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me2
10-10-2010, 07:13 AM
I have been doing some research on Cytoxan and I have found things that my pharmacy and doctors have not told me about protection from Cytoxan. I am currently getting 500 mg of Cytoxan IV twice a month with Mesna. I take Mesna the day of the infusion.

My story is long so I will skip over it and just say that I am concerned about getting maximum protection from the negative effects of Cytoxan.
The primary way that Cytoxan does bladder damage is through its metabolite by-product acrolien. Mesna neutralizes this quite effectively. The problem is that there are a couple of other chemicals that also do damage that are not neutralized by Mesna.

I did a search for natural methods to counteract the damage of Cytoxan and was surprised to find that there were several studies that showed effectiveness of safe natural supplements against the negative effects of Cytoxan. I assume that we are not told this because they are not drugs and no one gets paid to push them on doctors who then in turn tell us about them.
I would like to share the information I have found. Some of the studies and the substances are open to question in my mind because their safety is not known. However, two of the substances are very well known and I think quite safe. Vitamin E and melatonin.
For myself I want continued protection from the Cytoxan beyond the one day I take Mesna. Cytoxan has a half life which means my body is continually metabolizing it beyond the day that I get my infusion.
Also, I am concerned for people who take Cytoxan daily and need all the protection they can get.
I have the pdf. of the study. I got it from the library at the University where I get my treatment.
I would be glad to email a copy to anyone who would like it. If there is a better way to share it let me know.

Sangye
10-10-2010, 07:54 AM
I didn't know how ctx damaged the bladder-- very useful info! Can you post the pdf here?

me2
10-10-2010, 08:09 AM
Well, I will give it a try. Here goes:


Ibrahim Yildirim Ahmet Korkmaz Sukru Oter
Ayhan Ozcan Emin Oztas

Contribution of antioxidants to preventive effect of mesna
in cyclophosphamide-induced hemorrhagic cystitis in rats

Received: 9 February 2004 / Accepted: 18 March 2004 / Published online: 30 June 2004

 Springer-Verlag 2004

Abstract Purpose: The aim of this study was to evaluate
whether combination of antioxidants and mesna may
prevent cystitis induced by cyclophosphamide better
than mesna alone. Materials and methods: A total of 46
male Spraque-Dawley rats were divided into six groups.
Five groups received single dose of cyclophosphamide
(CP, 100 mg/kg) intraperitoneally with the same time
intervals: group 2 received CP only, group 3 received
mesna (21.5 mg/kg for three times), group 4 beta-carotene
(20 mg/kg for two times) and mesna, group 5
received alpha-tocopherol (20 mg/kg for two times) and
mesna, and group 6 received melatonin (5 mg/kg for two
times) and mesna on the day of CP injection. Group 1
served as control. Results: CP injection resulted in severe
cystitis. Mesna has showed meaningful but not full
protection against CP toxicity. Although beta-carotene
did not show any additional beneficial effect when
combined with mesna, alpha-tocopherol and especially
melatonin with mesna resulted full protection that the
pathologist, blinded to the slides, could not differ from
sham control. Conclusion: Oxidants may be important
in the pathogenesis of CP-induced cystitis. Melatonin
and alpha-tocopherol may help to ameliorate bladder
damage along with other drugs such as mesna and
diuretics.

Keywords Cyclophosphamide Mesna

Antioxidants Cystitis

Introduction

Cyclophosphamide (CP) is in the nitrogen mustard
group of alkylating antineoplastic chemotherapeutic
agents. It is used alone or in combination with other
chemotherapeutic agents for the treatment of many
neoplastic diseases [6]. Hemorrhagic cystitis (HC) is a
major potential toxicity and dose limiting side effect of
CP and ifosfamide, a synthetic analog of CP [19]. 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, including urinary
frequency, dysuria, urgency, suprapubic discomfort, and
stangury with microhematuria, to life-threatening HC
[8]. Bladder fibrosis, necrosis, contracture, and vesicoureteral
reflux and a 4% percent mortality rate among
patients with massive bladder hemorrhage have also
been reported [6, 19]. The Urotoxicity of these nitrogen
mustard group cytostatics is not based on a direct
alkylating activity on urinary system but the formation
of renally excreted 4-hydroxy metabolites, in particular
acrolein, which is formed from hepatic microsomal
enzymatic hydroxylation [10].
Mesna contains a sulfhydryl compound which binds
acrolein within the urinary collecting system and detoxifies
it; the resultant inert thioether is passed innocuously
in the urine and does not induce any damage to the
uroepithelium [7, 10]. Although mesna has been widely
used as an effective agent against CP-induced cystitis,
significant HC, defined as an episode of symptomatic
(burning, frequency and dysuria), microscopic or macroscopic
hematuria, has still been encountered clinically.

I. Yildirim
Department of Urology,
Gulhane Military Medical Academy,
Ankara, Turkey
A. Korkmaz S. Oter (&)
Department of Physiology,
Gulhane Military Medical Academy,
Ankara, Turkey
E-mail: [email protected]
Tel.: +90-312-3043606
Fax: +90-312-3042150
A. Ozcan
Department of Pathology,
Gulhane Military Medical Academy,
Ankara, Turkey
E. Oztas
Department of Medical Histology and Embryology,
Gulhane Military Medical Academy,
Ankara, Turkey
Cancer Chemother Pharmacol (2004) 54: 469–473
DOI 10.1007/s00280-004-0822-1

Recently, it has been shown that increasing nitric
oxide (NO) production is responsible for the detrimental
effects of CP on bladder [9, 15, 16]. This toxicity probably
comes from reactive nitrogen species (RNS), in
particular peroxynitrite (ONOO) overproduction by
reacting NO and superoxide (O2

•)which appears
abundantly inflammatory area [17]. Moreover it is clear
that, in biological systems the primary source of all RNS
is NO. The overproduction of reactive oxygen species
(ROS) and RNS during inflammation leads to a considerable
oxidant stress, cellular injury and necrosis via
several mechanisms including peroxidation of membrane
lipids, protein denaturation and DNA damage
[18].
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 (SOD), catalase (CAT)
and glutathione peroxidase (GPx) [12] and some of the
most commonly used and experimentally studied nonenzyme
antioxidants, such as b-carotene, a-tocopherol
and melatonin [4]. These agents are key elements in
reducing molecular damage due to reactive oxygen and
nitrogen species and there is extensive literature, which
describes their multiple actions.
Since detoxifying acrolein with mesna cannot
remove HC symptoms completely and NO has been
shown to involve 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 this study we examined,
whether combination of antioxidants with mesna may
show better result than mesna alone in CP induced
bladder damage.

Materials and methods

Animals
A total of 46 male rats, with body weights of 270–340 g
were divided into six groups by ‘‘simple random sampling
method’’ and given food and water ad libitum.
Amount of water consumed by each animal was measured
to avoid the hiperhydrative effect of water. The
Gulhane Military Medical Academy Animal Care and
Use Committee approved the experimental protocol.
Drug administrations
The drug administration schedule is presented in
Table 1.

Experimental induction of HC The animals were given
urotoxic dose of 100 mg/kg CP in 2 ml saline. Group 1
animals were injected with the same amount of saline
and served as control.

Mesna and antioxidants administrations 64.5 mg/kg
mesna was administred 20 min before CP injection, and
continued every 4 h for a total of three equal doses. Betacarotene
(220 mg/kg), alpha-tocopherol (220 mg/kg),
melatonin (25 mg/kg) were given 12 and 1 h before CP
administration. All injections were performed intraperitoneally
(i.p.).
Tissue preparation
Twenty-four hours after CP administration, animals
were sacrificed using high i.p. injection of ketamine HCl
and xylazine HCl to prevent inadvertent bladder puncture.
The bladders were removed intact, evacuated
residual urine, cleaned from connective and lipoid tissue
around the wall, weighed, and fixed for 24 h in 10%
buffered formalin. Standart paraffin blocks, as well as
hematoxylin and eosin-stained slides, were prepared.
The pathologist, who had no knowledge of which of the
six groups each slide belonged to, rated the mean histologic
damage, including ulceration, hemorrhage and
edema, on a scale of 1 (light) to 4 (severe changes).
Normal bladder of control group rated as 0.
Definitions of hematuria
Hematuria was graded on a scale of 0–3 by performing
dip-stick analysis in the urine specimens obtained by
abdominal massage at 6, 12 and 24 h after CP injection.
Statistics
The results are expressed as the median (min–max) and

p<0.05 was assessed as statistically significant. All of

Table 1 Cyclophosphamide,
mesna, b-carotene,

a-tocopherol and melatonin
treatment schedule
Groups Drug administration timing

12 h 1 h 20 min 0 +4 h +4 h
1. n=7 – – – Saline – –
2. n=7 – – – CP – –
3. n=8 – – Mesna CP Mesna Mesna
4. n=8 b-carotene b-carotene Mesna CP Mesna Mesna
5. n=8 a-tocopherol a-tocopherol Mesna CP Mesna Mesna
6. n=8 Melatonin Melatonin Mesna CP Mesna Mesna
470

the numeric data were analyzed first using nonparametric
Kruskal–Wallis test to find whether there is difference
between groups and then Mann–Whitney U-test
was performed to analyze two groups consecutively.

Results

The animals in control had cytologically normal bladders
with assigned scores of 0 for all three parameters,
namely edema, hemorrhage, and ulceration. Animals
receiving CP (group 2) had shown severe cytologic
changes and higher grades of hematuria. Unscored
cytologic features peculiar to the slides of this group
included mucosal sloughing and hemorrhagic areas
(Figs. 1, 2).Moreover, severe ulceration and erosion had
been encountered in five of seven bladders as shown in
Fig. 3. No ulceration was observed in any other slides.
In treatment group received mesna only, statistically
significant protection was observed for ulceration as
shown in Table 2 (p<0.05 compared with CP), histologic
damages were present for edema and hemorrhage
(p>0.05 compared with CP) (Figs. 1, 2).
Addition of beta-carotene to mesna did not show
more significant protection than mesna alone. Alphatocopherol
(p<0.05 for hemorrhage compared with
group 3) and melatonin (p<0.05 for hemorrhage and
edema compared with group 3) are both revealed protective
properties along with mesna. Hematuria was
continued in CP group and almost disappeared in
treatment groups.

