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Int Urol Nephrol (2014) 46:23012310DOI 10.1007/s11255-014-0833-8

UROLOGY - ORIGINAL PAPER

Prevention of cyclophosphamideinduced hemorrhagic cystitis by resveratrol: a comparative experimental study with mesna

Ibrahim Keles Mehmet Fatih Bozkurt Mustafa Cemek Mustafa Karalar Ahmet Hazini Saadet Alpdagtas Hikmet Keles Turan Yildiz Cavit Ceylan Mehmet Emin Buyukokuroglu

Received: 2 July 2014 / Accepted: 29 August 2014 / Published online: 24 September 2014 Springer Science+Business Media Dordrecht 2014

2, 3, 4, 5, and 6 received only CP, CP + 20 mg/kg RES, CP + 40 mg/kg RES, CP + 80 mg/kg RES, and CP + classi-cal protocol of three doses of mesna (30 mg/kg three times), respectively. Antioxidants, cytokines, and malondialdehyde levels were measured in all groups. In addition, histopatho-logical alterations in tissues were examined.Results CP administration induced severe HC with marked edema, hemorrhage, and inflammation in group 2. RES 20 mg/kg showed meaningful protection against blad-der damage compared to the control group. It was seen that RES 40 mg/kg gave weaker protection but RES 80 mg/kg was not found to be effective.Conclusion In conclusion, marked bladder protection was found in 20 and 40 mg/kg RES applications compared to the control group, but this protection was weaker than with mesna.

Keywords Hemorrhagic cystitis Cyclophosphamide Resveratrol Mesna Rat

Introduction

Hemorrhagic cystitis (HC) is commonly encountered in patients undergoing cyclophosphamide (CP) therapy, and this limits the high-dose clinical use of CP [1]. The inci-dence of HC ranges from 2 to 40 %, with high mortality reported with untreated massive hemorrhagia [2]. This may reach 68 % in patients undergoing bone marrow transplan-tation [3, 4]. The main features of HC are urothelial dam-age, edema, necrosis, ulceration, hemorrhage, neovascu-larization, leukocyte infiltration [5], and transitional cell carcinoma of the bladder [4, 6].

The alkylating agent CP is used alone or in combina-tion with other antineoplastic drugs for the treatment for

Abstract Purpose Hemorrhagic cystitis (HC) is the most common urotoxic side effect of cyclophosphamide (CP). The aim of this study was to compare the classical efficacy of mesna (2-mercaptoethane sulfonate sodium) with three different doses of resveratrol (RES) on cyclophosphamide-induced HC in rats.Methods Forty-six male SpragueDawley rats were divided into six groups. Group 1 served as a negative control (sham). Five groups received a single dose of cyclophospha-mide (150 mg/kg) intraperitoneally at the same time. Groups

I. Keles (*) M. Karalar Department of Urology, Faculty of Medicine, Afyon Kocatepe University, Adnan Kahveci Bulvar No:67/1 Seluklu Mah. Seluklu Konaklar A Blok Kat 3 daire:7 Uydukent, Afyonkarahisar, Turkeye-mail: drkeles@hotmail.com

M. F. Bozkurt H. Keles Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyonkarahisar, Turkey

M. Cemek A. Hazini S. Alpdagtas Department of Bioengineering (Biochemistry Division), Faculty of Chemical and Metallurgic Engineering, Yldz Technical University, Istanbul, Turkey

T. Yildiz Department of Pediatric Surgery, Faculty of Medicine, Sakarya University, Sakarya, Turkey

C. Ceylan Department of Urology, Ankara Yksek Ihtisas Training and Research Hospital, Ankara, Turkey

M. E. Buyukokuroglu Department of Pharmacology, Faculty of Medicine, Sakarya University, Sakarya, Turkey

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various solid tumors, hematological malignancies, auto-immune disorders, and for bone marrow transplantation [1, 7]. CP is a prodrug that is converted to its metabo-lites phosphoramide mustard (active) and acrolein by hepatic microsomal enzymes. Acrolein is highly toxic to the urinary bladder, promoting the release of inflamma-tory mediators that ultimately lead to HC [810]. Acr-olein enters rapidly into the uroepithelium because of its chemical nature and causes increased reactive oxygen species (ROS) production and both directly and indi-rectly iNOS induction leading to NO overproduction in the bladder epithelium. Acrolein induces intracellular transcription factors such as NF-B and AP-1. Activated NF-B and AP-1 cause cytokine (TNF-, IL-1) gene expression, iNOS induction, and ROS production. Thus, the production of harmful molecules (cytokines, ROS, NO) increases dramatically. ROS attacks cellular macro-molecules (lipids, proteins, and DNA) and causes dam-age [11].

Treatment for cystitis is usually performed with the use of mesna (2-mercaptoethane sulfonate sodium), which can bind to and inactivate acrolein in the urinary bladder or other parts of the urinary tract [11, 12]. The administra-tion of mesna with CP is a practice derived primarily from investigations with ifosfamide (IFO), a structural analog of CP [13]. The free sulfhydryl group found in mesna com-bines directly with the double bond of acrolein as well as other urotoxic 4-hydroxyoxazaphosphorine metabolites [14, 15]. These factors contribute to the inhibition of acr-olein binding to cell surface proteins in the bladder, thereby potentially limiting CP-associated toxicities such as cysti-tis and bladder cancer [13]. Detoxifying acrolein does not prevent HC symptoms completely [7, 16]. The application of plant extracts that contain antioxidants to scavenge the harmful effects of CP and ROS has attracted worldwide interest [17, 18].

