Journal of Experimental and Integrative Medicine 2012; 2(2):95-112

www.jeim.org 95

Chemotherapy-induced hemorrhagic cystitis:

pathogenesis, pharmacological approaches and new insights

Ronaldo A. Ribeiro1,2

, Roberto C.P. Lima-Junior1, Caio Abner V.G. Leite

1,

Jose Mauricio S.C. Mota1, Francisco Y.B. Macedo

1, Marcos V.A. Lima

3, Gerly A.C. Brito

4

1Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara;

2Department of Clinical Oncology, Haroldo Juacaba Hospital, Cancer Institute of Ceara;

3Department of Surgery, Haroldo Juacaba Hospital, Cancer Institute of Ceara;

4Department of Morphology, Faculty of Medicine, Federal University of Ceara; Fortaleza, Ceara, Brazil.

Abstract

Chemotherapy-induced hemorrhagic cystitis (HC) remains a common and life-

threatening clinical complication, mainly due to the increasing usage of alkylating agents during conditioning regimen for hematopoietic cell transplantation.

Currently, mesna and hyperhydration are the two more employed preventive measures.

However, these prophylactic approaches have been proven not to be completely effective, since cystoscopic and histopathologic bladder damage are evidenced. Therefore,

understanding the pathogenesis of HC must be the cornerstone for the development of

novel therapeutic strategies.

The purpose of this review is to examine the current knowledge regarding the

pathogenesis of HC, describing the importance of transcription factors (nuclear factor

kappaB), cytokines (tumor necrosis factor-α, interleukin-1β, -4, -6, and -8), enzymes (inducible nitric oxide synthase and cyclooxygenase-2), among other mediators, for the

bladder injury. We also discuss the currently available animal models and future

perspectives on the management of HC.

Key words:

Cancer; Chemotherapy;

Hemorrhagic cystitis; Pathogenesis

Correspondence:

R.A. Ribeiro / R.C.P. Lima-Junior Departamento de Fisiologia e

Farmacologia,

Faculdade de Medicina, Universidade Federal do Ceara,

Rua Cel Nunes de Melo, 1127,

Rodolfo Teofilo, 60430-270, Fortaleza, Ceara, Brazil.

ribeiror@ufc.br / robertocesar@ufc.br

Received: December 28, 2011

Accepted: January 11, 2012 Published online: March 8, 2012

J Exp Integr Med 2012; 2:95-112

DOI:10.5455/jeim.080312.ir.010

©2012 GESDAV. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License which

permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided that the work is properly cited.

Introduction Cystitis is a common clinical occurrence in

cancer patients. It can be separated into three broad

categories: cystitis consequent to bladder cancer or

adjacent cancers that encroach upon the bladder

from the prostate, uterus, cervix, or rectum;

infectious cystitis that occurs in immune-

compromised cancer patients; and chemotherapy-

or radiotherapy-induced cystitis [1].

Whatever the cause, patients often have

complaints typical of cystitis, which can be grouped

into a cluster of symptoms called “LUTS” (lower

urinary tract symptoms): dysuria, frequency,

urgency, nocturia, suprapubic pain, and

microscopic or gross hematuria [1].

In this context, hemorrhagic cystitis (HC) is

considered to be the most severe form in the

spectrum of the chemotherapy-induced bladder

injuries and is defined as the inflammation of the

urinary bladder leading to irritative LUTS up to

severe hemorrhage [1-3]. In patients with mild HC,

hematuria can cause iron deficiency anemia, but in

severe cases, it may lead to hemodynamic

instability due to hypovolemic shock. In addition to

the somatic symptoms, we hypothesize that HC

may also lead to a burden of suffering, depression,

limitations and a worse quality of life, although

there has been a complete absence of studies in this

area.

Although busulfan, adenovirus, BK virus,

cytomegalovirus, graft-versus-host disease and

radiation are causative factors of HC, it most often

occurs following chemotherapy, mainly after

regimens with oxazaphosphorines (cyclophos-

phamide or ifosfamide) [4]. Cyclophosphamide and

ifosfamide are alkylating agents with therapeutic

efficacies in multiple malignancies, including

testicular cancer, small-cell and non small-cell lung

cancer, soft tissue sarcoma, gynecological cancer,

bladder cancer, non-Hodgkin’s lymphoma, ad-

vanced breast cancer, and high-dose cyclophos-

phamide is often used as part of a conditioning

regimen for hematopoietic cell transplantation [5].

The incidence of HC is variable and appears to be

dependent on the type of treatment given. Before

the use of prophylactic regimens, HC occurred in

Invited

Review

Ribeiro et al: Chemotherapy-induced hemorrhagic cystitis

DOI:10.5455/jeim.080312.ir.010 96

10-40% of the patients exposed to high-dose

cyclophosphamide chemotherapy for solid tumors

[6] and up to 70% of patients following bone

marrow transplantation [7]. The incidence has been

reduced to 6-50% using prophylactic measures,

such as hyperhydration, bladder irrigation and

2-mercapto-ethane-sodium sulphonate (mesna)

[7-9]. Recently, Hadjibabaie et al performed a non-

randomized controlled clinical study and showed

that continuous bladder irrigation with mesna

treatment and hyperhydration can additionally

decrease the incidence of cyclophosphamide-

induced HC (by 32 to 50%) and its relative

complications in a conditioning regimen for allo-

geneic hematopoietic cell transplantation [10].

Despite the prophylactic approaches, HC remains

a common and life-threatening complication of

high-dose chemotherapy during bone marrow

transplantation, leading to prolonged hospitalization

[10, 11] and sometimes death [12]. It seems that the

standard preventive protocol does not completely

protect from the chemotherapy-induced bladder

injury, even when clinical signs and symptoms are

not reported. In a randomized controlled clinical

study of ifosfamide-based chemotherapy recipients,

we demonstrated that even with classical

prophylaxis with three doses of mesna, 66.7% of

patients presented with gross cystoscopic injuries,

and 100% had urothelial damage, such as edema,

exocytosis, and hemorrhage [13], possibly

revealing a novel concept of “subclinical

hemorrhagic cystitis.” It remains unknown if this

clinical occurrence may have any negative

consequences.

