Author's Accepted Manuscript The Protective Effect Of Apocynin On Testicular Ischemia-Reperfusion Injury Ozkan Ozbek , Ramazan Altintas , Alaaddin Polat , Nigar Vardi , Hakan Parlakpinar , Mustafa Sagir , Zeynep Rumeysa Duran , Azibe Yildiz

PII: DOI: Reference:

S0022-5347(14)04934-9 10.1016/j.juro.2014.11.086 JURO 12021

To appear in: The Journal of Urology Accepted Date: 6 November 2014 Please cite this article as: Ozbek O, Altintas R, Polat A, Vardi N, Parlakpinar H, Sagir M, Duran ZR, Yildiz A, The Protective Effect Of Apocynin On Testicular Ischemia-Reperfusion Injury, The Journal of Urology® (2014), doi: 10.1016/j.juro.2014.11.086. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.

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ACCEPTED MANUSCRIPT THE PROTECTIVE EFFECT OF APOCYNIN ON TESTICULAR ISCHEMIAREPERFUSION INJURY

Ozkan Ozbek a*, Ramazan Altintas a,Alaaddin Polat b, Nigar Vardi c, Hakan Parlakpinar d,

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Mustafa Sagir d, Zeynep Rumeysa Duran b, Azibe Yildiz c

Department of Urology, Inonu University School of Medicine, Malatya, Turkey.

b

Department of Physiology, Inonu University School of Medicine, Malatya, Turkey.

c

Department of Histology and Histology, Inonu University School of Medicine, Malatya, Turkey.

d

Department of Pharmacology, Inonu University School of Medicine, Malatya, Turkey.

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a

*Correspondence to:, Ozkan OZBEK M.D.,

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Department of Urology, Faculty of Medicine, Inonu University, 44280, Malatya, Turkey. Telephone: + 90 414 511 30 07

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Telefax: + 90 414 511 07 55

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E-mail: [email protected]

Ozkan Ozbek: [email protected] Alaaddin Polat: [email protected] Nigar Vardi: [email protected] Hakan Parlakpinar: [email protected] Mustafa Sagir: [email protected] Zeynep Rumeysa Duran: [email protected] Azibe Yildiz: [email protected]

ACCEPTED MANUSCRIPT Abstract Purpose: We investigated the protective effect of apocynin, NADPH-oxidase inhibitor, on testicular damage induced by ischemia and injury (I/R) in rats. Materials and Methods: Thirty-two rats were randomly divided into a control group and

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3 I/R groups (4-h torsion followed by 1-h detorsion): group 1- control, only left scrotal exploration; group 2- I/R, left testicular torsion&detorsion; group 3- I/R + saline, left testicular torsion + 10 ml/kg saline intraperitoneally (i.p.) given at 210th min of

ischemia+detorsion; group 4- I/R + apocynin, left testicular torsion + 20 mg/kg apocynin

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(i.p.) given at 210th min of ischemia+detorsion. The histopathological findings and specific biochemical analyses were determined.

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Results: In I/R and I/R+S groups, malondialdehyde (MDA), total oxidative capacity (TOC) and oxidative stress index (OSI) levels were significantly higher, whereas, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and reduced glutathione (GSH) levels were significantly lower. Apocynin significantly reduced MDA, TOC and OSI levels and significantly increased SOD and CAT levels.

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There were significantly increase in the number of giant, degenerated and desquamated cells in I/R group. Apocynin treatment significantly improved these histological alterations.

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Conclusion: The beneficial effects of apocynin on testicular I/R injury were evaluated with histopathological and biochemical findings.

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Keywords: Testis, ischemia-reperfusion injury, apocynin, rat.

ACCEPTED MANUSCRIPT Introduction: Testis torsion, which is among the disorders that require immediate treatment, is seen at a rate of 1/4000 among males under the age of 25 [1]. If an accurate diagnosis is not made and if it is not treated appropriately, it causes irreversible ischemia and may result in the loss of the affected testis within a short time [2]. Reactive oxygen species (ROS) produced during oxidative stress and

reperfusion may result in irreversible problems [3].