Resveratrol (RES), a natural grape-derived polyphe-nolic phytoalexin, possesses pleiotropic effects, including anticancer, anti-aging, anti-inflammatory, and antioxidant activities [19, 20]. It is both a free radical scavenger and a potent antioxidant because of its ability to promote the activities of a variety of antioxidant enzymes [21]. Previ-ous reports have shown that RES can ameliorate several types of renal injury, such as diabetic nephropathy, drug-induced injury, aldosterone-induced injury, ischemiareperfusion injury, sepsis-related injury, and unilateral ureteral obstruction in animal models through its anti-oxidant effect [22]. Again, antioxidants act against CP-induced HC by maintaining antioxidant enzyme activi-ties as well as by re-balancing redox status [2325]. The aim of this study was to compare the classical efficacy of mesna with three different doses of RES on CP-induced HC in rats.

Materials and methods

Animals

The Animal Care Committee of Afyon Kocatepe Univer-sity approved this experimental study. A total of 46 male SpragueDawley rats (225250 g) were divided into six groups by a simple random sampling method and were maintained on a 12:12-h light/dark cycle with free access to water and food.

Drugs and chemicals

Cyclophosphamide (CP) (Endoxan 150 mg/kg Eczacbas Istanbul, Turkey), RES (Sigma, Germany), and mesna (Uromitexan 400 mg, Eczacbas Baxter, Turkey) were used. All drugs were diluted in 0.9 % sterile saline.

Hydrogen peroxide, GSH, thiobarbituric acid, phos-phate buffer, butylated hydroxytoluene, trichloroacetic acid, EDTA, [5,5-dithiobis-(2-nitrobenzoic acid)], diso-dium hydrogen phosphate, phenylendiamine, sodium azide, 2,4-dinitrophenylhydrazine, ethanol, hexane, sodium nitrite, sodium nitrate, sulfanilamide, N-(1-Naphthyl) eth-ylenediamine dihydrochloride, and vanadium (III) chlo-ride were purchased from Sigma Chemical Co. All other chemicals and reagents used in this study were of analytical grade. In addition, TNF- (Invitrogen, USA), IL-10 (Cusa-bio, USA) superoxide dismutase (SOD), and glutathione peroxidase (GPx) commercial kits (Randox, UK) were used.

Hemorrhagic cystitis model

Animals were injected intraperitoneally (i.p) with 150 mg/kg CP in 2-ml saline according to the methods described by Botta et al. [26]. After 48 h of HC induction, rats were killed using i.p. injection of ketamine HCL 80 mg/kg and xylazine HCL 10 mg/kg. Following an abdominal incision, the bladders were removed and emptied of their urinary contents.

Experimental protocol

The animals were divided into six groups of eight rats each, only group 1 consisted of six rats. Group 1 (sham) animals were given an i.p injection of 2-ml saline while group 2 (control) animals received 2 ml of CP (150 mg/kg) intraperitoneally. RES (groups 3, 4, 5) at three different doses of 20, 40, and 80 mg/kg in 2-ml saline was adminis-tered 20 min before CP injection (i.p). A total of 90 mg/kg mesna (group 6) was administered in three equal doses of 30 mg/kg i.p in 2-ml saline. The first injection was given 20 min before the CP injection, and the second and third

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injections were administered 4 and 8 h after CP injection, respectively. The drug administration schedule is presented in Table 1.

Biochemical analysis

Blood samples for biochemical analysis were collected by cardiac puncture using heparinized, normal tube syringes. Whole blood was collected into heparinized tubes, and whole-blood malondialdehyde (MDA) and GSH levels were studied on the same day as taken. Blood was also col-lected into polystyrene microtubes, and after clotting, this was centrifuged at 1,000 g for 10 min at +4 C and the serum was removed using EDTA-washed Pasteur pipettes. Heparinized red blood cells were washed three times with phosphate-buffered saline pH 7.4 for erythrocyte sam-pling. The serum and erythrocyte samples were stored in polystyrene plastic tubes at 70 C until the time of anal-ysis. Serum ascorbic acid, retinol, and -carotene activi-ties were studied by spectrophotometer (Jenway 6305 UV/Vis).

The MDA levels were measured according to the method of Jain et al. [27]. The principle of the method is based on the spectrophotometric measurement of the color that occurs during the reaction of thiobarbituric acid with MDA. Concentrations of thiobarbituric acid-reactive sub-stances (TBARS) were calculated by the absorbance coef-ficient of MDAthiobarbituric acid complex and expressed in nmol/ml. GSH concentration was also measured by a spectrophotometric method. After lysing whole blood and removal of precipitate, disodium hydrogen phosphate and DTNB solution were added and the color formed was read at 412 nm. The results were expressed in mg/dl. Serum vitamin C (ascorbic acid) levels were determined after derivatization with 2,4-dinitrophenylhydrazine. The levels of -carotene at 425 nm and vitamin A (retinol) at 325 nm were detected after the reaction of serum/ethanol/hexane at a ratio of 1:1:3, respectively.

