al., 2007; Somosy et al., 2002). As reported before, the initial phase of the effects of ionised radiation arises in the first 1–3 days. However, life-threatening findings occur in the two weeks after ionised radiation exposure. Therefore, in the current study, we chose the 10-day interval for formation of maximum tissue damage like Cihan et al. (2013). Also, the dosage of AG (100 mg kg1) was selected according to related previous studies (Parlakpinar et al., 2005; Xu et al., 2008; Ozturk et al., 2012). Experimental procedure Before RT application, the rats were weighted and anaesthetised with ketamine (30 mg kg1) and xylazine (5 mg kg1) i.p. and placed in supine position. External APR irradiation was given with a 30 9 30 9 5 cm animal-fixing box, in which eight rats were kept for each irradiation, using 6 MV photons from the linear accelerator machine. The dose was calculated as Dmax dose at 1.5 cm depth for skin source distance (SSD) 100 cm. After scarification by an overdose of the anaesthesia, the kidney and testis tissue specimens were quickly and meticulously harvested for biochemical and histopathological analysis. Also, right kidney and testis were placed in liquid nitrogen and stored at 70 °C until assayed for thiobarbituric acid-reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), total antioxidant status (TAS), total oxidant status (TOS) contents and oxidative stress index (OSI), and the left kidney and testis were placed in formaldehyde solution for routine histopathological examination by light microscopy. Biochemical analyses Two hundred milligrams of frozen tissue specimens cut into pieces on dry ice was homogenised in 1.15% KCl buffer (1 : 9, w/v) using a manual glass homogeniser for approximately 5 min and flushed with centrifugation for approximately 10 s to remove large debris. The supernatant was used for analysis. Determination of serum thiobarbituric acid-reactive substances representing malondialdehyde content Thiobarbituric acid-reactive substances contents of the homogenates were determined by TBARS (Uchiyama & Mihara, 1978). Three millilitres of 1% phosphoric acid and 1 ml 0.6% thiobarbituric acid solution were added to 0.5 ml of plasma pipetted into a tube. The mixture was © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 683–692

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heated in boiling water for 45 min. After the mixture had cooled, the colour was extracted into 4 ml of n-butanol. The absorbance was measured by a spectrophotometer (UV-1601; Shimadzu, Kyoto, Japan) at 532 nm. The amount of lipid peroxides was calculated as TBARS of lipid peroxidation. The results were expressed in nanomoles per gram (nmol g1 tissue) according to a standard graph, which was prepared from the measurements of standard solutions (1,1,3,3-tetramethoxypropane). Determination of superoxide dismutase Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined based on the method of Sun et al. (1988). The principle of the method is the inhibition of nitrobluetetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide (O2•) generator. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was expressed as units per milligram protein (U g1 protein). Determination of catalase Catalase (EC 1.11.1.6) activity was determined with respect to Aebi’s method (Aebi, 1974). The principle of the assay is based on the determination of the rate constant (k, s1) or the H2O2 decomposition rate at 240 nm. Results were expressed as k per gram protein (k g1 protein). Determination of glutathione peroxidase Glutathione peroxidase activity (EC 1.6.4.2) was measured by the method of Paglia & Valentine (1967). An enzymatic reaction in a tube containing NADPH, GSH, sodium azide and glutathione reductase was initiated with the addition of H2O2, and the change in absorbance at 340 nm was monitored by a spectrophotometer. Activity was given in units per gram protein (U mg1 protein). Determination of glutathione content The GSH content in the tissues as nonprotein sulfhydryls was analysed following a previously described method (Ellman, 1959). Aliquots of tissue homogenate were mixed with distilled water and 50% trichloroacetic acid in glass tubes and centrifuged at 2200 g for 15 min. The supernatants were mixed with Tris buffer (0.4 M, pH 8.9), and 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB, 0.01 M) was added. After shaking the reaction mixture, its absorbance was measured at 412 nm within 5 min of the addition of DTNB against blank with no homogenate. The absorbance values were extrapolated from a glutathione standard curve and expressed as GSH (lmol g1 tissue). Measurement of total antioxidant capacity Total antioxidant capacity (TAC) levels were determined using a novel automated colorimetric measurement © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 683–692