Discussion

CP, an antineoplastic alkylating agent, is used to treat
neoplastic, immune mediated and transplant related
diseases and its use is likely to increase as new applications
are discovered. HC is a major therapy-limiting side
effect of CP. It is thought to be induced by acrolein, a
cytotoxic metabolite of CP, which is excreted in the
urine. The main futures of HC are urothelial damage,
transmural edema, hemorrhage, mucosal ulceration and
epithelial necrosis which could be demonstrated within
24 h of a single dose [8]. Mucosal sloughing has been
associated frequently with acute and chronic hemorrhage.
Within the first several hours, epithelial cells in
the superficial mucosal layer of the bladder begin undergo
degeneration and necrosis. At 18 h post-injection,
most of the mucosal lining is eroded or ulcerated, and
basal membrane damage is evident with subsequent

Fig. 2 Scale of rat bladder histology for hemorrhage from 1 (+;
light) to 4 (++++; severe) (H&E, approx. 100) (+ from group
6, ++ from group 5, +++ from group 3 and ++++ from
group 2)

Fig. 1 Scale of rat bladder histology for edema from 1 (+; light) to
4 (++++; severe) (H&E, approx. 100) (+ from group 6, ++
from group 5, +++ from group 3 and ++++ from group 2)

Fig. 3 Scale of rat bladder histology for ulceration from 1 (+;
light) to 4 (++++; severe) (H&E, approx. 100) (+ from group
5, ++ from group 4, +++ from group 3 and ++++ from
group 2)
471

damage of the surrounding capillaries. Thereafter,
healing began with evidence of mucosal hyperplasia and
bizarre papillary proliferation. Neovascularisation and
leukocyte infiltration may also be seen following days
[2].
Among various prophylactic and therapeutic measures
to treat HC, mesna showed the most promising
results. Its toxicity is negligible and oxidized to a stable
inactive disulfide within minutes of parenteral administration
and becomes active when excreted into the urine.
Mesna combines with acrolein in the urine to form an
inert, nontoxic thioester and allows maximum therapeutic
effect of the alkylating oxazaphosphorine drug. In
addition to neutralizing acrolein, it slows the degradation
of the 4-hydroxy-metobolites of the alkylating agent
[7, 10]. However, although mesna has showed an effective
uroprotection, HC still occurs in 10–40% of mesnatreated
patients.
CP induced HC is now known that NO is involved
the pathogenesis [9] 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, possible
through iNOS activation, is responsible the cystitis since
S-methylisothiourea (iNOS selective inhibitor) almost
abolished bladder damage [15]. In a recent study antioxidants
have been shown protective effect on bladder
damage [data not shown]. This histological improvement
is thought to be resulted from decreasing ROS and
RNS production.
Over the last few years, the overall picture of the
inflammatory process has been complicated by the potential
pathogenetic contribution of ROS and RNS. A
great variety of stimuli such as immune complexes, and
inflammatory cytokines are able to up-regulate expression
and synthesis of iNOS. Furthermore, it is now well
recognized that ROS and RNS may interact with each
other, resulting not only in the induction of further new
reactive species, but above all, in possible changes in the
concentration of these two classes of molecules [1].
There is no doubt that the overproduction of ROS and
the majority of NO produced during inflammation are
converted to peroxynitrite anion [13]. NO is the only
currently known biological molecule produced in
high enough concentrations to react fast enough with
superoxide (forming ONOO) to outcompete endogenous
SOD [3].
The formation of ONOO may be double edged
sword. First, NO neutralizes a potentially deleterious
species of oxygen radical, the superoxide radical. On the
other hand, the reaction consumes NO and produces a
potentially deleterious metabolite, ONOO. Inflammatory
cells such as PMNs and macrophages, but also
endothelial cells can release superoxide and NO,
potentially leading to peroxynitrite formation. Their
release can be mediated and regulated by several cytokines.
It is clear that overproduction or uncontrolled
formation of peroxynitrite is an important factor in the
tissue damaging mechanisms during pathological situations
[1]. There are several experimental reports suggesting
the formation of peroxynitrite during the
inflammatory process including ileitis, lung injury, and
endotoxemia via iNOS activation [13].
Results of this study suggest that antioxidants especially
alpha-tocopherol and melatonin may be helpful to
ameliorate CP induced cystitis when combined with
mesna. Moreover both melatonin and vitamin E alone
have also shown protective effect against CP induced
cystitis [unpublished data]. Melatonin is a newly discovered
antioxidant and not only itself but also chief
hepatic metabolite of melatonin, namely, 6-hydroxymelatonin,
is also reportedly an effective free radical
scavenger [11]. A recent study has also shown that
melatonin can directly scavenge the peroxynitrite [5].
Beside this antioxidant action, melatonin is an iNOS
inhibitor [4] and this feature may contribute to beneficial
effects of melatonin against bladder damage. The weaker
protective effect of b-carotene may be due to its limited
antioxidant effect that is, quenching of singlet oxygen
not superoxide anion [14].
In conclusion, a-tocopherol and melatonin ameliorated
bladder damage possibly through scavenging ROS
and RNS. Since a-tocopherol and melatonin are safe
and cheap, future studies may focus on the efficacy of
antioxidants alone or combination with other therapeutic
modalities such as mesna and diuretics on HC
caused by CP.

References

1. Beckman JS, Koppenol WH (1996) Nitric oxide, superoxide
and peroxynitrite: the good, the bad, and the ugly. Am
J Physiol 271C:1424–1437
2. Brock N, Pohl J, Stekar J (1981) Studies on the urotoxicity of
oxazaphosphorine cytostatics and its prevention. I. Experimental
studies on the urotoxicity of alkylating compounds. Eur
J Cancer 17(6):595–607
3. Crow JP, Beckman JS (1996) The importance of superoxide in
nitric oxide-dependent toxicity: evidence for peroxynitritemediated
injury. Adv Exp Med Biol 387:147–161
4. Cuzzocrea S, Reiter RJ (2001) Pharmacological action of
melatonin in shock, inflammation, and ischemia/reperfusion
injury. Eur J Pharm 426:1–10
5. Gilad E, Cuzzocrea S, Zingarelli B, Salzman AL, Szabo C
(1997) Melatonin is a scavenger of peroxynitrite. Life Sci
60:169–174

Table 2 Comparison of histologic damage of rat bladders [median
(min–max)]
Groups Edema Hemorrhage Ulceration
1. Control 0 (0–0) 0 (0–0) 0 (0–0)
2. CP 3* (1–4) 4* (2–4) 3* (1–4)
3. Mesna 2 (2–3) 2 (2–4) 1/ (1–2)
4. Mesna + b-carotene 2 (1–3) 2 (1–2) 1 (1–2)
5. Mesna + a-tocopherol 1,5 (1–4) 1d (1–1) 1 (1–1)
6. Mesna + melatonin 1d/ (0–2) 1d/ (0–1) 1/ (1–1)
*p<0.05 when compared with control group, /p<0.05 when
compared with CP group, dp<0.05 when compared with mesna
group.

me2
10-10-2010, 08:15 AM
Hey, it worked and Jack didn't have to tell me how this time. So, if you are still awake, here is another one:


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, Gulhane Askeri Tip Akademisi,
Fizyoloji Anabilim Dali, 06018–Etlik, Ankara, Turkiye (Turkey).
Tel: 90 312 3043606. Fax: 90 312 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 (n7) — Saline —
2. Cyclophosphamide; 100 mg/kg/day (n7) — CP —
3. b-carotene; 220 mg/kg/day (n8) b-carotene CPb-carotene b-carotene
4. a-tocopherol; 220 mg/kg (n8) a-tocopherol CPa-tocopherol a-tocopherol
5. Melatonin; 25 mg/kg/day (n8) melatonin CPmelatonin 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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13. Draper HH, Hadley M. Malondialdehyde determination as index
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14. Masuda H, Tsujii T, Okuno T, Kihara K, Goto M, Azuma H.
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Am J Physiol 2002; 282R: 1730–1738.
15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
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1951; 193: 265–275.
16. Ribeiro RA, Feritas HC, Campos MC, et al. Tumor necrosis
factor-a and interleukin-1b mediate the production of nitric
oxide involved in the pathogenesis of ifosfamide induced
hemorrhagic cystitis in mice. J Urol 2002; 167: 2229–2234.
17. Abd-Allah ARA, Gado AM, Al-Majed AA, Al-Yahya AA, Al-
Shabanah OA. Protective effect of taurine against cyclophosphamide-
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Pharmacol Physiol 2004; 31: 167–172.
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19. Achike FI, Kwan CY. Nitric oxide, human diseases and the
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20. Klotz LO, Sies H. Defenses against peroxynitrite: selenecompounds
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21. Korkmaz A, Oter S, Sadir S, et al. Peroxynitrite may be involved
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Copyright # 2006 John Wiley & Sons, Ltd. Cell Biochem Funct 2007; 25: 521–526

me2
10-10-2010, 08:25 AM
Toxic Effect of Cyclophosphamide on Sperm Morphology,
Testicular Histology and Blood Oxidant-Antioxidant Balance,
and Protective Roles of Lycopene and Ellagic Acid

Ali Osman eribas i1, Gaffari Trk2, Mustafa Snmez2, Fatih Sakin3 and Ahmet Ates s ahin4

1Department of Pathology, Faculty of Veterinary Medicine, Fırat University, Elazıg˘, Turkey, 2Department of Reproduction and Artificial
Insemination, Faculty of Veterinary Medicine, Fırat University, Elazıg˘, Turkey, 3Department of Pharmacology and Toxicology, Faculty of
Veterinary Medicine, Dicle University, Diyarbakır, Turkey, and 4Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine,
Fırat University, Elazıg˘, Turkey
(Received 17 November 2009; Accepted 26 January 2010)