The SOD and GPx activities were studied in hemolysates using commercial kits. CAT activity was measured accord-ing to the method of Aebi [28]. The principle of the assay is

based on the determination of the rate constant [k (s 1)] of hydrogen peroxide decomposition by catalase enzyme. The decomposition of the substrate hydrogen peroxide was monitored spectrophotometrically at 240 nm. The rate constant was calculated from the following formula: k = (2.3/t)(a/b) log(A1/A2).

Serum TNF- was measured by ELISA using an enzyme-linked immunoassay kit (Rat TNF- ELISA kit, Biosource International, Inc., Camarillo, CA, USA) accord-ing to the manufacturers protocol [29, 30]. TNF- content was expressed as pg/ml.

The IL-10 levels were measured using commercial kits (Cusabio, USA). This assay employs a quantitative sand-wich enzyme immunoassay technique. Antibody specific for IL-10 was pre-coated onto a microplate. Standards and samples were pipetted into the wells, and the immobilized antibody bound any IL-10 present [31].

Histopathological evaluation

Bladder specimens were fixed in 10 % buffered formalin, processed, blocked with paraffin, and then sectioned into 5-m sections and stained with hematoxylin and eosin (HE). These slides were examined under a light microscope (Nikon Eclipse Ci attached Kameram Digital Image Ana-lyze System) and graded as mild (+), moderate (++), and severe (+++) for hemorrhage, proprial vascularization, edema, inflammatory changes, basement membrane dam-age, epithelial hyperplasia, epithelial degeneration, and desquamation.

Immunohistochemical staining

In this study, the streptavidinbiotinperoxidase complex (VECTASTAIN Elite ABC Kit PK-6101/PK-6102) method was used. Briefly, 5-m tissue sections were mounted on silanized slides from paraffin blocks. Deparaffinized and rehydrated sections were transferred into hydrogen perox-ide for blocking endogenous peroxides, and then antigen retrieval processes were carried out. Nonspecific immuno-globulins were blocked with non-immune sera. Antibodies

Table 1 Cyclophosphamide, resveratrol 20, 40, 80 mg, and mesna treatment schedule

Sham saline, CP cyclophosphamide, RES resveratrol, MESNA 2-mercaptoethane sulfonate sodium

Drug administration timing

Groups 20 min ago Hemorrhagic cystitis induction 4 and 8 h later

Sham Saline (2 ml) Saline (2 ml) 2 Saline (2 ml)CP Saline (2 ml) CP (150 mg/kg) 2 Saline (2 ml)CP + RES 20 Resveratrol (20 mg/kg) CP (150 mg/kg) 2 Saline (2 ml)CP + RES 40 Resveratrol (40 mg/kg) CP (150 mg/kg) 2 Saline (2 ml)CP + RES 80 Resveratrol (80 mg/kg) CP (150 mg/kg) 2 Saline (2 ml)Mesna Mesna (30 mg/kg) CP (150 mg/kg) 2 Mesna (30 mg/kg)

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against NOS (Abcam, ab15203, C-terminal polyclonal, 1/40 dilution, 20 min, 800 watt microwave in citrate buffer, pH 6.0) and Up3 (Lifespan, LS-C40107, 1/20 dilution, 20 min, 800 watt microwave in citrate buffer, pH 6.0) were diluted and applied to the sections. For minimum back-ground and maximum positive reaction, optimization was done for each antibody.

Biotinylated secondary antibody and streptavidinper-oxidase was then applied to the tissue sections. After this process, sections were visualized with 3-amino-9-ethylcar-bazole chromogen (AEC, Invitrogen, 002007) and cover-slipped with aqua medium. Background was stained with Mayers hematoxylin. Except after the application of non-immune anti-goat sera, sections were washed with phos-phate buffer solution (3 2 min, pH 7.4) until background stages. For the positive control, tissues and methods pro-posed by the manufacturer were applied. Negative control staining was carried out similar to the main procedure; however, normal rabbit serum was used instead of primary antibody. Stained sections were stored at room temperature in the dark until inspection.

TUNEL assay

The 5-m sections from paraffin blocks prepared for his-topathological examination were transferred to silanized slides for the TUNEL assay. Nuclease-free Proteinase K (0.6 units/ml) incubation was applied (in 37 C oven, for 10 min) for antigen retrieval. After washing with PBS, 10-min 3 % hydrogen peroxide application was done at room temperature for blocking of endogenous peroxidase activities. Sections were transferred into a humidity cham-ber, and a commercial apoptosis kit (In Situ Cell Death Detection Kit, POD, Roche, Cat. No. 1 684 817) was used according to the manufacturers procedure. Visualization, background colorization, and covering were done with the same immunohistochemical method.

Statistical analysis

Statistical tests were performed using SPSS for Windows 20.0 software package (SPSS Inc., Chicago, Illinois, USA). Variables were investigated using visual (histogram, probability plots) and analytical methods (KolmogorovSmirnov test) to determine whether or not they were nor-mally distributed. Results are reported as mean SEM or as median (minmax). Data with normal distribution were analyzed using one-way analysis of variance (ANOVA) and Tukeys posttest. We used statistical evaluation by nonparametric KruskalWallis test for data with abnor-mal distribution. MannWhitney U test was performed to analyze two groups. p < 0.05 was assessed as statistically significant.

Results

Biochemical findings

The MDA was found at the highest level in the control group. Compared to the control, in the 20, 40, and 80 mg/kg RES treated and in the mesna-treated groups, MDA lev-els decreased significantly (p < 0.05, p < 0.01, p < 0.01, p < 0.05, respectively; Fig. 1a).