Thus, HC remains a clinical problem, and both

therapeutic and preventive measures have been

tested. Various therapeutic interventions are utilized

to produce hemostasis, such as continuous bladder

irrigation with isotonic saline, clot evacuation by

cystoscopy, electrocoagulation of bleeding vessels,

intravesical therapy with chemicals (formalin,

aluminum, phenol, silver nitrate, and prosta-

glandin), and hyperbaric oxygen therapy. In more

severe cases, the embolization or ligation of the

vesical [14] or internal iliac arteries, urinary

diversion, and cystectomy are used, although they

are usually reserved for refractory hemorrhage [15].

Therefore, understanding the pathogenesis of

oxazaphosphorine-induced HC must be the

cornerstone for the development of novel effective

preventive and therapeutic strategies. For that

reason, animal models of HC have been created

recently to aid in the studies of HC pathogenesis.

The most recent hypothesis implicates a number of

transcription factors, cytokines, and inducible

enzymes. This review will describe in detail the

ultimate mediators and pathways involved in the

generation of HC and will also discuss the currently

available animal models and future perspectives on

the management of HC.

Animal models for understanding HC

pathogenesis

Animal models have been developed to focus on

the morphological changes in the bladder and on

the pathogenic processes involved. The choice of an

animal model depends on various factors, such as

the accessibility to the animal, cost, administration

route, and the question under investigation. In

summary, there are two major models of

oxazaphosphorine-induced HC in rats or mice: the

systemic injections of cyclophosphamide or

ifosfamide and the intravesical injections of

acrolein (Fig.1).

Figure 1. Experimental hemorrhagic cystitis.

Routes of administration commonly used for the induction of experimental hemorrhagic cystitis.

(a) intravesical injection of acrolein;

(b) intraperitoneal injection of cyclophosphamide or ifosfamide;

(c) normal bladder;

(d) bladder presenting hemorrhagic cystitis;

(e) histopathologic aspect of

(e) normal

and (f) injured bladder.

Journal of Experimental and Integrative Medicine 2012; 2(2):95-112

www.jeim.org 97

Cyclophosphamide-induced HC

Experimental cyclophosphamide-induced bladder

damage has previously been shown in rats, dogs

[16] and mice [17]. However, the first rat model

was generated by Tolley and Castro in 1975 [18].

The animals received cyclophosphamide by

intraperitoneal or intravenous injections (50, 100,

150, 200 and 250 mg/kg). At different times

thereafter (from 2 h to 16 days), the animals were

killed, and their bladders were removed and

analyzed by histopathology. In this system, HC

occurred in a dose-dependent manner and was

measured by bladder wall edema, polymorpho-

nuclear infiltration and submucosal hemorrhage,

mainly identified 8 h following a single intravenous

injection of 100 mg/kg [18]. Furthermore, Gray et

al defined a system for the score evaluation of

macroscopic edema and bleeding and for histo-

logical changes [19]. Souza-Filho et al assessed

bladder edema by measuring the bladder wet

weight and vascular permeability by Evans blue

extravasation, with greater damage occurring 24 h

after the cyclophosphamide injection [20].

Ifosfamide-induced HC

Based on the evidence that HC is observed more

often in patients receiving ifosfamide than in those

receiving cyclophosphamide [21], our group

developed a model to study the ifosfamide-induced

HC in Swiss mice [22]. HC was induced by the

intraperitoneal injection of 100, 200 and 400 mg/kg

ifosfamide. At various times thereafter (6, 12, 24,

36 and 48 h), the animals were euthanized, and

their bladders were removed, emptied of urine and

evaluated for vesical edema (bladder wet weight),

macroscopic and microscopic Gray’s criteria [19].

Ifosfamide-induced vesical edema was greatest at

12 h after ifosfamide administration. The

microscopic analysis revealed vascular congestion,

edema, hemorrhage, fibrin deposition, neutrophil

infiltration, and epithelial denudation [22]. In a

similar study with rats, our group induced HC with

an intraperitoneal injection of 400 mg/kg

ifosfamide, and the bladders were removed 24 h

later [23].

Acrolein-induced HC

In 1985, Chaviano et al administered acrolein

into rat bladders and observed HC similar to that

found with cyclophosphamide injected

intraperitoneally [24]. Conversely, our group

showed for the first time a mouse model of

acrolein-induced HC using a pharmacological dose-

response curve (25, 75 and 225 μg/bladder) [25].

This work showed that the intravesical

administration of acrolein induced a dose- and

time-dependent (3, 6, 12, 24 h) increase in the

vascular permeability and bladder wet weight as

confirmed by histopathological Gray’s criteria, and

pretreatment with mesna inhibited all the changes

induced by acrolein [25]. The injury was greatest at

12 h. This model has a potential advantage over the

systemic administration of cyclophosphamide or

ifosfamide because it does not require the hepatic

metabolism to acrolein and could be a simple and

useful model for the evaluation of HC pathogenesis

and new uroprotective drugs.

Hemorrhagic cystitis pathogenesis

For a long time, the mechanisms involved in the

pathogenesis of HC were largely unknown and

were a matter of considerable interest for

researchers. Over the last two decades, several

studies have tried to elucidate these pathways. Most

of the published articles concentrated on the

cyclophosphamide-induced injury, and only a few

are related to ifosfamide; however, the underlying

mechanisms seem to be equivalent.

The first description of symptomatic bladder

damage with macro- and microscopic hematuria

after cyclophosphamide administration was

described shortly after its introduction as an

antineoplastic agent in 1958 [26, 27]. In addition,

cyclophosphamide was shown to induce bladder

lesions in rats and dogs [16].

Early studies focused on implicating the

alkylating agent itself. However, Philips et al

demonstrated that the effects were caused by the

metabolic products excreted into the urine [16].

They also found that the urine from the dogs treated

with cyclophosphamide could reproduce cystitis

when transplanted into the bladder of control dogs

[16]. Ten years later, acrolein was characterized as

a component present in the urine after cyclophos-

phamide metabolism [28]. In 1979, subsequent

work by Cox definitively identified acrolein as the

causative agent of HC lesions [29].

Another important hallmark was the first clinical

description of the preventive use of mesna in the

late 1970s [30]. Despite this knowledge, the first

demonstration of the involvement of pro-

inflammatory mediators, such as cytokines and

nitric oxide (NO), in the pathogenesis of

oxazaphosphorine-induced HC was described more

than fifteen years later by our group [20, 31]. Since

then, remarkable progress has been made.