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method developed by Erel (2004). In this method, the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction and reacts with the colourless substrate O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in colour. Upon the addition of sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mixture are suppressed by the antioxidant components of the sample, preventing the colour change and thereby providing an effective measure of the TAC of the sample. The assay has excellent precision values, which are lower than 3%. The results were expressed as mmol Trolox equivalent l1. Measurement of total oxidant status Total oxidant status was determined using a novel automated measurement method developed by Erel (2005). Oxidants present in the sample oxidise the ferrous ionO-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a coloured complex with xylenol orange in an acidic medium. The colour intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay was calibrated with hydrogen peroxide, and the results were expressed in terms of lmol H2O2 equivalent l1. Measurement of oxidative stress index The percentage ratio of the TOS to TAC yields the OSI, an indicator of the degree of oxidative stress. OSI (arbitrary unit) = TOS (mmol H2O2 equivalent l1)/TAC (mmolTrolox equivalent l1). The OSI value for the lung samples was also calculated as OSI (arbitrary unit) = TOS (mmol H2O2 equivalent g1 protein)/TAC (mmolTrolox equivalent g1 protein). Also, trunk blood was extracted to evaluate serum levels of blood urea nitrogen (BUN) and creatinine (Cr), and testosterone using an Olympus Autoanalyser (Olympus Instruments, Tokyo, Japan).

Histological evaluations Testis and kidney tissue samples were fixed 10% neutral formalin solution. After routine tissue processing paraffin-embedded tissues, sectioned at 5 lm and stained with haematoxylin and eosine (H&E). Renal tissues were also stained with Masson’s Trichrome stain for fibrosis. All tissue samples were examined under light microscope. The kidney damage was scored according to the presence of tubular atrophy, and the testes damage was scored the 685

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status of seminiferous tubule morphology and spermatogenesis as none (0), mild (1), moderate (2) and severe (Kaldir et al., 2008). Statistics For detecting even minor effects, the required sample sizes used in this experiment were identified using statistical power analysis. The sample sizes necessary for a power of 0.80 were estimated using NCSS software. Data were analysed using the SPSS software program for Windows, version 15.0 (SPSS Inc., Chicago, IL, USA). The normality of the distribution was confirmed using the Kolmogorov– Smirnov test. According to the results obtained from the normality test, one-way analysis of variance (ANOVA) and the Kruskal–Wallis H test were used for the statistical analysis, as appropriate. Multiple comparisons were carried out by Tukey’s test (for homogeneous variances) after the ANOVA test. The results were expressed as mean  standard deviation (SD) for biochemical evaluations. P-values less than 0.05 were regarded as statistically significant.

Results Body weights None of the animals died during the experimental period. There was no difference between the body weights before and after the experiments among the groups (data not shown). Biochemical assessment All of biochemical results obtained from renal and testis tissue were presented in the Tables 1 and 2. Briefly, in the testis tissue, malondialdehyde (MDA), TOS and OSI were significantly higher, whereas SOD, CAT, GPX and GSH were significantly lower in the RT group when compared to the control levels. Furthermore, AG significantly decreased MDA, TOS and OSI levels, whereas increased SOD, CAT, GPX and GSH levels in the treatment when compared to the RT-treated rats (Table 1). In the renal tissue, oxidative stress parameters including MDA, TOS and OSI were significantly higher the RT

Table 1 Comparison of tissue oxidative stress parameters among the study groups in the testis tissue Parameters