Abstract: In this study, the toxic effect of cyclophosphamide (CP) on sperm morphology, testicular histology and blood oxidant–
antioxidant balance, and protective roles of lycopene (LC) and ellagic acid (EA) were investigated. For this purpose, 48
healthy, adult, male Sprague-Dawley rats were divided into six groups; eight animals in each group. The control group was
treated with placebo. LC, EA and CP groups were given alone LC (10 mg ⁄ kg ⁄ every other day), EA (2 mg ⁄ kg ⁄ every other
day) and CP (15 mg⁄ kg ⁄week) respectively. One of the last two groups received CP + LC, and the other treated with
CP + EA. All treatments were maintained for 8 weeks. At the end of the treatment period, morphological abnormalities of
sperm, plasma malondialdehyde (MDA) levels and glutathione (GSH) levels, and GSH-peroxidase (GSH-Px), catalase (CAT)
and superoxide dismutase (SOD) activities in erythrocytes, and testicular histopathological changes were examined. CP administration
caused statistically significant increases in tail and total abnormality of sperm, plasma MDA level and erythrocyte
SOD activity, and decreases in erythtocyte CAT activity, diameters of seminiferous tubules, germinal cell layer thickness and
Johnsen’s Testicular Score along with degeneration, necrosis, immature germ cells, congestion and atrophy in testicular tissue.
However, LC or EA treatments to CP-treated rats markedly improved the CP-induced lipid peroxidation, and normalized
sperm morphology and testicular histopathology. In conclusion, CP-induced lipid peroxidation leads to the structural damages
in spermatozoa and testicular tissue of rats, and also LC or EA have a protective effect on these types of damage.

Most of the chemotherapeutic drugs used in the treatment of
neoplastic cells cause various sorts of damage to normal living
cells. One of these drugs is cyclophosphamide (CP;
C7H17Cl2N2O3P; MW: 279.10 g ⁄mol; N-bis(2-chloroethyl)-
1-oxo-6-oxa-2-aza-1k5-phosphacyclohexan-1-amine hydrate).
It has potent anticancer, and as well as immunosuppressive
effects for organ transplantation and autoimmune diseases.
CP therapy is a common continuing problem in the treatment
of a variety of glomerular diseases and leads to gonadal
toxicity as a side effect of the drug [1]. Previous studies have
shown that CP alters sperm chromatin structure, composition
of sperm head basic proteins and increases abnormal
sperm rate, and manifest biochemical and histological alterations
in testis [2–4]. It has been reported that oxidative
stress-induced biochemical and physiological damage is
responsible for CP toxicity in testis and spermatozoa [5–7].
The mitochondrial membrane of spermatozoa is more susceptible
to lipid peroxidation, as this compartment is rich in
polyunsaturated fatty acids and has been shown to contain
low amounts of antioxidants [8,9]. Additionally, mitochondria
and plasma membranes of morphologically abnormal
spermatozoa produce reactive oxygen species (ROS) [10].
Recently, there is growing interest in understanding the
roles and mechanisms of the carotenoids and phytochemicals
as inhibitors of oxidative stress. Lycopene (LC; C40H56;
MW: 536.87; w,w-Carotene, 2,6,10,14,19,23,27,31-Octamethyl-
dotriaconta-2,6,8,10,12,14,16,18,20,22,24,26,30-tridec
aene), a carotenoid occurring naturally in tomatoes, has
attracted considerable attention as an antioxidant. LC,
because of its high number of conjugated double bonds, has
been reported to exhibit high singlet oxygen (1O2)-quenching
ability and to act as a potent antioxidant, preventing the oxidative
damage of critical biomolecules including lipids, proteins
and DNA [11]. Ellagic acid (EA; C14H6O8; MW:
302.20; 3,7,8-tetrahydroxy[1]-benzopyrano[5,4,3-cde] [1]benzopyran-
5,10-dione) has potent radical scavenging and
chemopreventive properties [12,13]. Raspberries, strawberries,
walnuts, longan seed, mango kernel [14,15] and pomegranate
[16] are rich plants with respect to EA. It contains
four hydroxyl groups and two lactone groups in which the
hydroxyl group is known to increase antioxidant activity in
lipid peroxidation and protect cells from oxidative damage
[17]. It has been reported that the therapeutic antioxidant
effect of LC [18] and EA [19] on germ cells could serve as
promising intervention to oxidative stress-induced infertility

Author for correspondence: Gaffari Trk, Department of Reproduction
and Artificial Insemination, Faculty of Veterinary Medicine,
Fırat University, 23119 Elazıg˘, Turkey (fax +90 424 238 81 73,
e-mail [email protected]; [email protected]).

Basic & Clinical Pharmacology & Toxicology, 107, 730–736 Doi: 10.1111/j.1742-7843.2010.00571.x

2010 The Authors
Basic & Clinical Pharmacology & Toxicology 2010 Nordic Pharmacological Society

me2
10-10-2010, 08:34 AM
Protective Effect of Seleno-L-Methionine
on Cyclophosphamide-Induced Urinary
Bladder Toxicity in Rats

Adnan Ayhanci & Suzan Yaman & Varol Sahinturk &

Ruhi Uyar & Gokhan Bayramoglu & Hakan Senturk &

Yilmaz Altuner & Sila Appak & Sibel Gunes

Received: 28 June 2009 / Accepted: 3 July 2009 /
Published online: 24 July 2009

# Humana Press Inc. 2009

Abstract Cyclophosphamide (CP) is a widely used antineoplastic drug, which could cause
toxicity of the normal cells due to its toxic metabolites. Its urotoxicity may cause doselimiting
side effects like hemorrhagic cystitis. Overproduction of reactive oxygen species
(ROS) during inflammation is one of the reasons of the urothelial injury. Selenoproteins
play crucial roles in regulating ROS and redox status in nearly all tissues; therefore, in this
study, the urotoxicity of CP and the possible protective effects of seleno-L-methionine
(SLM) on urinary bladder of rats were investigated. Intraperitoneal (i.p.) administration of
50, 100, or 150 mg/kg CP induced cystitis, in a dose-dependent manner, as manifested by
marked congestion, edema and extravasation in rat urinary bladder, a marked desquamative
damage to the urothelium, severe inflammation in the lamina propria, focal erosions, and
polymorphonuclear (PMN) leukocytes associated with occasional lymphocyte infiltration
determined by macroscopic and histopathological examination. In rat urinary bladder tissue,
a significant decrease in the endogenous antioxidant compound glutathione, and elevation
of lipid peroxidation were also noted. Pretreatment with SLM (0.5 or 1 mg/kg) produced a
significant decrease in the bladder edema and caused a marked decrease in vascular
congestion and hemorrhage and a profound improvement in the histological structure.
Moreover, SLM pretreatment decreased lipid peroxide significantly in urinary bladder

Biol Trace Elem Res (2010) 134:98–108
DOI 10.1007/s12011-009-8458-y
A. Ayhanci (*) : S. Yaman : G. Bayramoglu : H. Senturk : Y. Altuner : S. Gunes
Faculty of Arts and Science, Department of Biology, Meselik Campus, Izmir Osmangazi University,
F5 26480 Eskisehir, Turkey
e-mail: [email protected]
V. Sahinturk
Department of Histology, Faculty of Medicine, Izmir Osmangazi University, Eskisehir, Turkey
R. Uyar
Department of Physiology, Faculty of Medicine, Izmir Osmangazi University, Eskisehir, Turkey
S. Appak
Izmir Institute of Technology Department of Molecular Biology & Genetics,
Biology & Genetics, Izmir Institute of Technology, Eskisehir, Turkey

tissue, and glutathione content was greatly restored. These results suggest that SLM offers
protective effects against CP-induced urinary bladder toxicity and could be used as a
protective agent against the drug toxicity.

Keywords Cyclophosphamide . Seleno-L-methionine . Urotoxicity . Cytoprotectivity . Rats

Introduction

Cyclophosphamide (CP) is extensively used as an antineoplastic agent for the treatment of
various cancers and as an immunosuppressive agent for organ transplantation [1]. CP has
been shown to induce severe hemorrhagic cystitis (HC) in laboratory animals [2] and in
patients receiving the drug as a part of their treatment [3]. HC is the major dose-limiting
side effect of CP [4]. The incidence of this side effect is dose dependent and can be as high
as 75% in patients receiving a high intravenous CP dose.
There have been many attempts to prevent CP-induced cystitis with the pretreatments of
different drugs, such as zinc or 2-mercaptoethane sulfonate (MESNA). This compound has
been shown to reduce, in part, the magnitude of CP-induced cystitis [5]. It is important to
elucidate the mechanism of CP-induced HC in order to minimize the toxic and doselimiting
side effects of CP. Elimination the side effects of CP can lead to better tolerance of
the drug and a more efficient and comfortable therapy for patients in need of CP treatment
[6]. The overproduction of reactive oxygen species (ROS) and reactive nitrogen species
(RNS) during inflammation leads to considerable oxidant stress, cellular injury, and
necrosis via several mechanisms including peroxidation of membrane lipids, protein
denaturation, and DNA damage [7].
It is suggested that CP treatment of rats induces oxidative stress in the urinary bladder and
depletion of antioxidant enzyme, and the oxidative stress contributes to neutrophil infiltration
into bladder and hence inflammation [6]. Furthermore, Das et al. [8] have reported that CPinduced
testicular oxidative stress was manifested by a significant inhibition of peroxidase
and catalase enzyme activities and with high levels of malondialdehyde (MDA) and
conjugated dienes in the testis.
Numerous studies have shown that CP exposure enhances intracellular ROS production,
suggesting that biochemical and physiological disturbances may result from oxidative stress
[9]. The antioxidative defense system includes enzymes, such as glutathione peroxidase
(GPx), superoxide dismutase, catalase [10], and non-enzyme antioxidants, like carotenoids
[11] and selenium [12]. The cellular antioxidant system helps to minimize ROS-induced
tissue injury [6]. Thus, the combination of the drug delivery together with potent
antioxidant may be the appropriate approach to reduce the side effects of CP [1].
Selenium (Se) is a nutritionally essential trace element with anticarcinogenic properties.
Overexpression of Gpx has been reported to block ROS-induced apoptosis in several cell
types, suggesting that inhibition of this enzyme is closely related to apoptotic cell death
[12]. Limited data from studies in humans suggest that Se supplementation may enhance
immunity, including both humoral and cell-mediated responses [13]. It was demonstrated
that Se is a highly effective modulator of the therapeutic efficacy and selectivity of
anticancer drugs in nude mice bearing human tumor xenografts of colon carcinoma and
squamous cell carcinoma of the head and neck. Thus, the use of Se as selective modulator
of the therapeutic efficacy of anticancer drugs is new and novel [14]. Therefore, the aim of
the present study was to investigate the potential protective effect of seleno-L-methionine
(SLM) against CP-induced cystitis in rats.