Based on the current information on the

pathophysiology of HC and taking into account the

four-stage model proposed by Sonis for alimentary

Ribeiro et al: Chemotherapy-induced hemorrhagic cystitis

DOI:10.5455/jeim.080312.ir.010 98

tract mucositis [32], we suggest a four-stage model

to describe the phases involved in HC (Fig.2). The

first stage (initiation phase) occurs after acrolein

accumulates in the bladder, leading to urothelial

damage. The following stage (inflammatory phase)

is marked by the up-regulation of transcription

factors, such as nuclear factor kappaB (NF-κB)

[33, 34], and the local release of inflammatory

cytokines by the epithelial and conjunctive resident

cells, such as macrophages [22, 31]. The amplified

response is marked by large amounts of cytokines,

reactive oxygen species and the expression of

inflammatory enzymes, such as the inducible nitric

oxide synthase (iNOS) isoform and

cyclooxygenase-2 (COX-2) [20, 22, 31, 35-37]. The

combination of NO and superoxide radicals can

lead to the production of the over-reactive free

radical peroxynitrite [38]. The third stage

(symptomatic phase) involves the urothelial

denudation and ulcer formation, also leading to

visceral pain and bladder dysfunction [39]. The

fourth stage (healing phase) involves the tissue

repair with possible signaling from the fibroblasts

and the local release of growth factors, such as

keratinocyte growth factor [40]. In the following

sections, we attempt to describe the main

pathological aspects of chemotherapy-induced HC.

Figure 2. Proposed sequential phases of the development of

hemorrhagic cystitis.

The role of transcription factors and

inflammatory mediators in the pathogenesis of

hemorrhagic cystitis

Along with other groups, our laboratory has

investigated the role of inflammatory mediators in

the pathogenesis of alkylating agent-induced HC.

Beginning with the demonstration of the

participation of tumor necrosis factor-α (TNF-α)

and interleukin-1β (IL-1β) [31] and through the

latest studies, the involvement of several cytokines

and other inflammatory mediators has been

suggested [20, 22, 35-39, 41-43]. Each of these

mediators can be up-regulated by the activation of

the NF-κB pathway in response to inflammatory

damage, which makes this transcription factor a

possible target for the prevention of chemotherapy-

induced HC [44].

Nuclear factor kappaB

The transcription factor NF-κB is one of the key

regulators of the genes involved in the immune and

inflammatory response [45]. Under most

circumstances, NF-κB homodimers or heterodimers

are bound to IκB inhibitory proteins in the

cytoplasm of unstimulated cells. Many cytokines

promote the classical NF-κB pathway, in which the

serine phosphorylation and degradation of the IκB

proteins leads to the dissociation of the inactive

cytosolic NF-κB/IκB complexes and the subsequent

NF-κB translocation to the nucleus for DNA

binding. Upon translocation to the nucleus, the

NF-κB dimer binds to the promoters of the NF-κB-

dependent genes and facilitates their transcription

[46]. NF-κB induces the expression of the cytokines

TNF-α and IL-1β, the acute phase proteins iNOS

and COX-2, and adhesion molecules [34]. The

inappropriate activation of NF-κB can lead to

diseases, such as HC.

The first experimental evidence for the role of

NF-κB in the pathogenesis of HC was assessed by

Kiuchi and co-workers [34] through the use of a

potent NF-κB inhibitor, parthenolide [47]. These

authors clearly showed by western blotting that

parthenolide prevents the phosphorylation of

NF-κB p65 and also blocks IκBα

phosphorylation/degradation and NF-κB nuclear

translocation [34]. They also demonstrated that

parthenolide causes the marked inhibition of

COX-2 expression in the bladders of cyclophos-

phamide-treated animals and inflammatory

urothelial cells. These in vitro and in vivo anti-

inflammatory effects of parthenolide were

suggested to be dependent on the inhibition of

NF-κB activity. NF-κB activity in the cyclophos-

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phamide-treated animals contributes to the

activation of downstream inflammatory genes,

leading to bladder hyperactivity and HC [34].

Tumor necrosis factor-α

TNF-α regulates a number of cell functions,

including cell proliferation, survival, differen-

tiation, and apoptosis. TNF-α has been shown to

play a pivotal role in orchestrating the cytokine

cascade in many inflammatory diseases. Because of

this role as a key regulator of inflammatory

cytokine production, it has been proposed as a

therapeutic target for a number of diseases [48].

Aberrant TNF-α production and TNF receptor

signaling have been associated with the

pathogenesis of several diseases, including

rheumatoid arthritis, psoriasis [49], Crohn’s disease

[50], sepsis [51] and diabetes [52]. The receptors

for TNF-α are either constitutively expressed in

most mammalian tissues (TNF-R1) or are highly

regulated and expressed in the cells of the immune

system [53]. TNF, through its interaction with

TNF-R1, causes various cellular events, including

the activation of the caspase cascade that leads to

apoptosis. The interaction between TNF and

TNF-R1 also leads to the activation of NF-κB.

TNF-R2 is known not to possess a death domain

and therefore cannot directly cause apoptosis [53].

Depending on the cell type, TNF-R1 and TNF-R2

may have both distinct and overlapping roles in

signal transduction and gene expression [48].

The role of TNF-α in the inflammatory cascade

of HC was previously investigated by Gomes et al

[31]. These authors clearly demonstrated that anti-

TNF-α serum inhibits the histopathologic

parameters of cyclophosphamide-induced tissue

damage, such as mucosal erosion, hemorrhage,

edema, leukocyte migration, fibrin deposition and

ulcerations. The gains in both the bladder wet

weight and vascular permeability were prevented in

the anti-TNF-α serum-treated mice. These results

are supported by the fact that thalidomide, which

has been reported to inhibit the production of

TNF-α by increasing its mRNA degradation [54],

also prevented the ifosfamide-induced bladder

injury [22]. Therefore, considering that anti-TNF-α

therapy has been shown to effectively manage

several malignances [55-58], clinical trials are

needed to investigate the efficacy of specific TNF-

modulating agents to treat HC.

Interleukin-1β

IL-1β is a highly inflammatory cytokine that

affects nearly every cell type and often works in

concert with other cytokines or small mediator

molecules. The IL-1 receptor family (IL-1R)

includes coreceptors, decoy receptors, binding

proteins, and inhibitory receptors [59]. IL-1β

interacts with two IL-1 receptors, type I (IL-1RI),

which transduces cell signaling, and type II

(IL-1RII), which lacks the signaling-competent

cytosolic domain and is a decoy receptor [60, 61].