Control

MDA (nmol g1 tissue) SOD (U mg1 protein) CAT (K g1 protein) GPX (U mg1 protein) GSH (l g1 tissue) TOS (lmol H2O2Eq l1) TAC (mmolTrolox Eq l1) OSI (Arbitrary unit)

15.74 0.76 2.78 230.66 1.25 2.54 0.84 3.01

RT        

3.01 0.06 0.68 35.22 0.17 0.63 0.12 0.75

39.44 0.54 1.52 134.23 0.81 6.12 0.98 6.47

RT+AG        

8.10* 0.03* 0.35* 52.51* 0.02* 1.04* 0.20 1.61*

22.64 0.80 2.49 228.07 1.25 3.19 0.94 3.57

AG+RT        

5.79** 0.11** 0.40** 35.35** 0.33** 0.50** 0.21 1.04**

22.97 0.77 2.44 224.48 1.17 3.17 1.00 3.27

AG        

6.67** 0.05** 0.20** 41.76** 0.27** 0.66** 0.22 0.89**

21.58 0.75 2.50 217.92 1.26 2.95 0.91 3.60

       

5.93** 0.09** 0.53** 27.02** 0.23** 0.78** 0.33 1.31**

Data were presented as meanSD. *Significant versus control. **Significant versus RT group.

Table 2 Comparison of tissue oxidative stress parameters among the study groups in the renal tissue Parameters

Control 1

MDA (nmol g tissue) SOD (U mg1 protein) CAT (K g1 protein) GSH (l g1 tissue) TOS (lmol H2O2Eq l1) TAC (mmmolTrolox Eq l1) OSI (Arbitrary unit)

6.92 1.33 21.62 4.80 5.81 0.64

     

RT 1.72 0.18 7.11 0.85 0.59 0.12

9.26  1.62

15.98 1.11 10.00 1.63 10.59 0.63

RT+AG      

2.15* 0.07* 2.96* 0.30* 1.14* 0.07

17.00  2.64*

8.03 1.18 21.19 4.70 5.95 0.65

     

AG+RT 0.70** 0.07 7.12** 0.54** 1.10** 0.13

9.32  1.89**

7.91 1.22 20.14 4.89 6.24 0.72

     

AG 1.47** 0.12 5.68** 0.31** 1.06** 0.13

8.76  0.87**

6.81 1.27 19.79 5.04 6.58 0.61

     

1.68** 0.13 4.43** 0.60** 1.10** 0.11

11.10  3.62**

Data were presented as mean  SD. *Significant versus control. **Significant versus RT group.

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group when compared with the control group. On the other hand, the antioxidants such as SOD, CAT and GSH were found as significantly lower in the RT group. In the both AG treatment groups (AG+RT and RT+AG), all of these oxidative stress markers were significantly lower in comparison of RT group levels (Table 2). Blood urea nitrogen and creatinine levels did not differ among the groups, whereas testosterone levels were found to be reduced in the RT-treated rats. AG treatment significantly augmented these hazardous effects of RT on testis tissue (Table 3). Histopathological results The testis tissue in both the control and AG given group showed normal histomorphology and spermatogenesis including spermatids and spermatozoa in the lumen (Fig. 1a and b). In the RT group of testis, the cells of the seminiferous tubules were disturbed. Vacuolisation, necrosis and multinucleated giant cell formation were observed in this group (Fig. 1c, d). The spermatid amount was revealed as reduced in the lumen of the tubules. The giant cell formation and vacuolisation of the tubular epithelial cells were found as reduced in the both AG+RT and RT+AG groups (Fig. 1e). Moreover, there were no differences for histomorphological findings in the both treatment groups. Renal tissues in the control (Fig. 2a and b) and AG-treated groups (Fig. 2c and d) had normal histomorphological appeared. On the other hand, renal tubular atrophy and fibrosis were seen in the RT group (Fig. 2e and f). However, there was no inflammation in this group. The histomorphological changes of AG+RT and RT+AG groups were similar. The fibrosis seen in the RT group slightly reduced in both AG+RT and RT+AG groups (Fig. 2g and h). However, tubular atrophy remained unchanged. Also, histopathological score of renal and testis damage was presented in the Table 4. Discussion In the current study, we established a kidney and tissue damage model by a single dose of radiation to the whole abdomen in the rats. Our results demonstrated that APR irradiation of 8 Gy as a single dose caused testis and