Protective Effect of SLM on CP-Induced Urinary Bladder Toxicity in Rats 99

Materials and Methods

Animals
A total of 84 either sex Sprague–Dawley rats weighing 190–220 g were used for the
intraperitoneal injection of CP (Endoxan, Sigma-Aldrich, Taufkirchen, Germany; C0768)
and SLM (Sigma, Germany; S3132). Animals were given food and water ad libitium. Local
institutional animal care and use committee approved the experimental protocol.
Experimental Design
The rats were randomly divided into the following experimental groups, each including
seven animals: groups 1, 2, and 3 treated with 50, 100, or 150 mg/kg CP, respectively [15].
Groups 4 and 5 treated with 0.5 and 1 mg/kg SLM, respectively. Groups 6, 7, or 8 treated
with respective CP plus 0.5 mg/kg SLM. Groups 9, 10, or 11 were treated with respective
CP plus 1 mg/kg SLM. Group 12 is the control group that only received saline. Animal
groups and their treatments are summarized in Table 1.
Tissue Preparation and Histopathology
At the end of the experiment, the rats were weighed. Under the deeper ether anesthesia, the
animals were killed at day 7. The bladders were removed intact, evacuated of residual urine,
washed with saline, blotted dry on a filter paper, and weighed to determine the presence of
edema. The bladders were then cut into two equal pieces from the dome to the bottom. One half
was stored at −80C to measure bladder MDA and glutathione (GSH) contents; the rest was
fixed for 24 h in 10% buffered formaldehyde. Tissues were embedded in paraffin and serial
cross-sections 4–5-μm thick were taken fromeach bladder and stained with hematoxylin–eosin.
All sections were examined under 40 objective and scored for edema, hemorrhage, and
inflammation on a scale of 0 (normal) to 3 (severe changes). Mucosal ulceration was scored as 0
(normal), 1 (epithelial denuding), 2 (focal ulceration), and 3 (widespread epithelial ulceration)
[5–7, 9–16].
Malondialdehyde
MDA content was measured as described by Ohkawa et al. in 1979 [17]. Bladder tissue was
homogenized in appropriate buffers and used for the following assays. The mixture consisted
of 0.8 ml of sample (1 mg), 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% glacial
acetic acid adjusted to pH3.5, and 1.5 ml of 0.8% aqueous solution of 2-thiobarbituric acid.
The mixture was made up to 4 ml with distilled water and heated at 95C for 60 min using a
glass ball as condenser. After cooling with tap water, 1 ml distilled water and 5 ml n-butanol
and pyridine mixture (15:1) were added, and the solution was shaken vigorously. After
centrifugation at 2,000g for 10 min, the absorbance of the organic layer was measured at
532 nm. Amount of thiobarbituric reacting substances formed is calculated from standard
curve prepared using 1,1′,3,3′ tetramethoxy propane and the values expressed as nanomole
MDA per gram of tissue.
Glutathione
Tissue levels of acid-soluble thiols, mainly GSH, were determined colourimetrically
at 412 nm. Briefly, 0.5 mL of the previously prepared homogenate was added to

100 Ayhanci et al.

Table 1 Groups of Rats (n=7) and their Treatments
Days
1 2 3 4 5 6 7
Group 1 Saline Saline Saline CP (50 mg/kg) Saline Saline X
Group 2 Saline Saline Saline CP (100 mg/kg) Saline Saline X
Group 3 Saline Saline Saline CP (150 mg/kg) Saline Saline X
Group 4 SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) X
Group 5 SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) X
Group 6 SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) + SLM (0.5 mg/kg) SLM (0.5 mg/kg) X
CP (50 mg/kg)
Group 7 SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) + SLM (0.5 mg/kg) SLM (0.5 mg/kg) X
CP (100 mg/kg)
Group 8 SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) SLM (0.5 mg/kg) + SLM (0.5 mg/kg) SLM (0.5 mg/kg) X
CP (150 mg/kg)
Group 9 SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) + SLM (1 mg/kg) SLM (1 mg/kg) X
CP (50 mg/kg)
Group 10 SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) + SLM (1 mg/kg) SLM (1 mg/kg) X
CP (100 mg/kg)
Group 11 SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) SLM (1 mg/kg) + SLM (1 mg/kg) SLM (1 mg/kg) X
CP (150 mg/kg)
Group 12 Saline Saline Saline Saline Saline Saline X

CP cyclophosphamide, SLM seleno-L-methionine, X killed
Protective Effect of SLM on CP-Induced Urinary Bladder Toxicity in Rats 101

0.5 mL of 5% trichloroacetic acid, and after centrifugation at 750g for 5 min, the
supernatant (200μL) was added to a tube containing 1,750μL of 0.1 mol/L potassium
phosphate buffer, pH8, and 50μL 5,5′-dithiobis-(2-nitrobenzoic acid) reagent. Tubes
were mixed, and the yellow color developed was measured against a standard curve of
GSH. The protein thiol (protein-SH) content was expressed as micromole per gram of
tissue [16].
Statistics
The results were expressed as meansSEM and p<0.05 accepted as statistically significant.
Statistical analysis was performed using one-way analysis of variance for MDA and GSH
levels, body, and bladder weights. Histopathological score points were analyzed first by
using the non-parametric Kruskal–Wallis test to discover whether there was any difference
between groups. The Tukey honestly significantly different test was then performed to
analyze two groups consecutively.

Results

All histological parameters and urinary bladder weights are summarized in Table 2. Three
doses of CP (groups 1, 2, and 3) induced acute urinary bladder damage. This was
manifested by a significant increase in urinary bladder weight as a ratio of bodyweight,
reaching a more than fourfold (exclude group 1) increase with marked edema. Control
animals had histologically normal bladders with assigned scores of “0” for all four
conditions, edema, hemorrhage, inflammation, and ulceration. Severe histological changes
and higher grades of hematuria were observed for animals receiving CP (groups 2 and 3),
and severe ulceration and erosion was encountered, as shown in Fig. 1. In addition,
histological examination revealed that CP induced extensive inflammation of the lining
urothelium, petechial hemorrhage in the lamina propria associated with proteinaceous

Table 2 Comparison of Histological Damage and Bladder/Body Weight (blw/bw) Ratio of Rat Bladders
[Median (Min–Max)]
Groups (mg/kg) Edema Hemorrhage Inflammation Ulceration Total damage blw/bw (mg/g)
Control 0 0 0 0 0 0.53 (0.42–0.68)
50 CP 7 0 3 3 13 0.77* (0.67–0.88)
100 CP 17* 5 11 11 42* 2.12* (1.89–2.43)
150 CP 19* 8* 12* 15* 54* 2.39* (1.99–2.77)
0.5 SLM 0 0 0 3 3 0.51 (0.40–0.66)
1 SLM 0 0 0 4 4 0.48 (0.43–0.71)
50 CP +0.5 SLM 0 0 0 0 0 0.56* (0.41–0.70)
100 CP+0.5 SLM 4 1 3 3 11 0.85** (0.68–1.12)
150 CP+0.5 SLM 3 3 4 6 16 0.83** (0.65–1.11)
50 CP+1 SLM 2 0 0 8 10 0.62* (0.47–0.84)
100 CP+1 SLM 7 3 7 8 25 0.72** (0.58–0.98)
150 CP+1 SLM 1 3 0** 5 9 0.67** (0.51–0.83)

*p<0.05 compared with control group; **p<0.05 compared with CP group
102 Ayhanci et al.

material infiltrating into the lumen and infiltration of erythrocytes, desquamative epithelium
as well as PMN leukocytes with focal erosions of the urinary bladder. Pretreatment with
SLM (groups 6, 7, 8, 9, 10, and 11) caused a significant decrease in urinary bladder weight
(Table 2) and markedly decreased the vascular congestion and hemorrhage and resulted in a
profound improvement in the histological structure of the urinary bladder (Figs. 2 and 3).
SLM alone caused slight histopathological changes (ulceration) in the normal structure of
the urinary bladder (Table 2).
CP injection resulted also in increased MDA levels (two- to fivefold) and decreased GSH
levels (by 64%), indicating that oxidative stress was present in the bladders. Pretreatment with
SLM ameliorated CP-induced cystitis, as indicated by a significant decrease in lipid peroxides
(MDA) and a marked increase of antioxidant (GSH) levels (Figs. 4 and 5).

