Experimental bladder carcinogenesis – rodent models
Several rodent models of bladder cancer development have been established. The aim of this review article is to provide a critical assessment of different animal models available for the study of bladder carcinogenesis, its chemoprevention and therapy. All, except for transgenic and knockout anima...
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irk-123456789-1341552018-06-13T03:03:05Z Experimental bladder carcinogenesis – rodent models Oliveira, P.A. Colaco, A. L.F. De la Cruz, P. Lopes, C. Reviews Several rodent models of bladder cancer development have been established. The aim of this review article is to provide a critical assessment of different animal models available for the study of bladder carcinogenesis, its chemoprevention and therapy. All, except for transgenic and knockout animals, require 8–12 months experimental periods in order to generate a high yield of neoplasias. Spontaneous bladder tumor models are extremely rare. The significance of the results from animal experiments is dependent upon the selection of a suitable animal model. There are no rules regarding the choice of a model, it is however very useful to have knowledge of relevant comparative medical aspects concerning this subject. We describe chemical carcinogens most commonly used to induce bladder cancer, pellet implantation and urinary calculi, agents that promote bladder cancer, and irradiation. We also evaluated other tools such as cell cultures, tumor implantation and transgenic models for bladder cancer, that have been developed to study the process. The review considers how several imaging techniques can be applied to study rodent bladder carcinogenesis. Для изучения механизмов развития рака мочевого пузыря было создано несколько экспериментальных моделей на гры- зунах. Целью обзора была сравнительная оценка различных экспериментальных моделей для изучения канцерогенеза мочевого пузыря, профилактики и терапии. За исключением трансгенных и нокаутных животных, для получения высокого выхода опухолей в любой экспериментальной модели требуется 8–12 мес. Модели спонтанного канцерогенеза мочевого пузыря крайне редки. Выбор экспериментальной модели с определенными параметрами определяет значимость получен- ных результатов. В статье описаны различные методики, используемые для индукции рака мочевого пузыря in vivo, ряд методических подходов, таких как культура клеток, имплантация опухоли и трансгенные модели рака мочевого пузыря и современные методы мониторинга опухолевой прогрессии. 2006 Article Experimental bladder carcinogenesis – rodent models / P.A. Oliveira, A. Colaco,P. L.F. De la Cruz, C. Lopes // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 2-11. — Бібліогр.: 99 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/134155 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Reviews Reviews Oliveira, P.A. Colaco, A. L.F. De la Cruz, P. Lopes, C. Experimental bladder carcinogenesis – rodent models Experimental Oncology |
| description |
Several rodent models of bladder cancer development have been established. The aim of this review article is to provide a critical
assessment of different animal models available for the study of bladder carcinogenesis, its chemoprevention and therapy. All, except
for transgenic and knockout animals, require 8–12 months experimental periods in order to generate a high yield of neoplasias.
Spontaneous bladder tumor models are extremely rare. The significance of the results from animal experiments is dependent upon the
selection of a suitable animal model. There are no rules regarding the choice of a model, it is however very useful to have knowledge
of relevant comparative medical aspects concerning this subject. We describe chemical carcinogens most commonly used to induce
bladder cancer, pellet implantation and urinary calculi, agents that promote bladder cancer, and irradiation. We also evaluated
other tools such as cell cultures, tumor implantation and transgenic models for bladder cancer, that have been developed to study
the process. The review considers how several imaging techniques can be applied to study rodent bladder carcinogenesis. |
| format |
Article |
| author |
Oliveira, P.A. Colaco, A. L.F. De la Cruz, P. Lopes, C. |
| author_facet |
Oliveira, P.A. Colaco, A. L.F. De la Cruz, P. Lopes, C. |
| author_sort |
Oliveira, P.A. |
| title |
Experimental bladder carcinogenesis – rodent models |
| title_short |
Experimental bladder carcinogenesis – rodent models |
| title_full |
Experimental bladder carcinogenesis – rodent models |
| title_fullStr |
Experimental bladder carcinogenesis – rodent models |
| title_full_unstemmed |
Experimental bladder carcinogenesis – rodent models |
| title_sort |
experimental bladder carcinogenesis – rodent models |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2006 |
| topic_facet |
Reviews |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/134155 |
| citation_txt |
Experimental bladder carcinogenesis – rodent models / P.A. Oliveira, A. Colaco,P. L.F. De la Cruz, C. Lopes // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 2-11. — Бібліогр.: 99 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT oliveirapa experimentalbladdercarcinogenesisrodentmodels AT colacoa experimentalbladdercarcinogenesisrodentmodels AT lfdelacruzp experimentalbladdercarcinogenesisrodentmodels AT lopesc experimentalbladdercarcinogenesisrodentmodels |
| first_indexed |
2025-07-09T20:25:04Z |
| last_indexed |
2025-07-09T20:25:04Z |
| _version_ |
1837202361126223872 |
| fulltext |
� Experimental Oncology �8, �–11, �006 (March)
The bladder is one of the most common sites of
cancer in the urinary tract. Bladder tumors are manifesta-
tions of a multifocal disease whose natural history has not
been completely elucidated and the response of bladder
tumors to radio — and chemotherapy is unpredictable.