The GSH levels, as endogenous agents that protect the body against oxidative stress, were found at the lowest level in the control group. However, 20, 40, and 80 mg/kg RES or mesna treatment significantly inhibited the reduc-tion in GSH levels, compared to control (p < 0.05, p < 0.05, p < 0.01, p < 0.05, respectively; Fig. 1b). The other endog-enous antioxidant, ascorbic acid, was lowest in the control group. Only the group treated with an 80 mg/kg dose of RES had a significant increase (p < 0.05; Fig. 1c). Compar-ing -carotene and retinol levels, there were no significant differences between the groups (Fig. 1d, e).

The CAT, SOD, and GPx antioxidant enzyme activities of all the groups are presented in Fig. 2 (ac, respectively). CAT enzyme activity increased significantly compared to control and RES 20 groups (p < 0.05) (Fig. 2a). Simi-larly, SOD enzyme activities were higher in sham, RES 40, RES 80, and mesna groups (p < 0.01, p < 0.05, p < 0.01, p < 0.05, respectively) (Fig. 2b). Also, in all study groups, GPx activities were statistically higher than the control group (p < 0.01, p < 0.05, p < 0.05, p < 0.01, p < 0.05 respectively) (Fig. 2c).

The TNF- and IL-10 levels in all subjects are shown in Fig. 3 (a, b, respectively). TNF- as a marker associated with inflammation was found in the sham group at the low-est level compared to the control group (p < 0.001). TNF- levels were significantly lower in the 20, 40, and 80 mg/kg RES and mesna-treated groups, compared to the control group (respectively, p < 0.01, p < 0.01, p < 0.01, p < 0.05) (Fig. 3a). IL-10 levels increased significantly compared to the control and RES 20 group (p < 0.001, p < 0.05, p < 0.01 and p < 0.05, respectively) (Fig. 3b).

Histopathologic findings

Mucosal, submucosal, and proprial pathological find-ings were observed in the experimental groups. Histo-pathologic findings are given in Table 2 in detail. Variable intense hemorrhage, which was one of the main findings, was seen in proprialsubmucosal located extravasated erythrocytes mostly in the CP group and in RES 80 both intraepithelial located exocytotic erythrocytes and/or intra-luminal accumulated erythrocytes. Four animals in the fifth group, in relation to severe hemorrhage, showed excessive bloody content in the bladder lumen. Luminal shrinkage

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was seen as a result of proprial edema and sometimes pro-prial fibrotic changes. Apparent capillary hyperemia in a few animals and thrombotic findings in one animal were observed in the RES 80 group.

Inflammatory cells, mostly neutrophil leukocytes, were present in every region of the bladder, including the lumen. Degeneration, desquamation, destruction of the basement membrane, and papillary hyperplasia of transitional epi-thelium were present mostly in the RES 80 and CP groups compared to the RES 40 group and the RES 20 group.

The TUNEL positivity was mostly detected in inflam-matory cells. Limited epithelial positivity was mainly seen in the exfoliate luminal epithelial cells and lesser positiv-ity in settled epithelial cells. Slightly more TUNEL posi-tivity was detected in the resveratrol groups compared to the mesna group. But there was no statistically significant

difference in TUNEL staining. Histopathological images (Figs. 4, 5) are given for visualization.

Discussion

The urotoxicity of CP originates from renal excretion of acrolein, a hepatic microsome-mediated metabolic prod-uct of CP. The accumulation of acrolein in the bladder can cause HC. This major side effect limits the use of CP in the clinical field [32]. Current knowledge provides informa-tion about the pathophysiological mechanism of HC. Sev-eral transcription factors and cytokines, free radicals, and non-radical-reactive molecules, as well as poly adenosine diphosphate-ribose polymerase (PARP) activation are now known to take part in its pathogenesis [11]. CP treatment

Fig. 1 Oxidant and non-enzymatic antioxidant levels for each group. MDA, malondialdehyde; GSH, reduced glutathione; ascor-bic acid, vitamin C; retinol, vitamin A. Whole-blood MDA levels (a). Whole-blood GSH levels (b). Serum ascorbic acid levels (c).

Serum -carotene levels (d). Serum retinol levels (e). All values are expressed as mean SD. ap < 0.05 with respect to control, bp < 0.01 with respect to control, cp < 0.001 with respect to control

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Fig. 2 Antioxidant enzyme activities for each group. CAT catalase, SOD superoxide dismutase, GPx glutathione peroxidase. Erythrocyte CAT activity (a). Erythrocyte SOD activity (b). Erythrocyte GPx activity (c). ap < 0.05 with respect to control, bp < 0.01 with respect to control

Fig. 3 Cytokine levels for each group. Tumor necrosis factor-, TNF-; Interleukin-10, IL-10. Serum TNF- levels (a). Serum IL-10 levels (b). ap < 0.05 with respect to control. bp < 0.01 with respect to control. cp < 0.001 with respect to control

Table 2 Histopathological evaluation of the bladder in the experimental groups

Sham saline, CP cyclophosphamide, RES resveratrol, MESNA 2-mercaptoethane sulfonate sodiuma p < 0.05 with respect to control, b p < 0.01 with respect to control, c p 0.001 with respect to control