kidney injury in the rats and AG treatment exerted beneficial effects on these hazardous changes. It has been shown in many studies that testis and kidneys are highly sensitive to radiation injuries. Previously reported that administration of radiation to male patients with malignant diseases may cause temporary or permanent sterility by disruption of the normal cyclic process of spermatogenesis and impairment of fertility (Tarladacalisir et al., 2009). Radiation nephropathy is one of the side effects of RT application, and it is even more important in patients with longer life expectancy, especially in children since clinical findings and the time elapsed in a wide spectrum (Kucuktulu, 2012). It is well known that ionising radiation exerted oxidative stress on the testis and induced apoptosis primarily in the germ cells. Sensitivity to radiation-induced apoptosis was highest in the spermatogonia and spermatocytes, of which Sertoli and Leydig cells were comparatively resistant (Kanter et al., 2010). Infertility after irradiation is caused by the spermatogonia undergoing apoptosis instead of differentiation. The ability of the testis to recover spermatogenesis depends on the survival of some stem spermatogonia and their ability to repopulate the testis with differentiating cells. In some rat models, it is reported that administration of L-carnitine enhanced the spermatogenic recovery after irradiation in rats (Boekelheide et al., 2005). Recent studies indicate that the cells are endowed with cytosolic amplification mechanisms involving reactive oxygen and nitrogen species (ROS/RNS) and being responsive to low doses of ionising radiation. Also, some studies have reported that NO is an important mediator of RT-induced acute tissue damage. NO has been shown to have a cytotoxic function, and the toxicity of NO is due to both NO itself and NO-derived reactive oxidants (Beckman & Koppenol, 1996). Under conditions where the superoxide anion (O2) is generated, NO is rapidly consumed to produce the highly reactive ONOO, a potent oxidising agent known to initiate lipid peroxidation of biological membranes, hydroxylation and nitration of aromatic amino acid residues and sulfhydryl oxidation of proteins (Haddad et al., 1999). Aminoguanidine inhibits inducible iNOS in a selective and competitive manner, leading to decreased generation

Table 3 Results of BUN, Cr and testosterone in serum specimens Parameters 1

BUN (mg dl ) Cr (mg dl1) Testosterone (ng dl1)

Control

RT

RT+AG

AG+RT

AG

19.79  1.59 0.53  0.03 2.92  0.92

18.80  0.74 0.51  0.05 1.02  0.24

19.64  1.24 0.47  0.03 2.69  0.87

18.47  1.52 0.48  0.04 2.68  0.61

18.56  1.41 0.48  0.40 2.76  0.90

Data were presented as mean  SD.

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(a)

(b)

(c)

(d)

(e) Fig. 1 (a) Normal testis histology (Control group). H&E 9 100. (b) Normal testis histology (AG-treated group). H&E 9 100. (c) Multinucleated giant cells and vacuolisation in distributed testicular tubules (RT group). H&E 9 100. (d) Necrosis of testicular tubules in the central of picture (RT group). H&E 9 100. (e) Decreased vacuolisation and giant cells of tubule epithelium (RT-AG and AG-RT groups). H&E 9 100.