The extracellular IL-1 receptor chains, also known

as soluble IL-1RI (sIL-1RI), sIL-1RII, and

sIL-1RAcP, are inducible negative regulators of

IL-1β signaling in the extracellular space [61].

IL-1β first binds to the ligand-binding chain,

IL-1RI, and then the coreceptor chain, termed the

accessory protein (IL-1RAcP), is recruited. The

signal is initiated with the recruitment of the

adaptor protein MyD88 to the Toll-IL-1 receptor

(TIR) domain [62]. Subsequently, several kinases

are phosphorylated, NF-κB translocates to the

nucleus, and several inflammatory genes are

expressed [62].

IL-1β is a pivotal pro-inflammatory cytokine

implicated in the pathogenesis of stroke, diabetes

and rheumatoid arthritis [62, 63]. Agents that

reduce the production and/or activity of IL-1 are

likely to impact the clinical management of

pathologies such as HC.

Gomes et al evaluated the effect of anti-IL-1β

serum on the animal model of hemorrhagic cystitis

[31]. This modulating approach was beneficial

against the cyclophosphamide-induced bladder

damage and vesical wet weight gain but to a milder

degree than the anti-TNF serum. In addition,

pentoxifylline exerts multiple beneficial immune-

modulatory effects by down-regulating IL-1β and

TNF-α synthesis, decreasing interferon-γ (IFN-γ),

granulocyte-macrophage colony-stimulating factor

and IL-6 secretion, and attenuating the cell surface

expression of the IL-2 receptor [64] and was also

shown to inhibit the vesical edema and microscopic

alterations induced by ifosfamide administration

[22]. In fact, IL-1β is highly expressed in the

epithelial and subepithelial bladder cells of the

ifosfamide-injected mice as demonstrated by

immunohistochemistry [43].

Considering the ability of IL-1β to orchestrate the

inflammatory process and the protective effect

achieved by its inhibition, the selective inhibition of

IL-1 function could also present interesting clinical

outcomes.

Interleukin-4

IL-4 is a cytokine that acts as an anti-

inflammatory compound under certain conditions

and is therefore responsible for the homeostasis of

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the immune system. It has a marked inhibitory

effect on the expression and release of pro-

inflammatory mediators, such as the monocyte-

derived cytokines TNF-α and IL-1β [65] and a

modulating effect on the expression of IL-1

receptor antagonist (IL-1ra) [66]. In addition to the

inhibition of the production of inflammatory

cytokines, IL-4 has been reported to inhibit the

induction of COX-2, with a consequent reduction in

the production of prostaglandins [67].

Macedo et al explored the anti-inflammatory

potential of IL-4 in the model of ifosfamide-

induced HC [43]. Interestingly, IL-4 production is

enhanced after ifosfamide administration. However,

the administration of exogenous IL-4 inhibits

inflammatory parameters, such as the expression of

pro-inflammatory cytokines (TNF-α and IL-1β) and

enzymes (iNOS and COX-2) [43]. In addition, it

was demonstrated that both IL-4 antiserum-treated

and genetically deficient mice have exacerbated

HC.

Interleukin-6 and interleukin-8

IL-6 is a member of a cytokine family sharing a

common signal transducer (gp130), which also

includes IL-11, IL-27, IL-31, leukemia inhibitory

factor, ciliary neurotrophic factor and

cardiotrophin-1 [68]. Some studies have reported an

increased local expression of IL-6 in the bladder or

in the serum after cyclophosphamide challenge

[69-71]. Nishii et al [69] showed that IL-6 gene

expression is significantly up-regulated in the

submucosal layer of the bladder after

cyclophosphamide administration in mice, peaking

at 6 h and declining thereafter. In addition, Girard

et al also showed that IL-6, IL-6 receptor α subunit

(IL-6Rα) and gp130 (β subunit) are highly

expressed in the urothelium and detrusor following

cyclophosphamide treatment [71]. In contrast, IL-8

expression and its role in HC have never been

investigated. IL-8 is a critical chemo-attractant

responsible for leukocyte recruitment, cancer

proliferation, and angiogenesis [72]. IL-8 has been

implicated in the pathogenesis of diseases,

including acute lung injury and acute respiratory

distress syndrome [73], osteoarthritis [74] and

cystic fibrosis [75].

We showed that IL-6 and IL-8 are involved in the

pathogenesis of HC. Mice intraperitoneally injected

with 200 mg/kg cyclophosphamide had significant

increases in bladder wet weight and vascular

permeability when compared to the saline-treated

group. Furthermore, treatment with anti-IL-6 and

anti-IL-8 sera (25 or 50 uL/animal) before the

cyclophosphamide injection markedly prevented

the development of edema (unpublished data).

As far as we know, this is the first report of an

experimental modulation of IL-6 and IL-8 and the

pathological contribution of these cytokines to the

cyclophosphamide-induced edema in HC. Further

studies are needed to precisely define the role of

these mediators in the inflammatory cascade

involved in this pathology.

Bradykinin

Bradykinin and its homologues, known

collectively as kinins, are produced by the action of

kallikrein enzymes [76]. The kinins are blood-

derived, local-acting peptides that have broad

effects mediated by two related G-protein-coupled

receptors, the B1 and B2 bradykinin receptors. The

endogenous kallikrein-kinin system controls blood

circulation and kidney function and promotes

inflammation and pain in pathological conditions

[77], including sepsis and cancer [78].

Maggi and co-workers demonstrated the

participation of bradykinin via B2 receptors in the

genesis of detrusor hyperreflexia during cyclophos-

phamide-induced cystitis [79]. In addition, a study

published by Chopra and colleagues showed that

the B2 receptor, but not the B1 receptor, is

expressed in the urothelium and detrusor smooth

muscle of bladders from control animals [80].

However, the expression of B1 receptors is highly

increased during cyclophosphamide-induced HC.

Convincingly, these authors demonstrated that the

urothelial expression of bradykinin receptors is

plastic and altered by pathology, indicating a role of

both bradykinin receptors in the cyclophosphamide-

induced bladder hyperactivity via the extracellular

release of urothelial-derived factors, such as ATP,

which act on the pelvic nerve afferents [80].

The participation of the B2 receptors in

cyclophosphamide-induced bladder edema

formation was also investigated. Rats had a

significant increase in their bladder wet weight and

vascular permeability 48 h after the administration

of cyclophosphamide (100 mg/kg) when compared

to the saline-treated group. Animals pre-treated

with Hoe-140 (2 mg/kg), a B2 receptor antagonist,

plus cyclophosphamide markedly ameliorated both

the vesical wet weight and vascular permeability in

comparison with the cyclophosphamide-injected

rats (unpublished data).