Discussion

HC is a major therapy-limiting side effect of CP. The main features of HC are urothelial
damage, transmural edema, hemorrhage, mucosal ulceration, and epithelial necrosis. These
can be demonstrated within 24 h of a single dose [18]. The urotoxicity of CP is thought to
be due to the formation of acrolein that damages the urothelium [19, 20]. In accordance

Fig. 1 Representative light micrographs showing bladder histology. a Bladder taken from the control rats
showing normal histology. b A bladder section of a CP 50-treated rat showing edema in the connective tissue
and condensed nuclei (arrow) in some epithelial cells. c A bladder section taken from a CP 100-treated rat
showing interruptions in the epithelium. There are hemorrhage and edema in the connective tissue. d A
bladder section of a CP 150-treated rat showing epithelial disappearance with edema and hemorrhage in the
connective tissue
Protective Effect of SLM on CP-Induced Urinary Bladder Toxicity in Rats 103

with other studies, the damage caused in this study by CP to the structure of the bladders
increased as the dose rose. Similarly structural damage was obtained when 100 mg/kg CP
was injected to rats [5]. In the present study, treatment with CP produced marked
congestion and edema in rat bladder. Histopathological examinations also showed petechial
hemorrhage and inflammatory reaction in the lamina propria of the urinary bladder and
proteinaceous material in the lumen. PMN leukocytes with occasional lymphocyte
infiltration and focal erosions were also observed in urinary bladders from CP-treated rats.
These results are consistent with those of previous studies reporting that CP caused
excessive protein extravasation, vascular congestion, and edema of the bladder and
extensive leukocyte production of NO [21, 22].
HC is now accepted as a non-microbial inflammation, and the pathogenesis of HC may
be summarized as cytokine production leading to inducable nitric oxide synthase (iNOS)
induction. There is evidence that urinary bladder epithelial cells express reactivity to iNOS
in the cytoplasm, leading to peroxynitrite production [22]. Increased NO production is
probably responsible for the cystitis because S-methylisothiourea (an iNOS-selective
inhibitor) almost abolishes CP-induced bladder damage [11]. This improvement is thought
to result from a decrease in NO production. In addition, CP produced a decrease in the
endogenous antioxidant compound GSH and an elevation of lipid peroxidation in urinary
bladder [16, 18–22]. Our findings are in agreement with those of these studies, in which
there was a significant depletion of GSH pool and a significant increase in lipid peroxides

Fig. 2 Representative light micrographs showing bladder histology. Sections showing normal histology
from; a SLM 0.5-treated, b a SLM 0.5-+CP 50-treated rat. c Bladder taken from a SLM 0.5-+CP 100-treated
rat showing a thin, discontinuous epithelium and edema in the connective tissue. d A bladder section from a
SLM 0.5-+CP 150-treated rat showing a partially thin, discontinuous epithelium but no hemorrhage and
edema in the connective tissue
104 Ayhanci et al.

Fig. 3 Representative lightmicrographs showing bladder histology. A section froma SLM 1-treated rat showing
a mild ulceration. b SLM 1-+CP 50-treated rat showing a mild discontinuous epithelium and a mild edema in
the connective tissue. c SLM 1-+CP 100-treated rat showing mild changes in the epithelium and edema in the
connective tissue. d SLM 1-+CP 150-treated rat showing a mild discontinuous epithelium and edema in the
connective tissue

0
200
400
600
800
1000
1200
1400
50 100 150

Doses (mg/kg)
MDA (nmol/g tissue)

CP
CP+0.5 mg/kg SLM
CP+1 mg/kg SLM
Control

Fig. 4 MDA levels of the bladder.
CP administration severely
increased MDA in groups 1, 2,
and 3 (51%, 77%, and 83%,
respectively, p<0.001). CP and
SLM decreased MDA level in
groups 6, 7, and 9 nearly to the
level of the control group's
(p>0.05). In group 8, MDA level
was increased by60% compared
to the control (p<0.001), whereas
decreased by 58% compared to
group 3 (p<0.001). In group 10,
MDA level was increased by
36% compared to the control

(p<0.01) and found to be decreased
by64% compared to
group 2 (p<0.001). In group 11,
MDA level was increased by66%
compared to the control
(p<0.001) and found to be decreased
by 51% compared to the
third group (p<0.001)
Protective Effect of SLM on CP-Induced Urinary Bladder Toxicity in Rats 105

(MDA) in urinary bladder tissues after CP injection. Similarly, it was reported that CP
produced a marked decrease in urinary bladder GSH content [23]. GSH is the major cellular
sulphydryl compound that serves as both a nucleophile and an effective reductant by
interacting with numerous electrophilic and oxidizing compounds. It can act as a nonenzymic
antioxidant by direct interaction of –SH group with ROS, or it can be involved in
the enzymatic detoxification reaction for ROS, as a cofactor or coenzyme. The depletion of
GSH content may be attributed to the direct conjugation of CP and its metabolites with free
or protein bound –SH groups [24], thereby interfering with the antioxidant functions.
Decreased GSH content can explain the additional decreased concentration of vitamin C,
which enters the cell mainly in the oxidized form where it is reduced by GSH [25]. Besides
other protective drugs, recently, amifostine and glutathione were reported to prevent
acrolein-induced HC in mouse bladder; however, the efficacy of these agents in humans has
yet to be determined [26].
Previous studies have shown that CP toxicity is associated with oxidative cell damage [27];
thus, therapeutic strategies aim to limit free radical-mediated urinary bladder injury [28].
Se is recognized to have a capacity for conferring tolerance to the toxic manifestations of
various heavy metal exposures, including cadmium, mercury, lead, and arsenic [29, 30] in
addition to role as an anticancer agent [31]. Moreover, Se has protective effects against
mercury toxicity in rat kidney and Se also used to diminish the toxic effects of the cadmium
on the antioxidant enzyme system, which in turn affects the membranes structures such as
mitochondria and endoplasmic reticulum. On the other hand, Se deficiency reducing GPx
activity has been reported in diet of glomerular disease and in diet of tubular epithelium in
normal rats [32]. Concurrent or prior injections of Se were found to protect against many of
the acute effects of cadmium, e.g., testicular necrosis, placental hemorrhage, teratogenecity,
and damage to the lactating mammary glands [14–16, 18–31, 33].

0
0,5
1
1,5
2
2,5
3
3,5
50 100 150

Doses (mg/kg)
GSH (μmol/g tissue)

CP
CP+0.5 mg/kg SLM
CP+1 mg/kg SLM
Control

Fig. 5 GSH levels of the bladder.
GSH levels were found to be
decreased dose dependently only
in the first three groups that
received CP alone compared to
the control. This reduction (25%)
was not statistically significant in
group 1 (p>0.05); in groups 2
(46%) and 3 (64%), the reduction
was statistically significant
(p<0.001). When administrated
with CP, SLM raised the antioxidant
GSH levels in group 6 by
53%, group 7 by 45%, group 9
by 51%, and in group 10 by 36%,
and these were found to be
statistically highly significant
(p<0.001); in group 8, it raised
the GSH level significantly (26%,

p<0.01). The increase in the
antioxidant GSH levels in group
11 (% 23) was not found to be
statistically significant (p>0.05)
106 Ayhanci et al.

Olas and Wachowicz [34] has reported that the administration of sodium selenite appears
to reduce cisplatin toxicity without inhibiting the antitumor activity of cisplatin. Weijl et al.
[35] have found that supplementation with a higher dose of Se in combination with other
antioxidants (vitamins C and E) could correlate with cisplatin-induced autotoxicity and
nefrotoxicity. While the deprivation of Se can reduce the protection against oxidative stress
and impair immunocompetence, certain cancer cells appear to have acquired a selective
survival advantage that is apparent under conditions of Se deficiency and oxidative stress
[36].
The evidences obtained from our study indicate that pretreatment with SLM ameliorates
CP-induced cystitis because it leads to a reduction in the macroscopic changes induced by
CP, such as hemorrhage and edema, as well as a profound improvement in the histological
structure and shape. Both SLM doses that we used improved bladder morphology in a dose
dependent manner. SLM also offers marked protection against extensive CP-induced
inflammation. Furthermore, pretreatment with SLM ameliorated CP-induced cystitis, as
indicated by a significant decrease in MDA and a marked increased of antioxidant GSH
levels in bladder tissue. All histopathological examinations and biochemical measurements
suggest that Se may have a role in two stages, namely, prevention and interception, of
defense against ROS and RNS production.
In essence, the results of the present study suggest that CP-induced cystitis is related to
oxidative stress and ROS formation owing to depletion of GSH and/or inflammation. Se
can also protect the structure and function of the urinary bladder against CP-induced
oxidative stress. Our findings suggest that, at convenient concentration, selenium could be a
potentially effective drug in the treatment of CP-induced cystitis. We believe that additional
experimentation should be performed to explore the underlying mechanism of Se protection
against CP toxicity.

References

1. Selvakumar E, Prahalathan C, Sudharsan PT, Varalakshmi P (2006) Chemoprotective effect of lipoic acid
against cyclophosphamide-induced changes in the rat sperm. Toxicology 217:71–78
2. Alfieri B, Gardner CJ (1997) The NK1 antagonist, GR203040, inhibits cyclophosphamide-induced
damage in the rat and ferret bladder. Br J Pharmacol 29:245–250
3. Frasier LU, Sarathchandra K, Kehrer JP (1991) Cyclophosphamide toxicity: characterising and avoiding
the problem. Drugs 42:781–795
4. West NJ (1997) Prevention and treatment of hemorrhagic cystitis. Pharmacother 17(4):696–706
5. zcan A, Korkmaz A, Oter S, Coskun O (2005) Contribution of flavonoid antioxidants to the preventive
effect of MESNA in cyclophosphamide-induced cystitis in rats. Arch Toxicol 79:461–465
6. Premila A, Indirani K, Preethi K (2008) Alterations in antioxidant enzyme activities and increased
oxidative stress in cyclophosphamide-induced hemorrhagic cystitis in the rat. J Cancer Therapy 6:563–

