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Alpha-tocopherol, beta-carotene and melatonin
administration protects cyclophosphamide-induced
oxidative damage to bladder tissue in rats
Serdar Sadir1, Salih Deveci2, Ahmet Korkmaz1 and Sukru Oter1*
1Department of Physiology, Gulhane Military Medical Academy, Ankara, Turkey
2Department of Pathology, Gulhane Military Medical Academy, Ankara, Turkey
Cyclophosphamide (CP) has potential urotoxicity such as hemorrhagic cystitis (HC). 2-Mercaptoethane sulfonate (mesna)
has been widely used as an effective agent against CP-induced cystitis, but significant HC has still been encountered
clinically. In recent studies, mesna was shown to be more effective if combined with antioxidants. The purpose of this study
was to evaluate the effects of antioxidants, a-tocopherol, b-carotene and melatonin on CP-induced bladder damage in rats,
even if used without mesna administration. Male Spraque–Dawley rats weighing 180–210 g were divided into 5 groups. Four
groups received a single dose of CP (100 mg/kg) intraperitoneally with the same time intervals. Group 2 received CP only,
group 3 received b-carotene (40 mg/kg/day), group 4 received a-tocopherol (40 mg/kg/day) and group 5 received melatonin
(10 mg/kg/day) both before and the day after CP injection. Group 1 served as control. Bladder histopathology, as well as
malondialdehyde (MDA) and iNOS levels, and excretion of nitrite-nitrates (NOx) in urine were evaluated. CP injection
resulted in severe histological changes and macroscopic hematuria. a-Tocopherol and melatonin showed meaningful
protection against bladder damage. Protection by b-carotene was also significant but weaker. MDA levels increased
significantly with CP injection and all antioxidants ameliorated this increase in bladder tissue. CP also elevated the NOx level
in urine and iNOS activity in bladder. Only melatonin was able to decrease these parameters. In conclusion, there is no doubt
that oxidants have a role in the pathogenesis of CP-cystitis. Antioxidants, especially melatonin and a-tocopherol, may help to
ameliorate bladder damage induced by CP. Copyright # 2006 John Wiley & Sons, Ltd.
key words—cyclophosphamide; cystitis; melatonin; alpha-tocopherol; beta-carotene
abbreviations—CP, cyclophosphamide; HC, hemorrhagic cystitis; NO, nitric oxide; NOS, nitric oxide synthase; eNOS,
endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide
synthase; NOx, nitrite and nitrate; MDA, malondialdehyde; Mesna, 2-mercaptoethane sulfonate; RNS,
reactive nitrogen species; ROS, reactive oxygen species
INTRODUCTION
Cyclophosphamide (CP), an oxazaphosphorine alkylating
agent introduced in 1958, currently is widely
used in the treatment of solid tumors and B-cell
malignant disease, such as lymphoma, myleloma,
chronic lymphocytic leukemia and Waldenstrom’s
macroglobulineamia. Hemorrhagic cystitis (HC) is a
major potential toxic and dose-limiting side effect of
CP.1 The incidence of this side effect is related to the
dosage and can be as high as 75% in patients receiving
a high-intravenous dose. The urological side effects
vary from transient irritative voiding symptoms to lifethreatening
HC.2 The urotoxicity of these nitrogen
mustard cytostatics is not based on a direct alkylating
activity on the urinary system but the formation of
renally excreted 4-hydroxy metabolites, in particular
acrolein, which is formed from hepatic microsomal
enzyme hydroxylation.3
cell biochemistry and function
Cell Biochem Funct 2007; 25: 521–526.
Published online 19 July 2006 in Wiley InterScience (Wiley Online Library (http://www.interscience.wiley.com)). DOI: 10.1027/cbf.1347
* Correspondence to: S. Oter, Gu¨lhane Askeri Tip Akademisi,
Fizyoloji Anabilim Dali, 06018–Etlik, Ankara, Tu¨rkiye (Turkey).
Tel: þ90 312 3043606. Fax: þ90 312 3043605.
E-mail:
[email protected],
[email protected]
Copyright # 2006 John Wiley & Sons, Ltd.