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inflammation play an important role on the damage caused by torsion. The damage incurred with

Apocynin obtained from the roots of the Apocynum cannabinum plant is a substance that has the effect of an NADPH oxidase (NOX) inhibitor. Apocynin (4-hydroxy-3- methoxyacetophenone) is one of the most promising selective NOXes, which plays a role in the production of superoxide

(I/R) in a rat model [4]. It was observed that apocynin

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inhibitors. Altintas et al. demonstrated the positive effects of apocynin on renal ischemia reperfusion

strengthens antioxidant defensive systems and enhances the reduced glutathione (GSH), and it

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limits cellular stress triggered by ischemia [5]. Moreover, it was revealed that in the ischemic cerebral cortex, apocynin decreases GSH consumption, hydrogen peroxide formation (H2O2), and DNA fragmentation [6].

The current study was designed to determine the protective effect of apocynin on testicular I/R damage induced by testicular torsion. Our hypothesis puts forward the idea that

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the apocynin has a protective effect in ischemic tissue.

ACCEPTED MANUSCRIPT Methods: Animals and Experimental Protocol: The experimental protocol in the current study was approved by the Ethics Committee on Animal Research at Inonu University (protocol number: 2012/136), and the Guidelines for Animal Research from the National Institutes of Health publication were followed in the all experimental procedures. Thirty-two male, post-pubertal (12–16 weeks) Wistar Albino rats weighing

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250–290 g were obtained from Inonu University Laboratory Animals Research Centre and maintained at a constant temperature (21±2°C), humidity (60 ± 5%), and light (12:12-h light and dark cycle) controlled room, on a standard diet and water ad libitum. The rats were randomly divided into four groups (n=8), as follows: group 1- control, only underwent left scrotal exploration; group 2- I/R, left

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testicular torsion + detorsion (4-h ischemia followed by 1-h reperfusion); group 3- I/R + S, left testicular torsion + 10 ml/kg % 0.9 saline intraperitoneally (i.p.) given at the 210th min of ischemia + detorsion (4-h ischemia

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followed by 1-h reperfusion); group 4- I/R + apocynin, left testicular torsion + 20 mg/kg apocynin (Sigma–Aldrich; St. Louis, MO, USA) (i.p.) given at the 210th min of ischemia + detorsion (4-h ischemia followed by 1-h reperfusion). Experimental procedures and the dose of apocynin were selected according to previous studies [4]. Short term effects of ischemia were observed by sacrificing the rats one hour after the reperfusion Surgical Procedure:

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Anesthesia was performed to all study groups throughout the study by administering ketamine (i.p. 75 mg/kg) and xylazine (i.p. 5 mg/kg) combination. All groups were kept under anesthesia for five hours along.(tüm gruplar 5 saat anestezi altında tutuldu) The left testis was accessed through a scrotal midline incision, and the vascular structures of the testis were revealed. In

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group 1, the testis and the vascular structures were raised and immediately replaced without creating torsion in the scrotal sac, while in groups 2, 3, and 4, the testis was rotated 720º in a counterclockwise direction and fixed to the scrotum with two 4/0 silk sutures. The

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scrotum was then closed with 2/0 silk sutures. Thirty minutes prior to detorsion, group 3 was administered normal saline, and in group 4 apocynin was administered i.p. The scrotum was reopened after four hours of torsion and the testis was detorsed. To prevent hypothermia, the testis was covered with gauze wetted with lukewarm normal saline. After 1 hour of reperfusion, the left orchiectomy was performed and the removed testis was divided longitudinally into two equal-sized pieces, one section was placed in formaldehyde solution for routine histopathological examination by light microscopy. The other half of the testis was placed in liquid nitrogen and stored at -70°C until assayed for malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), reduced glutathione (GSH), myeloperoxidase (MPO), total oxidative capacity (TOC), total antioxidant capacity (TAC), and oxidative stress index (OSI) levels.

ACCEPTED MANUSCRIPT Biochemical analysis Determination of MDA production: The MDA contents of the homogenates were evaluated spectrophotometrically by measuring the presence of thiobarbituric acid reactive substances (TBARS) [7]. Three ml of 1% phosphoric acid and 1 ml 0.6% thiobarbituric acid solution were added to 0.5 ml of homogenate pipetted into a tube. The substance was heated in boiling water for 45 min. After cooling of the mixture, the colored part

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was extracted into 4 ml of n-butanol. The absorbance was determined by a spectrophotometer (UV1601; Shimadzu, Kyoto, Japan) at 532 and 520 nm. The amount of lipid peroxides was calculated as TBARS of lipid peroxidation. The results were given in nmol/g tissue. Determination of SOD activity:

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Total SOD activity was assessed according to the method of Sun et al. [8]. The principle of this method is the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide generator. One unit of SOD was measured as the enzyme amount

Determination of CAT activity:

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inducing 50% inhibition in the NBT reduction rate. SOD activity was given as U/g protein.