570
7. Virag L, Szabo E, Gergely P, Szabo C (2003) Peroxynitrite-induced cytotoxicity: mechanism and
opportunities for intervention. Toxicol Let 141:113–124
8. Das UB, Mallick M, Debnath JM, Ghosh D (2002) Protective effect of ascorbic acid on cyclophosphamideinduced
testicular gametogenic and androgenic disorders in male rats. Asian J Androl 4:201–207
9. Manda K, Bhatia AI (2003) Prophylactic action of melatonin against Cyclophosphamide-induced
oxidative stress in mice. Cell Biol Toxicol 19:367–372
10. Mates JM, Perez-Gomez P, Nunez de Castro I (1999) Antioxidant enzymes and human disease. Clin
Biochem 32:595–603
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ischemia/reperfusion injury. Eur J Pharmacol 426:1–10

Psyborg
10-10-2010, 08:36 AM
I was told of this risk when I started on CTX. The doctor primarily wants me to drink a lot of liquids and empty the bladder frequently. Apparently the biggest risk is if it sits in your bladder for extended time. I got home and looked it up and found out the drug is actually derived from the same formula as mustard gas of all things. Weird stuff we have to do to get well.

me2
10-10-2010, 08:48 AM
Hi Psy,
I knew that about the mustard gas too. That is crazy.
My infusion nurse was there when my doc looked over the short abstracts on these studies. After he pooh poohed and left she came over to me and said "If I were you , I would be doing exactly the same thing" referring to my proactive effort to help myself and look for solutions beyond what I was being told. I did what the doctors told me thirty years ago and ended up with blood in my urine and a damaged bladder. (under the care of Dr Pooh Pooh ironically enough)
Thats not acceptable to me. In my opinion don't depend on a doctor to tell the whole story of anything.
Read the story of the man who discovered the cure for ulcers. (Great book. Reads like a fast novel) He spent years fighting conventional medical thought on what causes ulcers and what the cure is. Turns out it is a baceria that can be treated with anti biotics. You would think the medical establishment could pick up that idea quite easily- they didn't. Todays fringe is tomorrows common practice. Not all the time of course but enough that I think I have to be open minded and proactive.

me2
10-10-2010, 09:04 AM
As a side note , I know it sounds crazy, but I had been doing Cytoxan for several months and part of my ritual at home after infusion was to gorge on frozen blueberries, raspberries and mangos mixed with a little yogurt. I also had a craving for walnuts and dried apricots. This was before I looked up these studies. Was my body trying to steer me to the antioxidants mentioned above ? Dunno but after reading this material I didn't change my diet much. I have added the supplements mentioned. Melatonin, alpha tocopheral, selenium (I go real easy on that one), and lycopene from cooked tomatoes.

me2
10-10-2010, 10:24 AM
--I emailed one of the authors of this article from 2007 and asked if there are any articles he could refer me to in addition to this one. I'll post here if I get a reply.--

Cell Biol Toxicol 2007; 23: 303–312.
DOI: 10.1007/s10565-006-0078-0 C Springer 2007

Pathophysiological aspects of cyclophosphamide and ifosfamide induced
hemorrhagic cystitis; implication of reactive oxygen and nitrogen species as
well as PARP activation

A. Korkmaz, T. Topal and S. Oter

Gulhane Military Medical Academy, Department of Physiology, Ankara, Turkey

Received 21 December 2005; accepted 11 December 2006; Published online: 15 January 2007

Keywords: acrolein, cyclophoshamide, ifosfamide, hemorrhagic cystitis, peroxynitrite, PARP

Abstract

Cyclophosphamide (CP) and ifosfamide (IF) are widely used antineoplastic agents, but their side-effect
of hemorrhagic cystitis (HC) is still encountered as an important problem. Acrolein is the main molecule
responsible of this side-effect and mesna (2-mercaptoethane sulfonate) is the commonly used preventive
agent. Mesna binds acrolein and prevent its direct contact with uroepithelium. 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. There is no doubt that HC
is an inflammatory process, including when caused by CP. Thus, many cytokines such as tumor necrosis
factor (TNF) and the interleukin (IL) family and transcription factors such as nuclear factor-κB (NF-κB)
and activator protein-1 (AP-1) also play a role in its pathogenesis. When these molecular factors are
taken into account, pathogenesis of CP-induced bladder toxicity can be summarized in three steps: (1)
acrolein rapidly enters into the uroepithelial cells; (2) it then activates intracellular reactive oxygen species
and nitric oxide production (directly or through NF-κB and AP-1) leading to peroxynitrite production;
(3) finally, the increased peroxynitrite level damages lipids (lipid peroxidation), proteins (protein oxidation)
and DNA (strand breaks) leading to activation of PARP, a DNA repair enzyme. DNA damage
causes PARP overactivation, resulting in the depletion of oxidized nicotinamide–adenine dinucleotide
and adenosine triphosphate, and consequently in necrotic cell death. For more effective prevention against
HC, all pathophysiological mechanisms must be taken into consideration.

Abbreviations: AP-1, activator protein-1; CAT, catalase; CP, cyclophosphamide; eNOS, endothelial nitric
oxide synthase; EPCG, epigallocatechin 3-gallate; GSH, glutathione; GSH-Px, glutathione peroxidase;
HC, hemorrhagic cystitis; IF, ifosfamide; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase;MDA,
malondialdehyde; mesna, 2-mercaptoethane sulfonate; NAD+, nicotinamide–adenine dinucleotide; NF-

κB, nuclear factor-κB; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase;
O−

2 , superoxide anion (radical); ONOO−, peroxynitrite; ONOOH, peroxynitrous acid; PAF, plateletactivating
factor; PARP, poly(adenosine diphosphate-ribose) polymerase; ROS, reactive oxygen species;
SOD, superoxide dismutase; TNF-α, tumor necrosing factor alpha
304

Introduction

Cyclophosphamide (CP), an oxazaphosphorine
alkylating agent introduced in 1958, is widely
used in the treatment of solid tumors and B-cell
malignant disease, such as lymphoma, myeloma,
chronic lymphocytic leukemia and Waldenstrom
macroglobulinemia. Furthermore, CP and ifosfamide
(IF), a synthetic analogue of CP, have
had an increasing role in the treatment of nonneoplastic
diseases, such as thrombocytopenic
purpura, rheumatoid arthritis, systemic lupus erythematosis,
nephritic syndrome, and Wegener
granulomatosis, and as a conditioner before
bone marrow transplantation (Levine and Richie,
1989).
The first side-effects of CP were reported by
Coggins and co-workers as early as 1960. The urological
side-effects, a major limiting factor in its
use, vary from transient irritative voiding symptoms,
including urinary frequency, dysuria, urgency,
suprapubic discomfort and strangury with
microhematuria, to life-threatening hemorrhagic
cystitis. Bladder fibrosis, necrosis, contracture,
and vesicoureteral reflux also have been reported
(Coggins et al., 1960).
Later, other oxazaphosphorine alkylating
agents were found to have similar effects. In early
series the incidence of HC during and after treatment
was reported to be as high as 68%. Mortality
from uncontrolled hemorrhage has been reported
to be 4% and morbidity from severe hemorrhage
is extremely high (Gray et al., 1986). Hemorrhage
usually occurs during or immediately after treatment,
whether with short-term high or long-term
low dosages. When mesna (2-mercaptoethane
sulfonate) is given as prophylaxis, the incidence
is decreased to approximately 5%.
The urotoxicity of these cytostatics is not
based on a direct alkylating activity on the
urinary system but rather on the formation of
4-hydroxy metabolites, in particular, renal excretion
of acrolein, which is formed from hepatic microsomal
enzymatic hydroxylation (Brock et al.,
1981).

Toxicity of acrolein

Humans are exposed to acrolein in industrial, environmental,
and therapeutic situations. Industrially,
acrolein is mostly used as a herbicide. Environmentally,
acrolein occurs naturally in foods
and is formed during the combustion of organic
materials. Thus, acrolein is found in all types of
smoke including cigarette smoke. In vivo, acrolein
is a metabolic product of CP and IF (Kehrer and
Biswal, 2000).
In order to understand the pathophysiological
mechanism of CP-induced HC, the question “How
is acrolein toxic?” needs to be answered. Acrolein
is the most reactive of the α,β-unsaturated aldehydes,
and will rapidly bind to and deplete cellular
nucleophiles such as glutathione. It can also react
with some residues of proteins and with nucleophilic
sites in DNA. However, this reactivity is
the basis for the cytotoxicity evident in all cells
exposed to high concentrations of acrolein, and
monitoring of urinary acrolein concentration indicates
that in humans who are admitted to hospital
for treatment of solid tumors and hematological
diseases it cannot reach such high concentrations
(Takamoto et al., 2004). Thus, in case of CPinduced
bladder damage, the toxicity of acrolein
does not come from direct toxic effects.
At lower acrolein doses, other biological effects
become evident. One of the most important features
of acrolein is the ability to rapidly react at
many cellular sites, for example, in depletion of
cellular thiols or in gene activation, either directly
or subsequent to effects of transcription factors
such as nuclear factor-κB (NF-κB) (Horton et al.,
1999) and activator protein-1 (AP-1) (Biswal
et al., 2002). Furthermore, acrolein has also been
identified as a product and also an initiator of lipid
peroxidation (Adams and Klaidman, 1993). Alternately,
or in addition, a more direct action of
acrolein on various factors is possible. The direct
alkylation of DNA by acrolein, while possible,
seems unlikely at low doses, which would be expected
to react with the abundant levels of cellular
glutathione (GSH) or other nucleophiles prior
305
to reaching the nucleus. A lack of direct DNA
damage is supported by some experimental work
(Horton et al., 1997).
With CP or IF treatment, it seems possible that
a high enough concentration of acrolein is present
only in urine. Thus the toxicity of acrolein has
generally been encountered in the urinary system.
Further, mesna—the most trusted preventive
agent—binds the acrolein in the bladder or the
whole urinary system and does not allow it to
get into the uroepithelium. If acrolein does enter
the uroepithelium, it induces compounds such as
reactive oxygen species. Is there any mechanism
in the uroepithelium to resist acrolein? Given the
uroepithelial action of acrolein, we investigated
oxidative stress and the antioxidant status of the
cells.