Sham Control RES 20 RES 40 RES 80 Mesna

Hemorrhage 0.08 0.2c 3.0 0 1.25 0.27b 1.25 0.27b 2.25 0.98 0.08 0.2cEpithelial degeneration 0c 3.0 0 1.33 0.51b 2.0 0.44b 2.0 0.31b 1.0 0cEpithelial desquamation 0.08 0.2b 2.75 0.41 1.41 0.56b 1.58 0.2b 2.33 0.4 0.33 0.81bInflammation 0b 2.75 0.41 1.41 0.66b 2.0 0.44a 2.75 0.41 0bEdema 0c 3.0 0 2.5 0.54 2.91 0.2 2.75 0.27 0cEpithelial hyperplasia 1.25 0.52b 2.91 0.2 1.5 0c 1.58 0.37b 2.41 0.66 1.95 0.71aVascularization 0b 2.63 0.43 2.16 0.51 2.58 0.49 2.5 0.44 0.16 0.4bBasal membrane destruction 0c 3.0 0 1.33 0.25b 2.08 0.49a 2.0 0.44a 0c

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causes a significant increase in ROS level in bladder tis-sues. Administration of antioxidants ameliorates oxida-tive stress by reducing ROS levels in bladder tissues [32]. Several antioxidants, such as -tocopherol [24], -carotene [33], and melatonin [34, 35] have similar effects on cystitis symptoms [11]. In the present study, we tested experimen-tally whether treatment with RES had a protective effect against CP-induced injury.

The MDA, an indicator of lipid peroxidation, increases in body systems after administration of CP and leads to the eventual destruction of membrane lipids with the formation and propagation of lipid radicals, and increases the uptake of oxygen, causing rearrangement of the double bonds in unsaturated lipids [36]. Previous studies reported that tissue or blood MDA levels were significantly higher in the CP-treated group [3537]. The increase in MDA level may be a reflection of an insufficiency in enzymatic and non-enzy-matic antioxidants as defense mechanisms. In our study, MDA was found at the highest level in the control group; however, the MDA levels in the RES and mesna-treated

groups decreased significantly. As we know from previ-ous studies, acrolein can cause chain reactions that include lipid peroxidation. When we used mesna, MDA levels decreased. Because mesna can bind to acrolein and inhibit its activity, this potentially limits CP-associated toxicities [13]. Furthermore, the anti-lipid peroxidative activity of RES was shown to be as effective as mesna.

The GSH is the major cellular sulfhydryl compound that serves as both a nucleophile and an effective reductant by interacting with numerous electrophilic and oxidizing com-pounds. It can act as a non-enzymic antioxidant by direct interaction of SH groups with ROS or it can be involved in the enzymatic detoxification reaction of ROS, as a cofac-tor or coenzyme [38]. Acrolein causes the depletion of cellular antioxidant nucleophiles, such as glutathione, and it initiates the lipid peroxidation that results in HC [39]. Scientists have demonstrated a severe depletion in cellu-lar GSH content in urinary bladder homogenates of CP-treated groups [25, 40]. IFO caused significant reductions in GSH levels both in kidney and bladder, when compared

Fig. 4 Histopathological view of group 1 [Sham group], group 2 [150 mg/kg CP, ip.], and group 3 [20 mg/kg of res-veratrol + 150 mg/kg CP, ip.]. Images presented in column A were stained with HxE. The original magnification was 40, and the scale bars represent 250 m. Images presented in column B were stained with TUNEL (AEC used as chro-mogen and background colored with Meyers hematoxylin), the original magnification was 100, and the scale bars represent 100 m. Abbrevia-tions as shown by the arrows: h hemorrhage, hp hyperplasia, a apoptosis, i inflammation, o edema, d desquamation, nv neovascularization

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to control rats, while MDA levels in both tissues were found to be significantly higher than a control group. RES treatment accompanying IFO administration abolished the depletion of GSH levels and prevented the elevation in MDA levels in both tissues [41]. In the present study, it was seen that there was a significant decrease in GSH levels in the control group when compared to the sham group. How-ever, RES or mesna administration significantly prevented the decrease in GSH levels. Excessive lipid peroxidation can cause GSH consumption, or GSH depletion results in enhanced lipid peroxidation. The cause of high levels of GSH in RES or mesna-administered groups may be related to a decreased lipid peroxidation with RES or mesna. The effects of different doses of RES on GSH levels were found to be similar to mesna.

The antioxidant vitamins such as ascorbic acid, retinol, and -carotene play an important acute and chronic role in reducing or eliminating oxidative damage produced by ROS [42]. In the present study, serum retinol and -carotene lev-els in treatment groups were very similar, and there was no

significant difference between groups. Unlike retinol and -carotene, serum ascorbic acid levels were different, and there was a significant difference only between control and RES 80 groups. Thus, it may be suggested that the protective effect of RES against CP-induced oxidative stress may be partly related to the restoration of ascorbic acid availability.