of NO. It is well established that AG inhibits in vitro and in vivo, the formation of highly reactive advanced glycosylation end products (AGEs) associated with the pathogenesis of secondary complications in diabetes mellitus and cardiovascular changes in ageing. AG ameliorates various complications in diabetes and prevents age-related arterial stiffening and cardiac hypertrophy, effects probably dependent on inhibition of AGEs formation (Sugimoto & Yagihashi, 1997). AG inhibits diamine oxidase; binds to sites of nonenzymatic glycosylation and prevents further advanced glycosylation. Recent studies have pointed antioxidant effects of AG and peroxynitrite scavenger effects (Mansour et al., 2002; Tilton et al., 1993). The protective effect of AG against ionising radiation has already been shown in rats with whole-body irradiation with a single dose of 8 Gy using NO-synthase inhibitors (Babicova et al., 2013). Also in recent studies, the therapeutic effect of AG against radiation in rat models has been shown on lungs and small bowels using antioxidant enzymes (Eroglu et al., 2008; Huang et al., 2009). To the best of our knowledge, there is no study in the literature

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regarding the use of AG against RT-induced testis and kidney injury. It is well known that antioxidant enzymes protect tissues against oxidative injury. Tissue damage is reported to occur after the antioxidants are depleted (Abraham et al., 2009). In the current study, MDA, TOS and OSI were significantly higher in the RT group in the testis tissue, whereas SOD, CAT, GPX and GSH contents were significantly lower when compared to the control group. These findings confirm that radiation causes oxidative stress by increasing MDA and freeradical production in the testis tissue. MDA is a commonly used as a marker to indicate the level of lipid peroxidation in the tissue. Also production of MDA is increased as a result of ROS formation due to the oxidation of unsaturated fatty acids in the cell membrane (Halliwell & Chirico, 1993). AG significantly decreased MDA, TOS and OSI levels, whereas increased SOD, CAT, GPX and GSH levels when compared to the RT group (Table 1). Also, serum testosterone levels were reduced in the RT-treated rats. AG treatment

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Fig. 2 (a) Normal kidney histology (Control group). H&E 9 100. (b) Normal kidney histology (Control group). Masson’s Trichrome 9100. (c) Normal kidney histology (AG group). H&E 9 100. (d) Normal kidney histology (AG group). Masson’s Trichrome 9100. (e) Focal fibrosis and tubular atrophy foci (RT group). H&E 9 100. (f) Focal fibrosis and tubular atrophy foci (RT group). Masson’s Trichrome 9100. (g) Slightly decreased fibrosis (RT-AG and AG-RT groups). H&E 9 100. (h) Slightly decreased fibrosis (RT-AG and AG-RT groups). Masson’s Trichrome 9100.

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

significantly improved testosterone levels in the AG+RT and RT+AG groups. Another important finding in the present study is that in the renal tissue, oxidative stress parameters including MDA, TOS and OSI were significantly higher in the RT group when compared to the control rats. On the other hand, SOD, CAT and GSH were found as significantly lower in the RT-treated rats. These findings demonstrate the hazardous effect of radiation on the antioxidant system. In the both AG treatment groups (AG+RT and RT+AG), all of these detrimental effects of RT were © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 683–692

significantly lower when compared to the RT-treated rats (Table 2). However, serum BUN and Cr levels did not differ among the groups. In accordance with our results, the protective effect of AG has been previously addressed in other models of cell damage induced by drugs. Mansour et al. (2002) noted that AG protects against nephrotoxicity induced by cisplatin in rats. Guney et al. (2007) reported that AG has direct scavenging activities against hydroxyl radicals. Also, Giardino et al. (1998) found that AG acted as an antioxidant in vivo, preventing ROS formation 689

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Table 4 Histopathological score of kidney and testis damage Score 0 Kidney Control RT AG+RT RT+AG AG Testis Control RT AG+RT RT+AG AG

Score 1

Score 2

n=6 n=6

n=2 n=2

Score 3

n=8 n=8

n=8 n=8 n=7 n=7

n=8 n=1 n=1

n=8

n = Number of the rat in the groups.

