Int Urol Nephrol (2014) 46:23012310

DOI 10.1007/s11255-014-0833-8

UROLOGY - ORIGINAL PAPER

Prevention ofcyclophosphamideinduced hemorrhagic cystitis

byresveratrol: a comparative experimental study withmesna
IbrahimKeles MehmetFatihBozkurt MustafaCemek MustafaKaralar
AhmetHazini SaadetAlpdagtas HikmetKeles TuranYildiz CavitCeylan
MehmetEminBuyukokuroglu

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

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.
MethodsForty-six male SpragueDawley rats were
divided into six groups. Group 1 served as a negative control
(sham). Five groups received a single dose of cyclophosphamide (150mg/kg) intraperitoneally at the same time. Groups

I.Keles(*) M.Karalar
Department ofUrology, Faculty ofMedicine, Afyon Kocatepe
University, Adnan Kahveci Bulvar No:67/1 Seluklu
Mah. Seluklu Konaklar A Blok Kat 3 daire:7 Uydukent,
Afyonkarahisar, Turkey
e-mail: drkeles@hotmail.com
M.F.Bozkurt H.Keles
Department ofPathology, Faculty ofVeterinary Medicine, Afyon
Kocatepe University, Afyonkarahisar, Turkey
M.Cemek A.Hazini S.Alpdagtas
Department ofBioengineering (Biochemistry Division), Faculty
ofChemical andMetallurgic Engineering, Yldz Technical
University, Istanbul, Turkey
T.Yildiz
Department ofPediatric Surgery, Faculty ofMedicine, Sakarya
University, Sakarya, Turkey
C.Ceylan
Department ofUrology, Ankara Yksek Ihtisas Training
andResearch Hospital, Ankara, Turkey
M.E.Buyukokuroglu
Department ofPharmacology, Faculty ofMedicine, Sakarya
University, Sakarya, Turkey

2, 3, 4, 5, and 6 received only CP, CP+20mg/kg RES,

CP+40mg/kg RES, CP+80mg/kg RES, and CP+classical protocol of three doses of mesna (30mg/kg three times),
respectively. Antioxidants, cytokines, and malondialdehyde
levels were measured in all groups. In addition, histopathological alterations in tissues were examined.
Results CP administration induced severe HC with
marked edema, hemorrhage, and inflammation in group 2.
RES 20mg/kg showed meaningful protection against bladder damage compared to the control group. It was seen that
RES 40mg/kg gave weaker protection but RES 80mg/kg
was not found to be effective.
Conclusion In conclusion, marked bladder protection was
found in 20 and 40mg/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 incidence 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 transplantation [3, 4]. The main features of HC are urothelial damage, edema, necrosis, ulceration, hemorrhage, neovascularization, leukocyte infiltration [5], and transitional cell
carcinoma of the bladder [4, 6].
The alkylating agent CP is used alone or in combination with other antineoplastic drugs for the treatment for

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various solid tumors, hematological malignancies, autoimmune disorders, and for bone marrow transplantation
[1, 7]. CP is a prodrug that is converted to its metabolites phosphoramide mustard (active) and acrolein by
hepatic microsomal enzymes. Acrolein is highly toxic to
the urinary bladder, promoting the release of inflammatory mediators that ultimately lead to HC [810]. Acrolein enters rapidly into the uroepithelium because of its
chemical nature and causes increased reactive oxygen
species (ROS) production and both directly and indirectly 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 macromolecules (lipids, proteins, and DNA) and causes damage [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 administration 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 combines directly with the double bond of acrolein as well as
other urotoxic 4-hydroxyoxazaphosphorine metabolites
[14, 15]. These factors contribute to the inhibition of acrolein binding to cell surface proteins in the bladder, thereby
potentially limiting CP-associated toxicities such as cystitis 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 polyphenolic 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]. Previous reports have shown that RES can ameliorate several
types of renal injury, such as diabetic nephropathy, druginduced injury, aldosterone-induced injury, ischemia
reperfusion injury, sepsis-related injury, and unilateral
ureteral obstruction in animal models through its antioxidant effect [22]. Again, antioxidants act against CPinduced HC by maintaining antioxidant enzyme activities 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.

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Int Urol Nephrol (2014) 46:23012310