Received 31 January 2006
Revised 3 April 2006
Accepted 19 April 2006
Further, it has been shown that increased nitric
oxide (NO) production is responsible for the detrimental
effects of CP on bladder.4–6 Constitutive
expression of two nitric oxide synthase (NOS)
isoforms is responsible for a low basal level of NO
synthesis in neural cells (nNOS) and endothelial cells
(eNOS). Induction of the inducible isoform (iNOS) by
cytokines has been observed in virtually all cell types
including macrophages, fibroblasts, epithelial cells,
and results in the production of large amounts of NO.
The NO produced by iNOS is toxic, since in animal
models, selective iNOS inhibition decreases inflammatory
events.7 This toxicity probably comes from
reactive nitrogen species (RNS), in particular peroxynitrite
overproduction by reaction of NO with
superoxide which appears abundantly in the inflammatory
area.8 The overproduction of reactive oxygen
species (ROS) and RNS during inflammation leads to
considerable oxidative stress, cellular injury and
necrosis via several mechanisms including peroxidation
of membrane lipids, protein denaturation and
DNA damage.9
Thwarting the damage inflicted by free radicals and
reactive species is the function of a complex
antioxidative defense system. This system includes
some enzymes, such as superoxide dismutase, catalase
and glutathione peroxidase and some of the most
commonly used and experimentally studied nonenzyme
antioxidants, such as a-tocopherol, b-carotene
and melatonin.10 These agents are key elements
in reducing molecular damage due to ROS and RNS
and there is an extensive literature, which describes
their multiple actions.
2-Mercaptoethane sulfonate (mesna), an acrolein
binding and detoxifying compound within the urinary
collecting system, has been widely used as an effective
agent against CP-induced cystitis, but significant HC,
defined as an episode of microscopic or macroscopic
hematuria, has still been encountered clinically.3 Since
detoxifying acrolein with mesna cannot remove HC
symptoms completely and NO has been shown to be
involved in the pathogenesis, CP-induced HC is
probably not only due to direct contact of acrolein
with bladder mucosa but also related to increased ROS
and RNS production. In recent works we showed that
mesna is more effective in preventing CP-induced
bladder toxicity if combined with antioxidants.11,12 In
this study, we examined,whether antioxidantswere able
to diminish CP-induced bladder damage if used alone.
MATERIALS AND METHODS
Animals
Thirty-eight male Spraque–Dawley rats weighing
180–210 g were divided into five groups by ‘simple
random sampling method’ and given food and water
ad libitum. The amount of water intake consumed by
each animal was measured to avoid its hyperhydrative
effect. All animals received humane care according to
the criteria outlined in the ‘Guide for the Care and Use
of Laboratory Animals’ prepared by the National
Academy of Sciences and published by the National
Institutes of Health. The Gulhane Military Medical
Academy Animal Care and Use Committee approved
the experimental protocol.
Drug administrations
All antioxidants were obtained from Sigma-Aldrich
Co (b-carotene, C9750; a-tocopherol acetate, T3001;
melatonin, M5250). Their daily amount was first
dissolved in 0.5 ml ethanol and then diluted 10-fold
with saline. CP was used in its commercial form
(Endoxan1). HC induction was performed by a
urotoxic dose of 100 mg/kg CP in 2ml saline. Group 1
animals were injected with the same amount of saline
and served as control. Group 2 received CP only,
group 3 received b-carotene (220 mg/kg/day),
group 4 received a-tocopherol (220 mg/kg/day),
and group 5 received melatonin (25 mg/kg/day)
with CP, both before and the day after CP injection.
All drug administrations were performed intraperitoneally
(i.p.) as presented in Table 1.
Table 1. Cyclophosphamide, b-carotene, a-tocopherol, melatonin treatment schedule
Groups
Drug exposures
Day 1 Day 2 Day 3
1. Saline (n¼7) — Saline —
2. Cyclophosphamide; 100 mg/kg/day (n¼7) — CP —
3. b-carotene; 220 mg/kg/day (n¼8) b-carotene CPþb-carotene b-carotene
4. a-tocopherol; 220 mg/kg (n¼8) a-tocopherol CPþa-tocopherol a-tocopherol
5. Melatonin; 25 mg/kg/day (n¼8) melatonin CPþmelatonin melatonin
Copyright # 2006 John Wiley & Sons, Ltd. Cell Biochem Funct 2007; 25: 521–526.