Urothelial carcinogenesis in the rat goes through
a sequence of morphologic changes beginning as
simple hyperplasia. It then progresses to nodular and
papillary hyperplasia. These progress to papillomas
and can eventually progress to higher-grade, noninva-
sive carcinomas and ultimately to invasive neoplasms
(Fig. 1) [�0, 7�]. Many exophytic tumors induced in rats
are polypoid, often pedunculated and with an inverted
papillary growth pattern [7�]. Nodular hyperplasia, in
mice, is considerably more common than papillary
proliferations and nodular hyperplasia frequently oc-
curs with a complete absence of papillary hyperplasia
[3�]. Thus, the rat model strongly resembles papillary
neoplasms and the mouse model resembles flat uro-
thelial lesions, both identified in man [�8].
Since the Surgeon Dr. Rehn first suggested a role
for aniline dye in the etiology of bladder cancer in 1895,
this has became the neoplasic disease which has been
traditionally most strongly linked to occupational and envi-
ronmental exposure to chemicals [83]. To study urothelial
carcinogenesis it is possible to use dogs, rabbits, guinea
pigs, hamster, rats and mice. However, for investigation
of chemical carcinogenesis mice and rats are generally
used, for several reasons. For example, in addition to ben-
efits related to size, there is an abundance of information
concerning the biological characteristics of neoplastic de-
velopment in these species. This is why we have focused
this review on urothelial carcinogenesis in rats and mice.
However, other animals are also mentioned.
Fig. 1. Pathogenic pathways of rat and mice urinary bladder
carcinogenesis
This review focuses on several in vivo and in vitro ro-
dent urinary bladder cancers that have been established
over the past 40 years. In Fig. � we present the potential
applications of rodent bladder specimens obtained fol-
lowing chemical carcinogenesis of the urothelium.
The rodenT bladder
The structure and function of rodents’ lower urinary
tract is remarkably similar to that of humans [7�]. It ex-
tends from the renal pelvis through the ureters, urinary
bladder, and into the urethra [90]. Excluding the urethra,
the urinary tract consists of four layers: mucosa, lamina
propria, muscular and serosa [�1, �4, �5]. The mucosa
of the lower urinary tract has been referred to as a
transitional cell epithelium or as an urothelium. In the
urinary bladder it is made up of three cell layers: super-
experimenTal bladder carcinogenesis-rodenT models
P.A. Oliveira1,*, A. Colaço1, L.F. De la Cruz P 2, C. Lopes3,4
1Department of Veterinary Sciences, CECAV, University of Trás-os-Montes and Alto Douro, 5001-801,
Vila Real, Portugal
2Deparment of Physiology, Faculty of Veterinary, Santiago University, Spain
3Department of Pathology, Portuguese Institute of Oncology,
4200-072 Porto, Portugal
4Department of Molecular Biology and Immunology, ICBAS, Porto University, Porto, Portugal
Several rodent models of bladder cancer development have been established. The aim of this review article is to provide a critical
assessment of different animal models available for the study of bladder carcinogenesis, its chemoprevention and therapy. All, except
for transgenic and knockout animals, require 8–12 months experimental periods in order to generate a high yield of neoplasias.
Spontaneous bladder tumor models are extremely rare. The significance of the results from animal experiments is dependent upon the
selection of a suitable animal model. There are no rules regarding the choice of a model, it is however very useful to have knowledge
of relevant comparative medical aspects concerning this subject. We describe chemical carcinogens most commonly used to induce
bladder cancer, pellet implantation and urinary calculi, agents that promote bladder cancer, and irradiation. We also evaluated
other tools such as cell cultures, tumor implantation and transgenic models for bladder cancer, that have been developed to study
the process. The review considers how several imaging techniques can be applied to study rodent bladder carcinogenesis.
Key Words: bladder cancer, mice, rat, rodent model.
Received: February 14, 2006.