Regarding antioxidant enzymes, SOD, CAT, and GPx play an important role in protecting cells against the deleterious action of ROS. SOD catalyzes the conversion of superoxide radical to hydrogen peroxide and molecular oxygen. CAT catalyzes the reduction of hydrogen peroxides and protects tissues against reactive hydroxyl radicals. GPx is a seleno-protein that oxidizes GSH to glutathione disulfide (GSSG), which is then reduced to GSH by glutathione reductase, and reduces hydroperoxides. Decreased activity of the enzymatic antioxidants such as SOD, CAT and GPx has been well dem-onstrated in CP toxicity [25, 37, 43]. Treatment with mesna and antioxidants (lipoic acid) restored the enzyme activities to some extent [32]. In the present study, SOD, CAT, and GPx activities significantly decreased in CP-administered

Fig. 5 Histopathological view of group 4 [CP + RSV 40 group, 40 mg/kg resvera-trol +150 mg/kg CP, i.p.], group 5 [CP + RSV 80 group, 80 mg/kg resveratrol + 150 mg/kg CP, i.p.], and group 6 [Mesna group, 30 mg/kg Mesna 3 + 150 mg/kg CP, i.p.]. Images presented in column A were stained with H&E. The original magnifica-tion was 40, and the scale bars represent 250 m. Images presented in column B were stained with TUNEL (AEC used as chromogen and background colored with Meyers hematox-ylin), the original magnification was 100, and the scale bars represent 100 m. Abbrevia-tions as shown by the arrows: h hemorrhage, hp hyperplasia, a apoptosis, i inflammation, o edema, d desquamation, nv neovascularization

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rats when compared to the sham group. This is consistent with the previous reports. In rats treated with RES or mesna, however, the activity of these antioxidant enzymes was higher compared to rats exposed to CP alone. Forty and 80 mg/kg doses of RES were observed to be at least as effective as mesna. Thus, it may be suggested that the antioxidative activ-ity of RES is partly associated with the increased antioxidant enzyme capability that can scavenge ROS.

Recently, it has been shown that in the pathogenesis of HC, not only is acrolein involved, but also pro-inflamma-tory cytokines such as TNF- and IL-1 play a role as final mediators in NO synthesis [44]. Acrolein can rapidly react at many cellular sites, i.e., either directly or subsequent to the effects of transcription factors such as nuclear factor-B (NF-B) and activator protein-1 (AP-1). Activated NF-B and AP-1 cause cytokine (TNF-, IL-1) gene expression, iNOS induction, and again ROS production. Thus, the production of harmful molecules (cytokines, ROS, NO) increases dra-matically. Cytokines leave the uroepithelium and spread to other uroepithelial cells, detrussor smooth muscle, and the bloodstream. Cellular and tissue integrity are disrupted, and damage appears as edema, hemorrhage, and ulceration [45]. Hamsa and Kuttan [46] demonstrated that the serum level of TNF- is increased, whereas INF and IL-2 are decreased in CP-treated animals, showing its toxic nature. Sehirli et al. [41] have demonstrated that IFO treatment caused signifi-cant increases in the serum levels of the pro-inflammatory cytokines, TNF-, IL-1, and IL-6, while these elevations were significantly reduced by RES treatment. Interleukin-10 has been characterized as being a cytoprotective and potent anti-inflammatory cytokine that inhibits the production of other cytokines [47]. It reduces the activation of macrophages and inhibits the production of ROS and pro-inflammatory cytokines [48]. In the present study, TNF-, an inflammation marker, was found in the sham group at the lowest level com-pared to the control. Again, TNF- levels were significantly lower in RES and mesna-treated groups compared to the con-trol. Regarding IL-10 levels, they were found to be highest in the sham group, but lowest in the control. Likewise, RES (except for 20 mg/kg dose) and mesna significantly prevented the decrease in IL-10 levels, and 80 mg/kg RES was the most effective of these. Thus, CP administration caused an increase in TNF- levels, while mesna or RES administration inhib-ited this increase, and caused a decrease in IL-10 levels, while mesna or RES administration significantly prevented. Thus, it is suggested that the beneficial effect of RES against CP tox-icity may be partly associated with the modulation of the anti-inflammatory response.

It is known that HC is caused by direct contact of acr-olein with the uroepithelium. Among the pathogenetic pathways of CP toxicity, excessive production of ROS and release of cytokines and inflammatory mediators are sig-nificant. As a result, histopathological changes arise in the

bladder. In an experimental study of the protective effects of -tocopherol and melatonin against CP toxicity, it was demonstrated that CP causes severe histological changes and macroscopic hematuria [33]. In another study assess-ing the effects of spirulina in CP-induced nephrotoxic-ity and urotoxicity in rats, histopathologic changes were determined to be highest in the CP group. The histomor-phologic alteration in the urinary bladder in the spirulina-administered group was significantly lower compared to the CP group [36]. In our study, mucosal bleeding, epithe-lial degeneration, epithelial desquamation, inflammation, edema, epithelial hyperplasia, vascularization, and mem-brane degradation were found in the control group. These alterations were significantly reduced by mesna. This result is consistent with the previous reports. Again, the protec-tive effect of RES against CP toxicity was seen at 20 and 40 mg/kg doses. We think that the antioxidant and anti-inflammatory properties of RES contribute to preventing the histopathological deterioration induced by CP.

In conclusion, all biochemical measurements and histo-pathological examinations suggest that RES has a protec-tive effect against CP-induced HC. Marked bladder protec-tion was found in the 20 and 40 mg/kg RES-administered groups compared to the control. However, this protection was weaker than that of the mesna-administered group. The mechanism of this protection seems to be partly based on its antioxidant and anti-inflammatory properties. We hope that our results will shed light on future clinical trials.

Acknowledgments This study was supported by the Afyon Kocatepe University Scientific Research Projects Coordination Unit (Project No: 12.TIP.09).

Conflict of interest None.