Despite the knowledge concerning the

inflammatory importance of bradykinin, only one

bradykinin receptor antagonist, icatibant (Hoe-140),

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has reached the market for the treatment of

hereditary angioedema [81].

Cyclooxygenases

COX is a group of bifunctional enzymes that

have both cyclooxygenase and peroxidase

activities. COXs catalyze the complex conversion

of arachidonic acid to prostaglandins and

thromboxanes, which act as autacoids with

autocrine and paracrine biological effects to trigger

many physiological and pathophysiological

responses [82, 83]. Three isoforms of COX have

been described to date: COX-1, -2, and -3. COX-2

is an isoform whose expression is commonly up-

regulated during inflammatory processes. It plays

an important role in pain [84], tumor activation

[83], inflammation and neurodegenerative diseases

[85]. The expression of COX-2 was already

demonstrated during cyclophosphamide- [86],

ifosfamide-[36] and acrolein- [37] induced HC.

Our group assessed the role of COX-2 on the

pathogenesis of ifosfamide-related HC with a

selective inhibitor of COX-2, etoricoxib, and a non-

selective inhibitor, indomethacin. We observed that

COX-2 is highly expressed 24 h after a single

injection of ifosfamide to induce HC mainly in

myofibroblasts and, to a lesser extent, in mast cells

[36].

Hu et al also provided evidence for COX-2

mRNA expression due to cyclophosphamide

injection [86]. The demonstration that COX-2 is

observed a few hours after the induction of HC

suggests the participation of other mediators in the

first hours following ifosfamide administration

[20, 22]. We verified this hypothesis by the use of

non-selective cytokine inhibitors, thalidomide and

pentoxifylline [36]. It was shown that these agents

prevent some of the inflammatory parameters when

compared to the groups treated only with

ifosfamide, which is in accordance with the data

published by Ribeiro et al [22]. In addition, a lower

expression of COX-2 was observed when the

animals were given thalidomide and pentoxifylline

before ifosfamide administration, which supports a

role for cytokines on COX-2 expression in this

animal model of HC [26].

It is known that HC can arise as a complication of

both cyclophosphamide and ifosfamide treatment

and that it is caused by the urinary metabolite

acrolein. Our group showed in mice that acrolein

can cause severe edema and hemorrhage, similar to

cyclophosphamide and ifosfamide [25].

In another study published by our group, acrolein

injected into the rat bladder induced a dose- and

time-dependent HC, and the resulting lesion is

partially mediated by the increased expression of

COX-2 [37]. However, this study also showed that

even 3 and 6 h after the acrolein instillation into the

bladder, COX-2 expression is almost absent,

despite the presence of severe edema and

hemorrhage.

Macedo et al reported that these observations

suggest the participation of other inflammatory

mediators in the changes observed within the first

hours of acrolein-induced HC [37], as demonstrated

previously for ifosfamide- and cyclophosphamide-

induced HC [22, 31]. Macedo et al showed that

between 3 and 6 h, there is not sufficient expression

of the COX-2 enzyme to affect the increase of

bladder wet weight in animals injected with

acrolein [37]. The anti-inflammatory effect of

etoricoxib was only detected 12 h post-acrolein

injection.

Hu et al demonstrated that conscious cystometry

in rats injected with cyclophosphamide and the

COX-2 inhibitor 5,5-dimethyl-3-(3-fluorophenyl)-

4-(4-methylsulfonyl)phenyl2(5H)-furanone (DFU)

showed increased micturition intervals 4 and 48 h

after cyclophosphamide treatment and decreased

intravesical pressures during filling and micturition

compared with rats administered cyclophosphamide

[86]. This finding clearly suggested an involvement

of the urinary bladder COX-2 and its metabolites in

the altered micturition reflexes during cyclophos-

phamide-induced cystitis.

Additionally, a very recent article published by

our group showed that the HC experimentally

induced by ifosfamide promotes detrimental

changes in the motor functions of the rat lower

urinary tract, interferes with the urinary bladder

emptying process in anesthetized rats, and alters the

contractile behavior as assessed by in vitro methods

[39]. We also suggested that this phenomenon

involves the inflammatory events mediated by the

COX-2 expression within the urinary bladder with a

consequent rise in the plasma levels of PGE2 [39].

In addition, the selective pharmacological inhibition

of COX-2 activity partially ameliorated these

functional changes. We concluded that the

prevention of the inflammatory process ameliorates

the consequences of the ifosfamide-induced cystitis.

Maggi [87] reviewed the role of prostanoids, such

as PGE2, as activators of bladder afferents, which

changes the micturition reflex threshold. The

mechanism of PGE2-induced normal bladder

hyperactivity was investigated by Ishizuka and

colleagues [88], who showed that intravesical

PGE2 might stimulate micturition by releasing

Ribeiro et al: Chemotherapy-induced hemorrhagic cystitis

DOI:10.5455/jeim.080312.ir.010 102

tachykinins from the nerves in and/or immediately

below the urothelium. These tachykinins, in turn,

initiate a micturition reflex by stimulating the NK1

and NK2 receptors, contributing to both urge and

the bladder hyperactivity observed in the

inflammatory conditions of the lower urinary tract.

However, the precise mechanisms involved in the

detrimental effects of ifosfamide in HC deserve

further investigation. Possible explanations involve

the modification of the synaptic mechanisms in the

urinary bladder, e.g., those related to the muscarinic

receptor response to agonists, which still needs to

be proven.

Nitric oxide

NO is synthesized by a family of enzymes known

as nitric oxide synthases (NOSs). Three distinct

isoforms of NOS, namely endothelial nitric oxide

synthase (eNOS), neuronal nitric oxide synthase

(nNOS) and inducible nitric oxide synthase (iNOS),

have been described based on the cells in which

they were first identified, isolated, purified, and

cloned [89]. Over the past two decades, NO has

gained increasing recognition as an important

neurotransmitter and cell mediator with a broad

range of functions in the lower urinary tract.