Free oxygen radicals and antioxidant defense
mechanism

Reactive oxygen species (ROS) are constantly
generated under physiological conditions as a consequence
of aerobic metabolism. ROS include
free radicals such as the superoxide (O−•

2 ) anion,
hydroxyl radicals (OH•) and the non-radical
molecule hydrogen peroxide (H2O2). These are
particularly transient species due to their high
chemical reactivity and can react with DNA, proteins,
carbohydrates, and lipids in a destructive
manner. The cell is endowed with an extensive
antioxidant defense system to combat ROS, either
directly by interception or indirectly through
reversal of oxidative damage. When ROS overcome
the defense systems of the cell and redox
homeostasis is altered, the result is oxidative stress
(Sies, 1997) (Figure 1).

Antioxidant defense mechanisms against ROS

The endogen antioxidant defense system functions
to prevent oxidative damage directly by intercepting
ROS before they can damage intracellular
targets. It consists of superoxide dismutase
(SOD), glutathione peroxidase (GSH-Px) and
catalase (CAT) (Sies, 1997). SOD destroys the
free radical superoxide (O−•

2 ) by converting it to
H2O2. The primary defense mechanisms against
H2O2 are CAT and GSH-Px. CAT is one of the
most efficient enzymes known and cannot be saturated
byH2O2 at any concentration. GSH-Px acts
through the glutathione redox cycle (Sies, 1999)
(Figure 1).

Nitric oxide and the nitric-oxide synthase
family

Nitric oxide (NO) is produced by a family of
enzymes called nitric-oxide synthases (NOS).
Constitutive expression of two NOS isoforms is
responsible for a low basal level of NO synthesis
in neural cells (nNOS) and in endothelial
cells (eNOS). Induction of the inducible isoform
(iNOS) by cytokines (TNF-α, interleukins) bacterial
products (endotoxin) and chemical agents
has been observed in virtually all cell types
tested including macrophages, fibroblasts, chondrocytes,
osteoclasts, and epithelial cells and results
in the production of large amounts of NO
(Moncada et al., 1991). Controversy arises from
observations reporting both cytotoxic and cytoprotective
effects of NO. In cases where NO
was found to be cytotoxic, it was questioned
whether NO exerted these effects directly or indirectly
through the formation of more reactive
species such as peroxynitrite (ONOO−) (Szabo,
1996).

The activated “Devil Triangle” in the target cell

As both excess NO and excess O−•

2 decreases the
bioavailability of ONOO−, equimolar concentrations
of the radicals are ideal for ONOO− formation.
The ONOO− anion is in pH-dependent
protonation equilibrium with peroxynitrous acid
(ONOOH). Homolysis of ONOOH gives rise
to formation of the highly reactive OH• mediating
molecular and tissue damage associated
306

Figure 1. The activated “Devil Triangle” (NO–O−•

2 –ONOO−) leading to permanent cellular damage. Under normal circumstances, oxidants
and antioxidant defense mechanisms are in redox homeostasis. Additional oxidants may alter the equilibrium. Note that SOD is first in enzymatic
scavenging; if SOD does not work, neither GPx nor CAT will scavenge. Once acrolein has entered the uroepithelial cells, both ROS production
and iNOS activation increase. Excess NO can outcompete SOD for O−•

2 , resulting peroxynitrite formation. Once produced, peroxynitrite can
cause lipid peroxidation, protein oxidation, and DNA damage. DNA damage then causes PARP activation, leading to cellular energy crisis.

with ONOO− production (Radi et al., 2001).
ONOO− is formed when NO and O−•

2 react in a
near diffusion-limited reaction. The most powerful
cellular antioxidant system protecting against
the harmful effects of O−•

2 is represented by
SOD. However, it has been shown that NO efficiently
competes with SOD for O−•

2 (Figure 1).
Beckman et al. have therefore proposed that
under conditions of increased NO production
NO can outcompete SOD for O−•

2 , resulting
307
in ONOO− formation (Beckman and Koppenol,
1996).

How is peroxynitrite harmful?

ONOO− is not a radical but is a stronger oxidant
than its precursor radicals. It can directly
react with target biomolecules via one or twoelectron
oxidations. Higher concentrations and
the uncontrolled generation of ONOO− may result
in unwanted oxidation and consecutive destruction
of host cellular constituents. ONOO−

may oxidize and covalently modify all major
types of biomolecules. One of the most important
mechanisms of cellular injury is a ONOO−-
dependent increase in DNA strand breakage,
which triggers the activation of poly(adenosine
diphosphate-ribose) polymerase (PARP), a DNA
repair enzyme. DNA damage causes PARP overactivation,
resulting in the depletion of oxidized
nicotinamide–adenine dinucleotide (NAD+) and
adenosine triphosphate (ATP), and consequently
in necrotic cell death (Virag and Szabo,
2002).
DNA single-strand breakage is an obligatory
trigger for the activation of PARP. ONOO− and
OH• are the key pathophysiologically relevant
triggers of DNA single-strand breakage (Schraufstatter
et al., 1988). Moreover, nitroxyl anion, a
reactive molecule derived from nitric oxide, is
a potent activator of DNA single-strand breakage
and PARP activation in vitro (Schraufstatter
et al., 1988; Virag and Szabo, 2002). Subsequent
studies clarified that the actual trigger of
DNA single-strand breakage is ONOO−, rather
than NO (Szabo et al., 1996). The identification
of ONOO− as an important mediator of the cellular
damage in various forms of inflammation
stimulated significant interest in the role of the
PARP-related suicide pathway in various pathophysiological
conditions. Endogenous production
of ONOO− and other oxidants has been shown to
lead to DNA single-strand breakage and PARP
activation (Szabo, 2003).

NF-κB and cytokines involved in bladder
toxicity

NF-κB is a member of the Rel protein family and
resides in the cytoplasm. This factor is normally
bound to a member of the family of inhibitory
proteins known as inhibitor-κB (I-κB) (May and
Gosh, 1997). The exposure of cells to NF-κB activators,
including ROS and cytokines (e.g. TNF-

α, IL-1), degrades I-κB. Activated NF-κB then is
translocated to the nucleus where it is an important
mediator of transcription events associated with a
variety of stress conditions. The pro-inflammatory
cytokine TNF-α plays an important role in diverse
cellular events such as septic shock, obesity,
diabetes, cardiovascular events, cancer, induction
of other cytokines, cell proliferation, differentiation,
necrosis, and apoptosis (Liu, 2005).
In response to TNF, transcription factors such as
NF-κB are activated in most types of cells and,
in some cases, apoptosis or necrosis may also be
induced.
Cells are often under genotoxic stress induced
by both endogenous (e.g., ROS) and exogenous
sources (e.g., ultraviolet radiation, ionizing radiation,
DNA damaging chemicals, and acrolein).
The cellular response to genotoxic stress includes
damage sensing, activation of different signaling
pathways, and biological consequences such as
cell cycle arrest and apoptosis. Transcription factors
such as NF-κB have been suggested to play
critical roles in mediating cellular responses to
genotoxic responses (Canman and Kastan, 1996).
These transcription factors elicit various biological
responses by inducing expression of their
target genes. Because activation of NF-κB can
have anti-apoptotic or pro-apoptotic effects, the
engagement of these two pathways may be key
cellular responses that modulate the outcome of
cells exposed to radiation and genotoxic chemicals.
In most types of cells, inactive NF-κB is sequestered
in the cytoplasm through its interaction
with the inhibitory proteins. In response to various
stimuli, such as TNF-α and IL-1, inhibitory
308
proteins of NF-κB release NF-κB and allow its
translocation into the nucleus and the subsequent
activation of its target genes. In case of CPinduced
HC, acrolein itself, cytokines, and ROS
may lead to NF-κB activation and intensification
of the harmful effects of acrolein.

Possible mechanisms of CP-induced bladder
damage

The first step in the pathogenesis of CP-induced
bladder damage is the entry of acrolein into the
uroepithelium. Then the cascade is activated as
suggested below and summarized in Table 1.
First, acrolein rapidly enters into the uroepithelial
cells. Second, it activates intracellular ROS
and NO production (directly or through NF-κB
and AP-1), leading to ONOO− production. Third,
the increased ONOO− level damages lipids (lipid
peroxidation), proteins (protein oxidation), and
DNA (strand breaks), leading to PARP activation.
Figure 2 demonstrates the proposed mechanism
of acrolein-induced HC in detail.

Table 1. The proposed mechanism of acrolein-induced
hemorrhagic cystitis
1. Acrolein enters rapidly into the uroepithelium because of its
chemical nature.
a. Acrolein causes increased ROS production in the bladder
epithelium.
b. Acrolein causes both directly and/or indirectly iNOS
induction leading to NO overproduction.
c. Acrolein induces some intracellular transcription factors such
as NF-κB and AP-1.
d. Activated NF-κB and AP-1 cause cytokine (TNF-α, IL-1β)
gene expression, iNOS induction, and again ROS production.
Thus, the production of harmful molecules (cytokines, ROS,
NO) increases dramatically.
e. Cytokines leave the uroepithelium and spread to other
uroepithelial cells, detrussor smooth muscle, and
bloodstream.
2. ROS and NO form peroxynitrite in both uroepithelium and
detrussor smooth muscle.
3. Peroxynitrite attacks cellular macromolecules (lipids, proteins,
and DNA) and causes damage.
4. Cellular and tissue integrity are broken and damage appears as
edema, hemorrhage, and ulceration.