522 s. sadir ET AL.
Tissue preparation
After 48 h of cystitis induction, rats were killed using a
large i.p. injection of ketamine HCl (85 mg/kg) and
xylazine HCl (12.5 mg/kg) to prevent inadvertent
bladder puncture. The bladders were removed intact,
evacuated of residual urine, cleaned from connective
and lipoid tissue around the wall and weighed to
determine if edema was present. Then the bladders
were cut into two equal pieces from dome to bottom.
One half was stored at 808C to measure bladder
malondialdehyde (MDA), the end product of lipid
peroxidation and iNOS activity and the rest fixed for
24 h in 10%-buffered formalin for histopathological
evaluation.
Analyses
During experiments, urine specimens were obtained
by abdominal massage and hemorrhage was evaluated
via the dip-stick method. Bladder edema was
evaluated by an increase in bladder-wet weight versus
body weight ratio (blw/bw). Assays of bladder MDA
levels and iNOS activity were performed via the
methods described by Draper&Hadley13 and Masuda
et al,14 respectively, and summarized below. The
tissues were homogenized in buffers by means of an
Ultra Turrax T25 homogenizer (IKA-Labortechnik,
Staufen, Germany) for MDA and iNOS activity
determination; the soluble fraction was prepared by
centrifugation at 6000g for 10 min. The protein
content of bladder tissues was determined by the
method of Lowry et al.15
MDA assay
This method exploits spectrophotometric measurement
of the colour produced during the reaction of
thiobarbituric acid (TBA) with MDA.13 The absorbance
of the final solution was measured with a
Shimadzu UV-1601 spectrophotometer (Shimadzu
Corp., Kyoto, Japan) at 532 nm and MDA levels
were expressed as nanomoles per mg-protein (nmol/
mg-prot).
iNOS assay
The method is based on determining the conversion of
[3H]L-arginine to [3H]L-citrulline.14 iNOS activity
was measured in a system in which calcium was
removed by addition of 2mM ethylenediaminetetraacetic
acid (EDTA) to the incubation mixture. NOS
activity was expressed as picomoles citrulline per mgprotein
per min (pmol-citrulline/mg-prot/min).
Urinary NO metabolites
Urine samples used for nitrite-nitrate (NOx) measurement
were collected in metabolic cages for 12 h just
before killing and frozen at 808C until assayed.
Samples were assayed for NOx using a NO Colorimetric
Assay Kit (Merck Eurolab GmbH, Darmstadt,
Germany) according to the manufacturer’s instructions.
The results were expressed as micromoles
(mmol).
Histopathological evaluation
At least four, approximately 5-m thick, cross-sections
were taken from each bladder. Histopathological
examination was performed by a pathologist in a
single blind fashion and scored as follows; edema,
hemorrhage and inflammation on a scale of 0 (normal)
to 4 (severe changes). Mucosal ulceration was scored
as 0 (normal), 1 (epithelial denuding), 2 (focal
ulceration), 3 (widespread epithelial ulceration) and
4 (submucosal ulceration).
Statistical analyses
The histological results are expressed as median (minmax)
and others meanSEM; p<0.05 was assessed
statistically significant. All of the numerical data were
analyzed first using the nonparametric Kruskal–Wallis
test to test whether there was a difference between
groups and then the Mann–Whitney U-test was
performed to analyze two groups consecutively.
RESULTS
All histological parameters are summarized in Table 2.
Control animals had histologically normal bladders
with assigned scores of ‘0’ for all parameters. CP
(group 2) showed severe histological changes
(p<0.01 vs. control for all parameters), and
macroscopic hematuria continued until the end of
the study. a-Tocopherol and melatonin (groups 4 and
5) showed meaningful protection against bladder
damage. There was no significant difference between
the protection attended by a-tocopherol and melatonin
for all parameters (p>0.05; group 4 vs. 5) (Figure 1).
Protection by b-carotene (group 3) was also significant
but weaker than both a-tocopherol and melatonin.
CP injection resulted also in increased MDA levels
indicating that oxidative stress was present in the
Copyright # 2006 John Wiley & Sons, Ltd. Cell Biochem Funct 2007; 25: 521–526.
antioxidants against cyclophosphamide-cystitis 523
bladder. All antioxidants ameliorated MDA levels in
bladder tissue (Figure 2A) (p<0.05 for group 3 vs.
group 2; p<0.01 for group 4 and group 5 vs. group 2).
Furthermore, CP also elevated the NOx level in urine
(Figure 2B) and iNOS activity in bladder (Figure 2C).
Only melatonin (group 5) was able to decrease NOx
levels in urine (p<0.01 vs. group 2) and iNOS activity
in bladder tissue (p<0.05 vs. group 2).
DISCUSSION
In CP and ifosfamide-induced HC it is now known that
NO is involved in the pathogenesis4–6 and bladder
epithelial cells have also been shown to express intense
reactivity to iNOS in the cytoplasm leading to
peroxynitrite production.16 Studies suggest that
increased NO production is responsible for cystitis
since S-methylisothiourea, an iNOS selective inhibitor,
almost abolished bladder damage.5,6 This histological
improvement was thought to result from decrease in
NO production. Nevertheless, in recent studies,
antioxidants such as a-tocopherol, b-carotene, epigallocatechin,
quercetin and melatonin, have been shown
to protect against bladder damage when combined with
mesna,11,12 the widely used protective agent against
CP-induced cystitis.3 Moreover, Abd-Allah et al.17
Figure 1. Histological analysis of representative bladder walls in cross section. Saline; normal bladder (H&E, 100), CP; meaningful
edema, leukocyte infiltration, hemorrhage and severe epithelial ulceration (H&E, 200). a-Tocopherol and melatonin; significantly different
from CP group in all parameters (H&E, 50 for both); note that slight edema is present in the tocopherol group; E, epithelial cell layer; M,
muscular layer; L, lumen; U, ulceration.
Table 2. Comparison of histological damage and bladder/body weight (blw/bw) ratio of rat bladders [median (min-max)].
Groups Edema Hemorrhage Inflammation Ulceration blw/bw (mg/g)
1. Saline 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0.67 (0.53–0.89)
2. Cyclophosphamide 4 (3–4) 4 (3–4) 3 (3–4) 3.5 (3–4) 2.73 (2.54–2.97)
3. b-carotene 2 (2–3) 1 (1–1) 1 (1–1) 3 (2–3) 0.87 (0.66–1.26)
4. a-tocopherol 1.5 (1–2) 0 (0–1) 1 (0–1) 1 (1–1) 0.73 (0.57–1.13)
5. Melatonin 1 (0–1) 0.5 (0–1) 0.5 (0–1) 0.5 (0–2) 0.75 (0.55–0.98)
p<0.01 compared with control group.
p<0.05 compared with CP group.
p<0.01 compared with CP group.
Copyright # 2006 John Wiley & Sons, Ltd. Cell Biochem Funct 2007; 25: 521–526.
524 s. sadir ET AL.
reported that taurine, an amino acid antioxidant and
Viera et al.18 reported that ternatin, a flavonoid
antioxidant, also have preventive effects against CPand
ifosfamide-induced HC.
The above mentioned flavonoids quercetin and
catechin, are herbal-derived compounds that have
been shown to reduce iNOS expression.19 We
hypothesized that bladder damage might not be due
only to overproduction of NO but also of ROS and
RNS. It is well known that increased ROS and/or RNS
production lead to oxidative damage.9 In the present
study,MDA levels of the CP group have clearly shown
that oxidative stress is present in damaged bladder.