A convincing first report on the participation of

NO in the pathogenesis of chemotherapy-induced

HC was published by our group [20]. This study

showed that the injection of cyclophosphamide

increases iNOS activity. Additionally, two NOS

inhibitors, L-NAME and L-NOARG, dose-

dependently inhibited the cyclophosphamide-

induced plasma protein extravasation and bladder

wet weight, which was reversed by L-arginine, a

NO precursor. Such inhibitory actions on NOS

activation critically prevented bladder damage, as

shown both macroscopically and microscopically

by the reduced mucosal damage. Another study

performed by Oter and colleagues evaluated the

role of iNOS-derived NO on bladder injury due to

cyclophosphamide injection [35]. In that study, the

administration of S-methylisothiourea, a selective

iNOS inhibitor, led to similar results as those

previously reported.

We also investigated the role of constitutive NOS

enzymes and found that NOS activity in the normal

bladder was mainly due to the cNOS (calcium-

dependent) isoform (>95%). Cyclophosphamide

administration significantly increased the calcium-

independent activity of iNOS, which was evident

6 h after the injection and remained elevated for up

to 48 h. In contrast, cNOS decreased over a similar

time course in the presence of cyclophosphamide

[20]. Consistent with these findings, nicotinamide

adenine dinucleotide phosphate diaphorase

(NADPH-d), a marker for NOS-containing cells,

was expressed in the urothelium and in the lamina

propria of control rats. This expression decreased

dramatically 12 h following cyclophosphamide

administration and coincided with an intense

sloughing of the urothelium. Alfieri et al also

explored the effect of the selective inhibition of

nNOS, which did not result in a protective effect

[41]. These observations indicate that inducible, but

not constitutive, NOS-derived NO exerts a

fundamental role in the pathogenesis of HC.

TNF-α and IL-1β were shown to mediate the

production of NO through iNOS induction in the

pathogenesis of ifosfamide- and cyclophosphamide-

induced HC [22, 31]. The induction of iNOS in the

urothelium appeared to depend on the production of

the cytokines IL-1β and TNF-α because antisera

against these cytokines also reduced the expression

of iNOS in the urothelium [31].

Peroxynitrite

The cellular damage that commonly occurs

during conditions of oxidative stress has been

observed following the formation of peroxynitrite

(ONOO-), which originates from the reaction of NO

with superoxide free radicals (O2-) [90]. An

excessive formation of peroxynitrite represents an

important mechanism contributing to DNA damage,

the inactivation of metabolic enzymes, ionic pumps,

and structural proteins, and the disruption of cell

membranes. Peroxynitrite reacts with the tyrosine

in proteins to create nitrotyrosines [91]. Because of

its ability to oxidize biomolecules, peroxynitrite is

implicated in an increasing number of diseases,

including neurodegenerative and cardiovascular

disorders, inflammation, pain, autoimmunity,

cancer, and aging [92].

A first report published by Korkmaz et al showed

that peroxynitrite might contribute to the

pathogenesis of cyclophosphamide-induced HC

[38]; Korkmaz et al reported that ebselen, which

has potent scavenging properties and was shown to

react with peroxynitrite [93], markedly protects

against the development of HC. The results of this

study suggest that the scavenging of peroxynitrite

and the inhibition of iNOS have similar protective

effects.

Platelet activating factor

Platelet-activating factor (PAF; 1-O-alkyl-2-

acetyl-sn-glycero-3-phosphocholine) is a phospho-

lipid mediator that acts largely by binding to its

receptor (PAFR), a G-protein-coupled receptor

Journal of Experimental and Integrative Medicine 2012; 2(2):95-112

www.jeim.org 103

found on most cells, such as monocytes,

neutrophils, B lymphocytes, and keratinocytes

[94-96]. PAF is involved in many biological

processes, including cellular activation, cytoskeletal

reorganization and intracellular signaling, and is a

potent mediator in many inflammatory processes,

including inflammatory bowel disease [97],

multiple sclerosis [98], and asthma [99].

In a study by Rickard et al, the combination of

increased prostacyclin and PAF in the bladder

circulation resulted in vasodilation and increased

polymorphonuclear leukocyte adherence to the

endothelium and facilitated the recruitment of

polymorphonuclear leukocytes to the bladder wall

of patients with interstitial cystitis [100]. In

addition, we found that PAF is one of the

inflammatory mediators contributing to the

activation of the L-arginine–NO pathway in HC

[20]. This conclusion was reached by the use of the

PAF antagonist BN52021, which markedly

protected rats from the cyclophosphamide-induced

plasma protein extravasation and bladder wet

weight gain as well as further prevented the

increase in bladder iNOS activity without

modifying cNOS activity. The maintenance of

cNOS activity during the PAFR blockade implies

that the integrity of the epithelial surface was also

maintained, corroborating the deleterious effect of

PAF in HC.

Substance P

Neurogenic inflammation, components of which

include arteriolar vasodilation (flare) and edema

caused by the extravasation of plasma from post-

capillary venules, is triggered by the release of

substances, such as the proinflammatory

neuropeptides substance P (SP) and calcitonin

gene-related peptide (CGRP), from sensory nerve

terminals [101]. In the urinary tract, such responses

include hyperemia, plasma protein leakage,

contraction of the ureter and bladder and

hyperactivity of the bladder.

Acting through the tachykinin NK1 receptors, SP

may induce tissue inflammation [102] due to the

increased production of NO [103]. Structurally

unrelated antagonist of the NK1 receptors has been

shown to ameliorate the increase in the plasma

permeability extravasation and the histological

damage of the bladder induced by

cyclophosphamide both in rats and ferrets [104]. It

is possible that SP released from the primary

afferent capsaicin-sensitive fibers stimulated by

cyclophosphamide or its metabolite acrolein may

increase NO levels, thereby inducing inflammatory

changes [41].

Alpha1-adrenoceptor

Sympathetic efferents are known to modulate the

neurogenic inflammatory responses by interacting

with primary afferent terminals. α1-Adrenoceptor

antagonists have been successfully used for the

treatment of the symptoms in patients with

indwelling double-J urethral stents [105], ureteral

colic [106], prostatitis and benign prostatic

hyperplasia [107].

Trevisani et al investigated whether α1-adreno-

ceptors are expressed in primary sensory neurons

and whether they contribute to the neurogenic

inflammatory responses that originated in the

urinary tract following cyclophosphamide treatment

[108]. These authors suggested that the α1-adreno-

ceptors contribute to neurogenic inflammation in

the bladder based on the finding that locally applied

phenylephrine increases plasma extravasation in a

manner abolished by alfuzosin, an α1-adrenoceptor

antagonist, and by NK1 receptor blockade. In

addition, these agents were equally effective at

inhibiting the bladder plasma extravasation due to

cyclophosphamide injection. However, alfuzosin

failed to affect the plasma extravasation evoked by

the direct stimulation of the endothelial NK1

receptor by [Sar9,Met(O2)

11]-SP, indicating

selectivity. These results suggest that α1-adreno-

ceptor activation at the terminals of primary

sensory neurons releases SP, which in turn

stimulates the vascular NK1 receptors, promoting

plasma protein leakage in the bladder tissue [108].

Pharmacological approaches to hemorrhagic

cystitis

Table 1 shows a broad review of the experimental

tools, natural products, and clinically available

drugs that have already been tested in

acrolein/chemotherapy-induced HC models.

Among them, mesna and hyperhydration are

currently the only measures used clinically to

prevent HC. From our point of view, the literature

lacks studies that test the effectiveness of anti-

cytokine therapy of HC. However, some anti-

inflammatory cytokines, such as IL-4, IL-11 and

keratinocyte growth factor, have been

experimentally evaluated with important

contributions.

Interleukin-4

As discussed above, exogenous IL-4

administration was able to prevent the development

of HC. Additionally, IL-4 seems to play an

important role in HC pathogenesis, possibly in

counteracting the inflammatory pathways, because

Ribeiro et al: Chemotherapy-induced hemorrhagic cystitis

DOI:10.5455/jeim.080312.ir.010 104

HC was exacerbated in IL-4-deficient mice and in

anti-IL-4 serum-treated mice [43].

Interleukin-11

IL-11 is a pleiotropic 178-amino acid polypeptide

with a molecular weight of 18 kDa and is secreted

by osteoblasts, synoviocytes, fibroblasts,

chondrocytes, trophoblasts, and many other cell

types in culture [109].

Recombinant human IL-11 (rhIL-11, oprevelkin)

has a number of biological activities, including the

inhibition of pro-inflammatory cytokines (TNF-α

and IL-1β), IL-12, NO synthesis and apoptosis

[110, 111] as well as the stimulation of cell

proliferation, differentiation and connective tissue

protection [111]. Several studies have shown a

protective role for IL-11 in a variety of models of

inflammatory diseases, such as inflammatory bowel

disease, psoriasis, and autoimmune joint disease

[112, 113].

IL-11 affects various stages of megakaryo-

cytopoiesis and thrombopoiesis, and its clinical use

has been shown to shorten the duration of

chemotherapy-induced thrombocytopenia [114].

Additionally, the potential anti-inflammatory

modulating effect of IL-11 was further investigated

in a study published by our group [115] using an

animal model of ifosfamide-induced HC. This

study showed that rhIL-11 inhibits ifosfamide-

induced vascular permeability and edema formation

in a dose-dependent manner, which was confirmed

by macroscopic and microscopic analysis. These

results indicate that the therapeutic manipulation of

IL-11 signaling may provide clinical benefits for

HC.

Keratinocyte growth factor

Keratinocyte growth factor (KGF), a heparin-

binding fibroblast growth factor also known as

FGF-7, has been suggested to play an important

role in the mesenchymal stimulation of normal

epithelial cell proliferation [116]. The

administration of KGF has been shown to protect

animals from various insults, such as radiation- and

bleomycin-induced lung injury [117] and mucositis

[118, 119]. Furthermore, Ulich et al showed that

KGF protects against cyclophosphamide-induced

HC with a marked trophic action on urothelial cells

and minor histological changes, such as edema and

acute inflammation of the urothelium and

submucosa [40].

Figure 3.

Proposed pathways

for the pathogenesis of hemorrhagic cystitis.

Journal of Experimental and Integrative Medicine 2012; 2(2):95-112

www.jeim.org 105

Table 1. Pharmacological approaches in acrolein- or cyclophosphamide(CYP)/ifosfamide(IFO)-induced hemorrhagic cystitis

Experimental

model Compounds Mechanisms of action Improved outcomes References

CYP-induced HC N-acetylcysteine Antioxidant activity Microscopic [18]

CYP-induced HC Mesna Chemical antagonist of

Acrolein Macro and microscopic [122]

CYP-induced HC Penicillamine Anti-inflammatory activity Macro and microscopic [123]

CYP-induced HC Misoprostol Prostaglandin E1 analog Macro and microscopic [19]

CYP-induced HC Prostaglandin F2 alpha Prostaglandin F2 alpha Macro and microscopic [124]

CYP-induced HC Carboprost

tromethamine

Prostaglandin F2 alpha

analog Macro and microscopic [125]

CYP-induced HC L-NAME/L-NOARG NOS inhibitor Macro and microscopic [20]

CYP-induced HC BN-52021 Platelet activating factor

antagonist Macro and microscopic [20]

CYP-induced HC Keratinocyte growth

factor epithelial growth factor Macro and microscopic [40]

CYP-induced HC L-2-oxothiazolidine-4-

carboxylate Antioxidant activity Macro and microscopic [126]

CYP-induced HC Dexamethasone Anti-inflammatory activity Macro and microscopic [121]

CYP-induced HC Glucose-mannose

binding lectins Anti-inflammatory activity Macro and microscopic [127]

CYP-induced HC Berberine Anti-inflammatory activity Macroscopic [128]

IFO-induced HC Thalidomide Inhibitor of TNF

production

Macro and microscopic and

permeability, and iNOS

inhibition

[22]

IFO-induced HC Pentoxifylline

Inhibitor of pro-

inflammatory cytokine

synthesis

Macro and microscopic and

permeability, and iNOS

inhibition

[22]

CYP-induced HC Eviprostat Anti-inflammatory activity Macro and microscopic and

function [129]

IFO-induced HC Ternatin Anti-inflammatory activity Macro and microscopic [130]

IFO-induced HC Latex from Calotropis

procera Antioxidant activity Macro and microscopic [131]

CYP-induced HC Taurine Antioxidant activity

Macro and

microscopic and oxidative

stress

[132]

CYP-induced HC Melatonin Antioxidant, iNOS inhibitor

and peroxynitrite scavenger

Macro and microscopic and

iNOS inhibitor [133]

CYP-induced HC REN1820 Nerve growth factor

recombinant Function [134]

CYP-induced HC Cyclopentenone

prostaglandin (PG) J2

Ligand for the peroxisome

proliferator-activated

receptor-gamma (PPAR-γ)

Macro and microscopic, pro-

inflammatory cytokines and

iNOS inhibition

[135]

CYP-induced HC Alpha-tocopherol Antioxidant activity Macro and microscopic [136]

Acrolein-induced

HC/ IFO-induced

HC

Amifostine Antioxidant activity Macro and microscopic [137]

Acrolein-induced

HC/ IFO-induced

HC

Glutathione Antioxidant activity Macro and microscopic [137]

IFO-induced HC Oprevelkin (rIL-11) Anti-inflammatory activity Macro, microscopic and

permeability [115]

CYP-induced HC Hemin Chemical inducer of HO Macro and microscopic and

iNOS inhibitor [138]

CYP-induced HC Epinephrine Alpha- and beta-adrenergic

receptor agonist Macro and microscopic

[139]

CYP-induced HC Ebselen Seleno compound

Peroxynitrite scavenger Macro and microscopic [140]

CYP-induced HC 1400W Selective iNOS inhibitor Macro and microscopic [140]

CYP-induced HC 3-aminobenzamide Poly(ADP-ribose)

polymerase inhibition Macro and microscopic [140]

CYP-induced HC S-allylcysteine Antioxidant activity Macro and microscopic [141]

Ribeiro et al: Chemotherapy-induced hemorrhagic cystitis

DOI:10.5455/jeim.080312.ir.010 106

Experimental

model Compounds Mechanisms of action Improved outcomes References

Acrolein-induced

HC and IFO-

induced HC

Etoricoxib COX2 inhibitor Macro and microscopic, COX2

inhibition and function

[36, 37, 39]

Acrolein-induced

HC and IFO-

induced HC

Indomethacin COX inhibitor Macro and microscopic, COX2

inhibition and function

[26, 37, 39]

CYP-induced HC A-317491 P2X3/P2X2/3 antagonist Function [142]

CYP-induced HC Curcumin Antioxidant and anti-

inflammatory activity Macro and microscopic [143]

CYP-induced HC Acacia Senegal gum

exudate

Antioxidant and anti-

inflammatory activity Macro and microscopic [144]

CYP-induced HC Aminoguanidine, Selective iNOS inhibitor Macro and microscopic [145]

CYP-induced HC Parthenolide Nuclear factor-kappaB

inhibitor

Macro, microscopic and

function, COX2 and TNF

inhibition

[34]

CYP-induced HC Rolipram Specific type-4

phosphodiesterase inhibitor

Macro and microscopic and

iNOS inhibition [146]

CYP-induced HC Simvastatin Anti-inflammatory activity

Macro and microscopic, and

pro-inflammatory cytokines

inhibition

[70]

CYP-induced HC Ipomoea obscura (L.) Antioxidant activity

Macro and microscopic, and

pro-inflammatory cytokines

inhibition

[147]

CYP-induced HC Glutamine Antioxidant activity Oxidative stress and neutrophil

infiltration inhibition [148]

CYP-induced HC Seleno-L-methionine Antioxidant activity Macro and microscopic,

oxidative stress inhibition [149]

CYP-induced HC MV8608 and MV8612 Anti-inflammatory and

antinociceptive activity

Macro, microscopic,

hypernociception, and

neutrophil infiltration inhibition

[150]

CYP-induced HC Tacrolimus Inhibitor of interleukin-2

production by T-cells

Microscopic and function, and

PGE2 and EP4 inhibition [151]

CYP-induced HC ISO-1 Macrophage migration

inhibitory factor

Macro, microscopic, function

and hypernociception, nerve

growth factor and pro-

inflammatory cytokines

inhibition

[152]

CYP-induced HC HC-067047 Selective TRPV4

antagonist Function [18]

CYP-induced HC ONO-8130 Selective prostanoid EP1

antagonist

Permeability and

hypernociception [153]

CYP-induced HC Hydroalcoholic extract

of Phyllanthus niruri Unknown

Macro, microscopic,

hypernociception and oxidative

stress

[154]

CYP-induced HC A-438079 P2X7 receptor antagonist

Macro, microscopic, neutrophil

and macrophage infiltration,

and inflammatory cytokines

inhibition

[155]

Chemotherapy-induced HC was considered to be

an unsolved problem in the oncology patient

population. Currently, clinical studies with

palifermin (recombinant KGF) have indicated that

hematopoietic stem cell transplant patients treated

with high-dose cytotoxic regimens present with a

reduced severity of lesions in the oral mucosa

[120]. Considering that mucosal ulceration seems to

be a key event in the most symptomatic phase of

HC, further studies are needed to better investigate

the potential protective effect of KGF on this

disease.

Corticosteroid therapy

Taking into consideration the unquestionable

participation of various inflammatory mediators in

the pathogenesis of HC, there is a question of

whether corticosteroids would be effective at

preventing HC-associated bladder damage. This

Journal of Experimental and Integrative Medicine 2012; 2(2):95-112

www.jeim.org 107

hypothesis was experimentally tested by our group

[23, 121]. In these studies, we verified that

dexamethasone in combination with mesna was

efficient in attenuating cyclophosphamide- or

ifosfamide-induced HC. However, the replacement

of the last two doses of mesna with saline or all of

the mesna doses with dexamethasone did not

prevent HC. This is a clear demonstration that

mesna is still needed to prevent the initial epithelial

bladder damage, which is not achieved by

dexamethasone alone. However, the corticosteroid

inhibition of cytokine activation prevents the

underlying inflammatory aspect of HC. Therefore,

clinical trials are needed to prove the experimental

findings using corticosteroids.

Conclusions

Hemorrhagic cystitis is an important clinical

issue that has been recently brought to attention due

to an augmented incidence mainly following the

increase in treatment regimens of high-dose

alkylating agents in the context of hematopoietic

stem cell transplantation and cancer chemotherapy.

Several inflammatory mediators have been

implicated in the development of hemorrhagic

cystitis due to chemotherapy agents (Fig.3). Despite

the recent advances in the knowledge of the

pathogenesis of this clinical condition, further

investigation is needed to determine the precise

inflammatory cascade involved. The enhanced

understanding of the role played by these

inflammatory pathways may allow the

identification of novel pharmacologic targets for

this pathology.

Acknowledgements.

R.A.R. is a researcher from Conselho Nacional de

Desenvolvimento Cientifico e Tecnologico (CNPq),

Brazil. This work was supported by research grants from

the CNPq and Fundacao Cearense de Apoio ao

Desenvolvimento Cientifico (FUNCAP) –Nucleo de

Estudos da Toxicidade do Tratamento Oncologico

(NETTO) - Processo 21.01.00/08.

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