Increased ROS production in the bladder
epithelium and smooth muscle

Several studies have investigated whether scavenging
of ROS with antioxidants may ameliorate
HC symptoms. Ternatin, a flavonoid, is popular
in Brazilian folk medicine and is known to exhibit
antioxidant properties. Vieira et al. showed
that in CP- or IF-induced HC, substitution of the
last two doses of mesna by ternatin was as effective
in preventing HC as the classical protocol
using three doses of mesna (Vieira et al.,
2004). Other flavonoids such as quercetin and epigallocatechin
3-gallate (EGCG), also have protective
effects against CP-induced bladder damage
(Ozcan et al., 2005). Several antioxidants
such as α-tocopherol (Yildirim et al., 2004), β-
carotene (Sadir et al., 2006) and melatonin (Sener
et al., 2004; Topal et al., 2005) have similar effects
on cystitis symptoms. It was also shown that
the antioxidants glutathione and amifostine prevented
IF- and acrolein-inducedHC(Batista et al.,
2007).

iNOS induction leading to NO overproduction

Souza-Filho et al. first reported that NO is involved
in the inflammatory events leading to HC
(Souza-Filho et al., 1997). The authors found
that NOS inhibitors dose-dependently inhibited
the CP-induced increase in plasma protein extravasation
and bladder wet weight. NOS inhibition
significantly reduced the mucosal damage,
hemorrhage, edema, and leukocyte infiltration in
the bladders of CP-treated rats. CP markedly
increased iNOS activity in the bladder with a
time course similar to that of the histopathological
alterations observed. Several experimental
studies performed in our laboratory have also
shown that NO produced by iNOS was involved
in CP-induced HC (Korkmaz et al., 2003; Oter
et al., 2004). Furthermore, platelet-activating factor
(PAF) was found to be one of the inflammatory
mediators contributing to the activation of
309

Figure 2. The overall mechanism regarding acrolein-inducedHCpathogenesis. (I) Acrolein enters the uroepithelium and causesROSproduction,
iNOS induction, and activation of transcription factors (e.g. NF-κB and AP-1). Activated NF-κB and AP-1 cause cytokine (TNF-α, IL-1β) gene
expression, iNOS induction and again ROS production. (II–III) Cytokine produced spreads out into other uroepithelial cells, the bloodstream,
and detrusor smooth muscle. ROS and NO form peroxynitrite in both uroepithelium and detrusor, leading to lipid peroxidation, protein oxidation,
and DNA damage. DNA damage causes PARP activation and energy crisis and eventually cellular necrosis. (IV) During necrotic cell death, the
cellular content is released into the tissue, exposing neighboring cells to potentially harmful attack by intracellular proteases and other released
factors.

the L-arginine–NO pathway (Souza-Filho et al.,
1997). Besides PAF, other inflammatory mediators
such as TNF-α and IL-1 were shown to
mediate the production of NO (through iNOS
induction) involved in the pathogenesis of IF- and
CP-inducedHC(Gomes et al., 1995; Ribeiro et al.,
2002). The induction of iNOS in the urothelium
appeared to depend on production of the cytokines
IL-1β and TNF-α since antiserum against these
cytokines reduced the inflammatory events as well
as the expression of iNOS in the urothelium. This
finding is supported by the fact that pentoxifylline
(IL-1β inhibitor) and thalidomide (TNF-α inhibitor)
reduced inflammatory events induced in
the bladder by IF administration (Gomes et al.,
1995). In cases where NO was found cytotoxic
(e.g. CP-induced HC), it was questioned whether
NO exerted its cytotoxic effects directly or indirectly
through the formation of more reactive
species such as ONOO−.
310

Table 2. Drugs used experimentally in acrolein-induced cystitis
Drug Action Main outcome References
Ternatin Vieira et al. (2004)
Glutathione Antioxidant Histological improvement Sener et al. (2004); Batista et al. (2007)
Amifostine Batista et al. (2007); Kanat et al. (2006)

α-Tocopherol and β-carotene Antioxidant Decreased oxidative stress, histological
improvement
Sadir et al. (2006); Yildirim et al. (2005)
Catechin and quercetin Ozcan et al. (2005)
Melatonin Antioxidant, iNOS inhibitor, peroxynitrite
scavenger
Decreased iNOS activation, decreased GSH
depletion, histological improvement
Sener et al. (2004); Topal et al. (2005);
Sadir et al. (2006)
Thalidomide and pentoxyfilline TNF-α and IL-1β inhibitors iNOS inhibition, histological improvement Gomes et al. (1995); Ribeiro et al. (2002)

L-NAME Non-selective NOS inhibitor NOS inhibition, histological improvement Souza-Filho et al. (1997); Korkmaz et al.
(2003)

S-Methylthiourea Korkmaz et al. (2003); Oter et al. (2004)
Aminoguanidine Selective iNOS inhibitor iNOS inhibition, histological improvement Korkmaz et al. (2005)
1400W Korkmaz et al., unpublished
Ebselen Peroxynitrite scavenger Histological improvement Korkmaz et al. (2005)
3-Aminobenzamide PARP inhibitor Histological improvement Korkmaz et al., unpublished

Peroxynitrite formation

A preliminary study in our laboratory showed that
ONOO− may contribute to the pathogenesis of
bladder damage caused by CP (Korkmaz et al.,
2005). In this work, acrolein was not blocked with
mesna nor was iNOS inhibited with a NOS inhibitor;
only ebselen was used to scavenge the
ONOO− produced. The results were promising
and bladder damage was clearly decreased. The
results of this study suggest that scavenging of
ONOO− and inhibition of iNOS have similar protective
effects. Thus, ONOO− may also be involved
in bladder damage caused by CP.

Macromolecular (lipids, proteins, and DNA)
damage leading to cellular necrosis

Increased malondialdehyde (MDA) levels, an indicator
of lipid peroxidation, have been observed
in several studies (Korkmaz et al., 2005; Sener
et al., 2004; Topal et al., 2005). This increase indicates
that lipid peroxidation is present in damaged
bladder tissue. Both ROS and ONOO− may cause
lipid peroxidation and scavenging them could lead
to decreased MDA levels in bladder tissue. Melatonin
is known as an antioxidant but it also has
iNOS-inhibitory and ONOO−-scavenging properties.
Recently, Topal et al. has shown that melatonin
may ameliorate bladder damage and decrease
MDA levels, possibly through scavenging
of ROS and ONOO− and inhibition of iNOS activity
in bladder tissue (Topal et al., 2005). Sener
et al. also showed that melatonin was capable of
reducing IF-induced nephrotic and bladder toxicity
(Sener et al., 2004). In this work, melatonin
acted as antioxidant and anti-inflammatory
and enhanced cell ATPase activity. Furthermore,
PARP activation caused by DNA damage also involves
CP-inducedHCleading to cellular necrosis
(unpublished data).
Table 2 summarizes the outcome of experimental
studies using several drugs against acrolein
cystitis.
311

Conclusions

Acrolein is the main compound responsible for
CP- and IF-induced cystitis and mesna is the agent
commonly used to protect against this side-effect.
Nevertheless, our current knowledge led us to
seek more information about the pathophysiological
mechanism of HC in detail: many cytokines,
free radicals and non-radical reactive molecules,
as well as PARP activation, are now known to take
part in the pathogenesis of CP- and IF-induced
HC. In addition, there is no doubt that HC is an
inflammatory process, including when caused by
CP or IF. Thus, many cytokines play a role in its
pathogenesis, such as the TNF and IL families.
Cytokines may trigger activation of transcription
factors such as NF-κBand AP-1, leading to further
events associated with a variety of stress conditions.
Thus, we suggest that for more effective protection
against CP- or IF-cystitis, all pathophysiological
mechanisms must be taken into consideration.
Possible alternative preventive methods may
be discussed in a separate article.

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Theresa
10-10-2010, 12:27 PM
Me2,
Thanks for posting this information. I'm quite concerned about the effects of cytoxan on Justin -my 15 year old son. He may be getting off it at the end of october and moving to celcept. From my initial reading on it, it can cause lymphoma. All these drugs that weggies need have so many scarey possibilities. I'll read your postings in detail. Thanks!

me2
10-10-2010, 12:44 PM
Hi Theresa,
I look forward to input from others here more knowlegable about what we can use from these studies. A lot of it seems easy to apply and doesn't replace conventional treatment. Your son is lucky to have you looking out for him. I was very young when I was sick 30 years ago and the doctors were not in the practice of explaining things. I had no family or friends to take an interest and so I was really on my own. No internet. Young people may act like they understand things but in my own case I was clueless. I wish had someone who cared to ask questions.

The good news I would like to share with you is that even under those circumstances the only ill effects I suffered from the Cytoxan was cystitis.
Bad enough yes, but it doesn't affect my quality of life. I was 5 years older than your son and I went on to enjoy twenty years of remission after becoming extremely ill and hospitalized. Treatments have improved and evolved. The future is bright... let me get my sun glasses.

Sangye
10-10-2010, 12:46 PM
That's great info, me2. The company Biotics makes a product called Dismuzyme Plus. It's SOD and catalase. It's a great source of antioxidant protection, and it won't boost the immune system like some other antioxidant formulas will. You can only get it through health care professionals--well-known brand. I use it myself. Don't get the more powerful version, though (Dismuzyme Plus 5000) as it contains other nutrients which can boost immunity.

Sangye
10-10-2010, 12:54 PM
Theresa, all immunosuppressants we take can cause lymphoma, including Cellcept. Suppressing the immune system also suppressed the blood cells that scout out cancer cells and destroy them. There's no way around this--we need the drugs. But you can help lower your son's chances of getting cancer by providing a very healthy diet and limiting his exposure to unnecessary radiation and other toxins.

me2
10-10-2010, 12:57 PM
Hi Sangye,
I use some Biotics products but I hadn't heard of Dismuzyme Plus. I will have to look into it. One of the studies I found and did not post used berberine. Although the results seemed promising I am concerned about some of its other possible effects. One source of berberine is goldenseal. You probably know this is an immune booster. Great if you are taking Cytoxan for cancer but should probably be avoided by us. I also found other things that are 'immune modulators' . I'm too cautious about anything that affects the immune system to want to try them myself. If my 'experiment' fails I would be very dissapointed to say the least and certainly cut off from further funding.

Sangye
10-10-2010, 01:07 PM
Yeah, I don't use any immune modulators, either. It's just too risky. Berberine and goldenseal are definitely off my list. Really sucks. :sad: