--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-
κ
B, 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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