References

1. Levine LA, Richie JP (1989) Urological complications of cyclo-phosphamide. J Urol 141:10631069

2. Kranc DM, Kim J, Straus F, Levine LA (1992) Prophylactic and therapeutic carboprost tromethamine bladder irrigation in rats with cyclophosphamide-induced hemorrhagic cystitis. J Urol 148:13261330

3. Levine LA, Jarrard DF (1993) Treatment of cyclophospha-mide-induced hemorrhagic cystitis with intravesical carboprost tromethamine. J Urol 149:719723

4. Chow YC, Yang S, Huang CJ, Tzen CY, Huang PL, Su YH et al (2006) Epinephrine promotes hemostasis in rats with cyclophos-phamide-induced hemorrhagic cystitis. Urology 67:636641

5. Assreuy AM, Martins GJ, Moreira ME, Brito GA, Cavada BS, Ribeiro RA et al (1999) Prevention of cyclophosphamide-induced hemorrhagic cystitis by glucosemannose binding plant lectins. J Urol 161:19881993

6. Wong TM, Yeo W, Chan LW, Mok TS (2000) Hemorrhagic pyeli-tis, ureteritis, and cystitis secondary to cyclophosphamide: case report and review of the literature. Gynecol Oncol 76:223225

2310 Int Urol Nephrol (2014) 46:23012310

1 3

7. West NJ (1997) Prevention and treatment of hemorrhagic cystitis. Pharmacotherapy. 17:696706

8. Traxer O, Desgrandchamps F, Sebe P, Haab F, Le Duc A, Gat-tegno B et al (2001) Hemorrhagic cystitis: etiology and treatment. Prog Urol 11:591601

9. Batista CK, Brito GA, Souza ML, Leito BT, Cunha FQ, Ribeiro RA (2006) A model of hemorrhagic cystitis induced with acrolein in mice. Braz J Med Biol Res 39:14751481

10. Manikandan R, Kumar S, Dorairajan LN (2010) Hemorrhagic cystitis: a challenge to the urologist. Indian J Urol 26:159166

11. Korkmaz A, Topal T, Oter S (2007) Pathophysiological aspects of cyclophosphamide and ifosfamide induced hemorrhagic cysti-tis; implication of reactive oxygen and nitrogen species as well as PARP activation. Cell Biol Toxicol 23:303312

12. Kehrer JP, Biswal SS (2000) The molecular effects of acrolein. Toxicol Sci 57:615

13. Monach PA, Arnold LM, Merkel PA (2010) Incidence and preven-tion of bladder toxicity from cyclophosphamide in the treatment of rheumatic diseases: a data-driven review. Arthritis Rheum 62:921

14. Dechant KL, Brogden RN, Pilkington T, Faulds D (1991) Ifosfamide/mesna. A review of its antineoplastic activity, phar-macokinetic properties and therapeutic efficacy in cancer. Drugs 42:428467

15. Hensley ML, Schuchter LM, Lindley C, Meropol NJ, Cohen GI, Broder G et al (1999) American Society of Clinical Oncol-ogy clinical practice guidelines for the use of chemotherapy and radiotherapy protectants. J Clin Oncol 17:33333355

16. Brock N, Pohl J, Stekar J (1981) Studies on the urotoxicity of oxazaphosphorine cytostatics and its preventionI. Experimen-tal studies on the urotoxicity of alkylating compounds. Eur J Can-cer 17:595607

17. Oboh G, Akomolafe TL, Adefegha SA, Adetuyi AO (2012) Atten-uation of cyclophosphamide-induced neurotoxicity in rat by yel-low dye extract from root of Brimstone tree (Morinda lucida). Exp Toxicol Pathol 64:591596

18. Rehman MU, Tahir M, Ali F, Qamar W, Lateef A, Khan R et al (2012) Cyclophosphamide-induced nephrotoxicity, genotoxicity, and damage in kidney genomic DNA of Swiss albino mice: the protective effect of Ellagic acid. Mol Cell Biochem 365:119127

19. Fulda S, Debatin KM (2006) Resveratrol modulation of signal transduction in apoptosis and cell survival: a mini-review. Cancer Detect Prev 30:217223

20. Pervaiz S, Holme AL (2009) Resveratrol: its biologic targets and functional activity. Antioxid Redox Signal 11:28512897

21. De la Lastra CA, Villegas I (2007) Resveratrol as an antioxidant and pro-oxidant agent: mechanisms and clinical implications. Biochem Soc Trans 35:11561160

22. Kitada M, Koya D (2013) Renal protective effects of resveratrol. Oxid Med Cell Longev. doi:10.1155/2013/568093

23. Vieira MM, Macdo FY, Filho JN, Costa AC, Cunha AN, Silveira ER et al (2004) Ternatin, a flavonoid, prevents cyclophosphamide and ifosfamide-induced hemorrhagic cystitis in rats. Phytother Res 18:135141

24. Yildirim I, Korkmaz A, Oter S, Ozcan A, Oztas E (2004) Contri-bution of antioxidants to preventive effect of mesna in cyclophos-phamide-induced hemorrhagic cystitis in rats. Cancer Chemother Pharmacol 54:469473

25. Bhatia K, Ahmad F, Rashid H, Raisuddin S (2008) Protective effect of S-allylcysteine against cyclophosphamide-induced bladder hem-orrhagic cystitis in mice. Food Chem Toxicol 46:33683374

26. Botta JA Jr, Nelson LW, Weikel JH Jr (1973) Acetylcysteine in the prevention of cyclophosphamide-induced cystitis in rats. J Natl Cancer Inst 51:10511058

27. Jain SK, McVie R, Duett J, Herbst JJ (1989) Erythrocyte mem-brane lipid peroxidation and glycosylated hemoglobin in diabe-tes. Diabetes 38:15391543

28. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121126 29. Ooboshi H, Ibayashi S, Shichita T, Kumai Y, Takada J, Ago T

et al (2005) Postischemic gene transfer of interleukin-10 pro-tects against both focal and global brain ischemia. Circulation 111:913919

30. Sentrk S (2005) Evaluation of the anti-endotoxic effects of pol-ymyxin-E (colistin) in dogs with naturally occurred endotoxic shock. J Vet Pharmacol Ther 28:5763

31. Zhang Y, Zhao M, Jin M, Xu C, Wang C, Liu W (2010) Immuno-toxicity of pyrethroid metabolites in an in vitro model. Environ Toxicol Chem 29:25052510

32. Song J, Liu L, Li L, Liu J, Song E, Song Y (2014) Protective effects of lipoic acid and mesna on cyclophosphamide-induced haemorrhagic cystitis in mice. Cell Biochem Funct 32:125132

33. Sadir S, Deveci S, Korkmaz A, Oter S (2007) Alpha-tocopherol, beta-carotene and melatonin administration protects cyclophos-phamide-induced oxidative damage to bladder tissue in rats. Cell Biochem Funct 25:521526

34. Sener G, Sehirli O, Yegen BC, Cetinel S, Gedik N, Sakarcan A (2004) Melatonin attenuates ifosfamide-induced Fanconi syn-drome in rats. J Pineal Res 37:1725

35. Topal T, Oztas Y, Korkmaz A, Sadir S, Oter S, Coskun O et al (2005) Melatonin ameliorates bladder damage induced by cyclo-phosphamide in rats. J Pineal Res 38:272277

36. Sinanoglu O, Yener AN, Ekici S, Midi A, Aksungar FB (2012) The protective effects of spirulina in cyclophosphamide induced nephrotoxicity and urotoxicity in rats. Urology 80:13921396

37. Al-Malki AL (2012) Synergestic effect of lycopene and Mela-tonin against the genesis of oxidative stress induced by cyclo-phosphamide in rats. Toxicol Ind Health 30:570575

38. Yuan ZM, Smith PB, Brundrett RB, Colvin M, Fenselau C (1991) Glutathione conjugation with phosphoramide mustard and cyclo-phosphamide. A mechanistic study using tandem mass spectrom-etry. Drug Metab Dispos 19:625629

39. Adams JD Jr, Klaidman LK (1993) Acrolein-induced oxygen rad-ical formation. Free Radic Biol Med 15:187193

40. Al-Yahya AA, Al-Majed AA, Gado AM, Daba MH, Al-Shabanah OA, Abd-Allah AR (2009) Acacia Senegal gum exudate offers protection against cyclophosphamide-induced urinary bladder cytotoxicity. Oxid Med Cell Longev 2:207213

41. Sehirli O, Sakarcan A, Velioglu-Ogn A, Cetinel S, Gedik N, Yegen BC et al (2007) Resveratrol improves ifosfamide-induced Fanconi syndrome in rats. Toxicol Appl Pharmacol 222:3341

42. Halliwell B (1996) Antioxidants in human health and disease. Annu Rev Nutr 16:3350

43. Zareia M, Shivanandappa T (2013) Amelioration of cyclophos-phamide-induced hepatotoxicity by the root extract of Decalepis hamiltonii in mice. Food Chem Toxicol 57:179184

44. Oter S, Korkmaz A, Oztas E, Yildirim I, Topal T, Bilgic H (2004) Inducible nitric oxide synthase inhibition in cyclophosphamide induced hemorrhagic cystitis in rats. Urol Res 32:185189

45. Korkmaz A, Oter S, Sadir S, Coskun O, Topal T, Ozler M, Bilgic H et al (2005) Peroxynitrite may be involved in bladder damage caused by cyclophosphamide in rats. J Urol 173:17931796

46. Hamsa TP, Kuttan G (2011) Protective role of Ipomoea obscura (L.) on cyclophosphamide-induced uro- and nephro-toxicities by modulating antioxidant status and pro-inflammatory cytokine lev-els. Inflammopharmacology 19:155167

47. Moore KW, OGarra A, Malefyt RW, Vieira P, Mosmann TR (1993) Interleukin-10. Annu Rev Immunol 11:165190

48. Huet O, Laemmel E, Fu Y, Dupic L, Aprico A, Andrews KL, Moore SL, Harrois A, Meikle PL, Vicaut E, Chin-Dusting JP, Duranteau J (2013) Interleukin 10 antioxidant effect decreases leukocytes/endothelial interaction induced by tumor necrosis fac-tor . Shock 39:8388

Prevention ofcyclophosphamide-induced hemorrhagic cystitis byresveratrol: a comparative experimental study withmesnaAbstract Purpose Methods Results Conclusion

IntroductionMaterials andmethodsAnimalsDrugs andchemicals

Hemorrhagic cystitis modelExperimental protocolBiochemical analysisHistopathological evaluationImmunohistochemical stainingTUNEL assayStatistical analysis

ResultsBiochemical findingsHistopathologic findings

DiscussionAcknowledgments References