Materials andmethods
Animals
The Animal Care Committee of Afyon Kocatepe University approved this experimental study. A total of 46 male
SpragueDawley rats (225250g) 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 andchemicals
Cyclophosphamide (CP) (Endoxan 150mg/kg Eczacbas
Istanbul, Turkey), RES (Sigma, Germany), and mesna
(Uromitexan 400mg, Eczacbas Baxter, Turkey) were
used. All drugs were diluted in 0.9% sterile saline.
Hydrogen peroxide, GSH, thiobarbituric acid, phosphate buffer, butylated hydroxytoluene, trichloroacetic
acid, EDTA, [5,5-dithiobis-(2-nitrobenzoic acid)], disodium hydrogen phosphate, phenylendiamine, sodium
azide, 2,4-dinitrophenylhydrazine, ethanol, hexane, sodium
nitrite, sodium nitrate, sulfanilamide, N-(1-Naphthyl) ethylenediamine dihydrochloride, and vanadium (III) chloride 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 (Cusabio, USA) superoxide dismutase (SOD), and glutathione
peroxidase (GPx) commercial kits (Randox, UK) were
used.
Hemorrhagic cystitis model
Animals were injected intraperitoneally (i.p) with 150mg/
kg CP in 2-ml saline according to the methods described
by Botta etal. [26]. After 48h of HC induction, rats were
killed using i.p. injection of ketamine HCL 80mg/kg and
xylazine HCL 10mg/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 2ml of CP (150mg/
kg) intraperitoneally. RES (groups 3, 4, 5) at three different
doses of 20, 40, and 80mg/kg in 2-ml saline was administered 20min before CP injection (i.p). A total of 90mg/kg
mesna (group 6) was administered in three equal doses of
30mg/kg i.p in 2-ml saline. The first injection was given
20min before the CP injection, and the second and third

Int Urol Nephrol (2014) 46:23012310

Table1Cyclophosphamide,
resveratrol 20, 40, 80mg, and
mesna treatment schedule

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

2303

Drug administration timing

Groups

20min ago

Hemorrhagic cystitis induction

4 and 8h later

Sham
CP
CP+RES 20
CP+RES 40
CP+RES 80

Saline (2ml)
Saline (2ml)
Resveratrol (20mg/kg)
Resveratrol (40mg/kg)
Resveratrol (80mg/kg)

Saline (2ml)
CP (150mg/kg)
CP (150mg/kg)
CP (150mg/kg)
CP (150mg/kg)

2 Saline (2ml)
2 Saline (2ml)
2 Saline (2ml)
2 Saline (2ml)
2 Saline (2ml)

Mesna

Mesna (30mg/kg)

CP (150mg/kg)

2 Mesna (30mg/kg)

injections were administered 4 and 8h after CP injection,

respectively. The drug administration schedule is presented
in Table1.
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 collected into polystyrene microtubes, and after clotting, this
was centrifuged at 1,000g for 10min at +4C 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 sampling. The serum and erythrocyte samples were stored in
polystyrene plastic tubes at 70C until the time of analysis. Serum ascorbic acid, retinol, and -carotene activities were studied by spectrophotometer (Jenway 6305 UV/
Vis).
The MDA levels were measured according to the
method of Jain etal. [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 substances (TBARS) were calculated by the absorbance coefficient 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 412nm. 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 425nm and vitamin A (retinol) at 325nm
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 according to the method of Aebi [28]. The principle of the assay is

based on the determination of the rate constant [k (s1)]

of hydrogen peroxide decomposition by catalase enzyme.
The decomposition of the substrate hydrogen peroxide
was monitored spectrophotometrically at 240nm. 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) according 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 sandwich 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 Analyze System) and graded as mild (+), moderate (++), and
severe (+++) for hemorrhage, proprial vascularization,
edema, inflammatory changes, basement membrane damage, 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 peroxide for blocking endogenous peroxides, and then antigen
retrieval processes were carried out. Nonspecific immunoglobulins were blocked with non-immune sera. Antibodies

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against NOS (Abcam, ab15203, C-terminal polyclonal,

1/40 dilution, 20min, 800 watt microwave in citrate buffer,
pH 6.0) and Up3 (Lifespan, LS-C40107, 1/20 dilution,
20min, 800 watt microwave in citrate buffer, pH 6.0) were
diluted and applied to the sections. For minimum background and maximum positive reaction, optimization was
done for each antibody.
Biotinylated secondary antibody and streptavidinperoxidase was then applied to the tissue sections. After this
process, sections were visualized with 3-amino-9-ethylcarbazole chromogen (AEC, Invitrogen, 002007) and coverslipped with aqua medium. Background was stained with
Mayers hematoxylin. Except after the application of nonimmune anti-goat sera, sections were washed with phosphate buffer solution (32min, pH 7.4) until background
stages. For the positive control, tissues and methods proposed 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 histopathological examination were transferred to silanized
slides for the TUNEL assay. Nuclease-free Proteinase K
(0.6 units/ml) incubation was applied (in 37C oven, for
10min) 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 chamber, 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 (Kolmogorov
Smirnov test) to determine whether or not they were normally distributed. Results are reported as meanSEM 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 abnormal distribution. MannWhitney U test was performed to
analyze two groups. p<0.05 was assessed as statistically
significant.