There is no doubt that the overproduction of ROS and
the majority of NO produced by during inflammation
is converted to peroxynitrite anion.20 There are several
experimental reports suggesting the formation of
peroxynitrite during the inflammatory process itself
including ileitis, lung injury, nephritis, myocardial
infarction, cerebral ischaemia, and endotoxemia via
iNOS activation.8 It is believed that peroxynitrite is
responsible for the harmful effects of iNOS-produced
NO during inflammation. A more recent study
suggested that peroxynitrite may also be involved in
CP-induced bladder damage.21
Three categories of defense against peroxynitritemay
be listed as prevention, interception and repair.20
Prevention of the exposure of cells to peroxynitrite
can simply be prevention of its formation. In the case of
HC, generation of peroxynitrite can be prevented by
inhibiting the formation of NO4–6,16 and/or of superoxide
anion11 by either inhibiting enzyme systems
responsible for the generation of these two radicals or
scavenging prior to the generation of peroxynitrite. In
our work, bladder iNOS and urine NOx measurements
indicated that neither b-carotene nor a-tocopherol
significantly diminished NO production. On the other
hand, accumulating evidence shows that a-tocopherol is
capable of decreasing the production and/or availability
of not only superoxide, but also of NO and
peroxynitrite.22 Moreover, decreased MDA levels of
tissue with these antioxidants indicate that b-carotene
and a-tocopherol protect by scavenging oxidants such
as superoxide; this may result in decrease of
peroxynitrite production. The weaker protective effect
of b-carotene may be due to its limited antioxidant
effect that is, quenching of singlet oxygen not superoxide
anion.23 According to defense categories against
peroxynitrite, these antioxidants may have a role in
prevention of peroxynitrite production.
Melatonin is not only an antioxidant but also an
iNOS inhibitor (prevention).24 Recent studies have
also shown that melatonin can directly scavenge the
peroxynitrite (interception).25 In our work, melatonin
showed a rather greater protective effect against CPinduced
damage than a-tocopherol or b-carotene and
diminished the bladder MDA levels, iNOS induction
and urinary NOx excretion significantly. All histopathological
examinations and biochemical measurements
suggest that melatonin may have a role in two
stages, namely prevention and interception, of defense
against peroxynitrite production.
It is believed that ulceration is caused by direct
contact of acrolein with uroepithelium.3 In our
Figure 2. MDA and iNOS levels of bladder tissue and NOx in urine. (A)MDA levels of bladder tissue. CP administration severely increased
and all of the antioxidants significantly lowered MDA to nearly saline level (p<0.01 for CP vs. control; p<0.05 for car, and p<0.01 for toc
and mel vs. CP). (B) Only melatonin decreased NOx in urine. b-carotene and a-tocopherol did not affect urinary nitrite and nitrate excretion
(p<0.01 for CP vs. control; p<0.01 for mel, and p>0.05 for car and toc vs. CP). (C) iNOS activity in control group was almost
undetectable. CP significantly induced iNOS (p<0.01 for CP vs. control). Only melatonin was able to inhibit iNOS induction (p<0.05 for
mel vs. CP). CP; cyclophosphamide, car; b-carotene, toc; a-tocopherol, mel; melatonin
Copyright # 2006 John Wiley & Sons, Ltd. Cell Biochem Funct 2007; 25: 521–526.
antioxidants against cyclophosphamide-cystitis 525
experiments, histological examination clearly demonstrated
that uroepithelial ulceration was also prevented
in spite of acrolein being not blocked with an agent
such as mesna. This protection may again be due to
decreased peroxynitrite production. It was shown that
exposure to high concentrations of peroxynitrite leads
to rapid cell death associated with rapid energetic
derangements.26 Moreover, our previous studies
performed with hyperbaric oxygen, an extremely
different treatment modality, also showed protective
effect against necrosis5,27 possibly via relieving the
uroepithelial energy crisis caused by the peroxynitrite
overproduction.
CONCLUSIONS
In the light of these data the suggestion that
peroxynitrite may be responsible, at least in part,
for CP-induced bladder damage is now more
powerful. Furthermore, melatonin and a-tocopherol
may ameliorate bladder damage through scavenging
ROS and RNS, even if used without mesna. Since
randomized controlled trials have an important place
in the assessment of the efficacy of complementary
medicine and a-tocopherol and melatonin are
safe and cheap agents, clinicians may be encouraged
to try such antioxidants as adjuvants in trial
studies.
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