and lipid peroxidation in cells and tissues, preventing oxidant-induced apoptosis. When all observations were considered together, we reached to conclusion that AG can alleviate the detrimental effects associated with RT-exposure. The beneficial effect of AG is probably due to its antioxidant, free-radical scavenger, and protective lipid peroxidation effects; however, the exact mechanism(s) is not clear. AG may directly eliminate free oxygen radicals such as peroxynitrite or directly increase antioxidant enzyme activity and prevent inhibition of these enzymes. Our histopathological findings were parallel to the biochemical results. In particular, in the RT group of testis, the cells of the seminiferous tubules were disturbed and vacuolisation, necrosis and multinucleated giant cell formation were observed. Also, the spermatid amount was found as reduced in the lumen of the tubules. The giant cell formation and vacuolisation of the tubular epithelial cells were found as reduced in the both AG+RT and RT+AG groups AG treatment significantly reduced all of this hazardous tissue changes. Moreover, there were no differences for histomorphological findings in the both treatment groups. In the kidneys, renal tubular atrophy and fibrosis were seen in the RT group. The fibrosis seen in the RT group slightly reduced in the both AG+RT and RT+AG groups. However, tubular atrophy remained unchanged. Conclusions Testis and kidneys are highly sensitive to radiation, and dose-dependent toxicity occurs in this tissue (van Beek et al., 1986; Perez & Halperin, 2008; Kucuktulu, 2012). It is well established that after radiation exposure, elevated levels of free radicals in mitochondria cause DNA, protein and lipid damage (Xiao & Whitnall, 2009). In the current

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study, AG treatment significantly improved testis and renal histology and biochemical parameters when compared to the RT-exposed rats. This reflects the beneficial effect of AG treatment in limiting or preventing the oxidative damage in the tissue caused by RT-exposure. According to our results, it is possible to say that the antioxidant properties of AG treatment can help protect against the toxic effects of RT in rat testis and renal tissue. Despite the benefits of acute administration of AG, its long-term usage with larger samples and groups should be investigated with further studies. Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the manuscript. References Abo-Salem OM (2012) The protective effect of aminoguanidine on doxorubicin-induced nephropathy in rats. J Bıochem Molecul Toxicol 26:1–9. Abraham P, Rabi S, Selvakumar D (2009) Protective effect of aminoguanidine against oxidative stress and bladder injury in cyclophosphamide-induced hemorrhagic cystitis in rat. Cell Biochem Funct 27:56–62. Aebi H (1974) Catalase. In: Methods of Enzymatic Analysis. Bergmeyer HU (ed). Academic Press, New York, pp. 673– 677. Babicova A, Havlınova Z, Hroch M, Rezacova M, Pejchal J, Vavrova J, Chladek J (2013) In vivo study of radioprotective effect of NO-synthase inhibitors and acetyl-L-carnitine. Physiol Res 62:701–710. Beckman JS, Koppenol WH (1996) Nitric oxide, superoxide and peroxynitrite, the good, the bad and the ugly. Am J Physiol Cell Physiol 271:1424–1437. van Beek ME, Davids JA, de Rooij DG (1986) Variation in the sensitivity of the mouse spermatogonial stem cell population to fission neutron irradiation during the cycle of the seminiferous epithelium. Radiat Res 108:282–295. Boekelheide K, Schoenfeld HA, Hall SJ, Weng CC, Shetty G, Leith J, Harper J, Sigman M, Hess DL, Meistrich ML (2005) Gonadotropin releasing hormone antogonist (cetrorelix) therapy fails to protect nonhuman primates (Macaca arctoids) from radiation-induced spermatogenic failure. J Androl 26:222–234. Cagin YF, Parlakpinar H, Polat A, Vardi N, Atayan Y, Erdogan MA, Ekici K, Sarihan ME, Aladag H (2015) The protective effects of apocynin on ionizing radiationinduced intestinal damage in rats. Drug Dev Ind Pharm 22:1–8. Cassady JR (1995) Clinical radiation nephropathy. Int J Radiat Oncol Biol Phys 31:1249–1256.

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