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Int Urol Nephrol (2014) 46:23012310

Results
Biochemical findings
The MDA was found at the highest level in the control
group. Compared to the control, in the 20, 40, and 80mg/
kg RES treated and in the mesna-treated groups, MDA levels 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 80mg/kg
RES or mesna treatment significantly inhibited the reduction in GSH levels, compared to control (p<0.05, p<0.05,
p<0.01, p<0.05, respectively; Fig.1b). The other endogenous antioxidant, ascorbic acid, was lowest in the control
group. Only the group treated with an 80mg/kg dose of
RES had a significant increase (p<0.05; Fig.1c). Comparing -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). Similarly, 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 lowest level compared to the control group (p<0.001). TNF levels were significantly lower in the 20, 40, and 80mg/
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 findings were observed in the experimental groups. Histopathologic findings are given in Table2 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 intraluminal accumulated erythrocytes. Four animals in the fifth
group, in relation to severe hemorrhage, showed excessive
bloody content in the bladder lumen. Luminal shrinkage

Int Urol Nephrol (2014) 46:23012310

2305

Fig.1Oxidant and non-enzymatic antioxidant levels for each

group. MDA, malondialdehyde; GSH, reduced glutathione; ascorbic 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 meanSD. ap<0.05 with respect to control, bp<0.01
with respect to control, cp<0.001 with respect to control

was seen as a result of proprial edema and sometimes proprial 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 epithelium 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 inflammatory cells. Limited epithelial positivity was mainly seen
in the exfoliate luminal epithelial cells and lesser positivity in settled epithelial cells. Slightly more TUNEL positivity 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 product 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 information about the pathophysiological mechanism of HC. Several 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

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Int Urol Nephrol (2014) 46:23012310

Fig.2Antioxidant 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.3Cytokine levels for each group. Tumor necrosis factor-, TNF-; Interleukin-10, IL-10. Serum TNF- levels (a). Serum IL-10 levels (b).
a
p<0.05 with respect to control. bp<0.01 with respect to control. cp<0.001 with respect to control

Table2Histopathological evaluation of the bladder in the experimental groups

Sham

Control

RES 20

RES 40

RES 80

Mesna

Hemorrhage
Epithelial degeneration
Epithelial desquamation
Inflammation
Edema
Epithelial hyperplasia
Vascularization

0.080.2c
0c
0.080.2b
0b
0c
1.250.52b
0b

3.00
3.00
2.750.41
2.750.41
3.00
2.910.2
2.630.43

1.250.27b
1.330.51b
1.410.56b
1.410.66b
2.50.54
1.50c
2.160.51

1.250.27b
2.00.44b
1.580.2b
2.00.44a
2.910.2
1.580.37b
2.580.49

2.250.98
2.00.31b
2.330.4
2.750.41
2.750.27
2.410.66
2.50.44

0.080.2c
1.00c
0.330.81b
0b
0c
1.950.71a
0.160.4b

Basal membrane destruction

0c

1.330.25b

2.080.49a

2.00.44a

3.00

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

a

p<0.05 with respect to control, bp<0.01 with respect to control, cp0.001 with respect to control

13

0c

Int Urol Nephrol (2014) 46:23012310

2307

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

causes a significant increase in ROS level in bladder tissues. Administration of antioxidants ameliorates oxidative 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 experimentally 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 CPtreated group [3537]. The increase in MDA level may be
a reflection of an insufficiency in enzymatic and non-enzymatic 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 previous 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 compounds. 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 cofactor 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 cellular GSH content in urinary bladder homogenates of CPtreated groups [25, 40]. IFO caused significant reductions
in GSH levels both in kidney and bladder, when compared

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

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. However, 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 levels in treatment groups were very similar, and there was no

13

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 selenoprotein 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 demonstrated 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

Int Urol Nephrol (2014) 46:23012310

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 80mg/kg
doses of RES were observed to be at least as effective as
mesna. Thus, it may be suggested that the antioxidative activity 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-inflammatory 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 (NFB) 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 dramatically. 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 etal.
[41] have demonstrated that IFO treatment caused significant 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 compared to the control. Again, TNF- levels were significantly
lower in RES and mesna-treated groups compared to the control. Regarding IL-10 levels, they were found to be highest
in the sham group, but lowest in the control. Likewise, RES
(except for 20mg/kg dose) and mesna significantly prevented
the decrease in IL-10 levels, and 80mg/kg RES was the most
effective of these. Thus, CP administration caused an increase
in TNF- levels, while mesna or RES administration inhibited 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 toxicity may be partly associated with the modulation of the antiinflammatory response.
It is known that HC is caused by direct contact of acrolein with the uroepithelium. Among the pathogenetic
pathways of CP toxicity, excessive production of ROS and
release of cytokines and inflammatory mediators are significant. As a result, histopathological changes arise in the

2309

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 assessing the effects of spirulina in CP-induced nephrotoxicity and urotoxicity in rats, histopathologic changes were
determined to be highest in the CP group. The histomorphologic alteration in the urinary bladder in the spirulinaadministered group was significantly lower compared to
the CP group [36]. In our study, mucosal bleeding, epithelial degeneration, epithelial desquamation, inflammation,
edema, epithelial hyperplasia, vascularization, and membrane degradation were found in the control group. These
alterations were significantly reduced by mesna. This result
is consistent with the previous reports. Again, the protective effect of RES against CP toxicity was seen at 20 and
40mg/kg doses. We think that the antioxidant and antiinflammatory properties of RES contribute to preventing
the histopathological deterioration induced by CP.
In conclusion, all biochemical measurements and histopathological examinations suggest that RES has a protective effect against CP-induced HC. Marked bladder protection was found in the 20 and 40mg/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 interestNone.

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