CAT activity was defined according to Aebi’s method [9]. The principle of this assay is based on the definition of the rate constant (k, s−1) or the H2O2 decomposition rate at 240 nm. The results were given as k/g protein. Determination of GPX activity:

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GPX activity was assessed by the method of Paglia and Valentine [10]. An enzymatic reaction in a tube containing NADPH, GSH, sodium azide, and glutathione reductase was initiated by the addition of H2O2, and the change in the absorbance at 340 nm was observed by a spectrophotometer. GPX activity was given as U/mg protein.

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Determination of GSH:

GSH concentration in the homogenate was defined spectrophotometrically according to the method of Ellman [11]. Each homogenate sample was mixed with 10 mM 5,5′-dithiobis (2acid)

in

100

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nitrobenzoic

mM

potassium

phosphate

buffer

(pH

7.5)

and

17.5

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ethylenediaminetetraacetic acid (EDTA). The reaction was started by adding 0.5 units of GSH reductase and 0.4 mM NADPH. The absorbance was determined at 410 nm after 5 min. The concentration of GSH was measured against a standard curve. The results were given in µmol/g tissue. Determination of MPO activity: MPO (EC 1.11.1.7) activity was defined by using a 4-aminoantipyrine/phenol solution as the substrate for MPO-mediated oxidation by H2O2 and a change in the absorbance at 510 nm was recorded. One unit of MPO activity was determined as the amount inducing degradation of 1 µmol H2O2/min at 25°C. The results were given in U/g protein [12].

ACCEPTED MANUSCRIPT Determination of TAC: TAC levels were defined spectrophotometrically with commercial kits (Rel Assay Diagnostics, Gaziantep, Turkey) [13]. The method is based on the disappearance/fading of the dark blue-green color of the stable 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical in proportion to the amount of antioxidants within the environment. The disappearance of this

according to the standard kit, and the results were given as mmol Trolox Eq/L. Determination of TOC:

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color increases parallel to the amount of antioxidants in the environment. Calculations were made

TOC levels were defined spectrophotometrically by using commercial kits (Rel Assay

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Diagnostics, Gaziantep, Turkey) [14]. This new method is based on the oxidation of ferrous ion-odianisidine complexes into ferric ions by the oxidant compounds within the samples. Spectrophotometric measurements were performed according to the color density that was obtained.

were given in mmol H2O2 Eq/L. Calculation of OSI:

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This measurement indicated the total amount of oxidant molecules within the environment. The results

OSI is the ratio of the TOC value expressed as mmol H2O2 Eq/L to the TAC value expressed as mmol Trolox Eq/L [15]. Histological evaluation

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The testis tissues obtained for histopathological examination were fixed in 10% formaldehyde. After fixation, the cleaned tissues were subject to dehydration and buffing, and then embedded in paraffin. Sections of 4-5 µm thickness were obtained from the paraffin blocks and stained with hematoxylin and eosin (H-E). The stained sections were examined under a Leica DFC-

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280 research microscope. In each section, 10 different areas were examined and the diameter of 20 tubules on each section was measured at x20 magnification, and the classification of the tubules was performed according to the spelling of germ cells into

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the lumen and the presence of multinucleated giant and degenerated cells. The separation of germinal epithelial cells and thereby falling off into the lumen is accepted as the sign of the disassociation of the intercellular connections [16]. Statistical Analysis

Histopathological data were analyzed using the SPSS software program for Windows,

version 17.0, (SPSS Inc., MedCalc 2007 Belgium). The normality of the distribution in the study 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 with Tukey's test for SOD, CAT, MPO, GPX, TAC, and TOC, and Tamhane’s test for MDA and GSH after the ANOVA test. The values are given as mean ± standard deviation (S.D.). Following a significant Kruskal–Wallis H-test,

ACCEPTED MANUSCRIPT Mann-Whitney U-test with Bonferroni correction was used as post-hoc for OSI. The results were expressed as median (minimum-maximum). For multiple comparisons, a value of p

The protective effect of apocynin on testicular ischemia-reperfusion injury.

We investigated the protective effect of the NADPH oxidase inhibitor apocynin on testicular damage induced by ischemia-reperfusion injury in rats...
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