*Correspondence author: E-mail: pamo@utad.pt
Abbreviations used: AAF — 2-acetylaminofluorene; BBN – N-bu-
tyl-N- (4-hydroxybutyl) nitrosamine; BCPN – N-butyl-N-(3 car-
boxybutyl) nitrosamine; BW — body weight; CIS — carcinoma
in situ; DBN — dibutylnitrosamine; EHBN — N-Ethyl-N-(4- hydro
xybutyl)nitrosamine; ENS — 4-ethylsulfonylnaphthalene-1-sulfo-
namide; FANFT — N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide;
MNU — N-Methyl-N-nitrosourea; SCC — squamous cell carcinoma;
TCC — transitional cell carcinoma; X — times; W — weeks; ♀ — fe-
male; ♂ — male.
Exp Oncol �006
�8, 1, �–11
review
Experimental Oncology �8, �–11, �006 (March) 3
ficial, intermediate and basal [�1, 7�]. Since the entire
urinary tract is lined with urothelium, theoretically the
urethra and ureters could develop similar proliferative
lesions. However, induced ureter and urethral prolifera-
tive lesions are rare in the rat. The reason for this may
be explained by the quick passage of urine containing
toxicants, compared to the more lengthy contact taking
place in the urinary bladder [30].
Fig. 2. Potential applications of bladder samples
sponTaneous Tumors
The incidence of spontaneous tumors in rodents
plays an important practical role in the design and in-
terpretation of carcinogenicity bioassays. The effect of
a weak carcinogen may be more readily detected if it is
not obscured by a background incidence of spontaneous
bladder tumors, so the choice of test strain is important
[51]. However, most naturally occurring strains of rodent
do not develop spontaneous bladder cancer, more than
99% are predominantly associated with advancing age
[11, 16, 18]. An exceptionally high incidence of urothelial
and ureteric neoplasms have been reported in two rat
strains, Brown/Norway (BN/RijHsd) and Dark Agouti
(DA/OlaHsd), which were associated with the presence
of calculi [10, 79]. Lesions identified in those animals
were classified as papillary and multifocal, with simulta-
neous carcinoma in situ (CIS) [�4, 77]. Other spontane-
ous non-neoplasic changes such as inflammation and
epithelial hyperplasia occur infrequently in the bladder.
Some spontaneous bladder tumors in rats can generally
be explained by infection by the bladder parasite Tricho-
somoides crassicauda [19]. The complications induced
by this parasite in experiments on rat bladder carcinogen-
esis are uncommon, since noninfected rats maintained
under optimal conditions do not become infected [14].
There are no known reports of parasitic infection of the
mouse bladder. Spontaneous bladder tumors in other
rodent species including hamsters, guinea pigs, and
rabbits appear equally as rarely as in mice [18].
requiremenTs For appropriaTe
animal models oF urinary bladder
carcinogenesis
Appropriate and valid animal models for urinary
bladder carcinogenesis must be similar to human blad-
der cancer in their histology, biochemical properties,
molecular and genetic characteristics, natural history,
and biological behaviour [96]. A simple and natural
method for the administration of the carcinogen is re-
quired; the carcinogen ought to be not toxic and should
affect only the urothelium. The incidence of tumors
should be reliable and high and the tumor induction
time should be relatively short [69]. The investigator
should be able to monitor the experiment or be able
to predict results. Some of the requirements to animal
models are listed in Table 1.
Table 1. Requirements for appropriate animal models to study
experimental bladder carcinogenesis
Histology The tumor should be of urothelial origin, with different
stages of disease progression. We should identify the flat
urothelial lesions and the papillary tumor growth
Biologic
behaviour
The tumor should grow intravesical, so that the tumor can
be directly exposed to intravesical anticancer drugs in its
natural environment
Natural
history
Mimic pathogenesis of human urinary bladder cancer, with
identifiable precursor lesions
Genetic
alterations
Stable molecular and genetic alterations similar to those
found in human bladder cancer
Feasibility Simple induction procedure, predictable and adjustable
incidence
There are several advantages to using rodents in
medical research, their physiology and genetics are
well understood, they are relatively easy and cheap to
maintain, and like humans, they are mammals. Rodents
develop cancer relatively easily in response to chemical
carcinogens. Their tumors also develop rather quickly,
over months rather than years [69]. A major difference
may be a lower rate of distant metastases in rats than in
humans, although this is not certain [90]. Metastases and
invasions of bladder cancer are more common in mice
than in rats. The lack of metastasis in experimental ani-
mals may be due to the time factor. Animals are killed at
the end of experimental period, or even earlier, to prevent
suffering and for optimal tissue collection, which may be
too soon for metastases development [11]. Concerning
the selection of animals, we may choose between inbred
or outbred strains of rodents. An important benefit of the
use of inbred animals is that many can be supplied which
develops virtually identical tumors at the same stage of
growth. However, an important concern is the degree to
which tumor heterogeneity may be lost [77]. Since the
aim of laboratory animal experiments is to elucidate the
pathological process in humans as well as to test chemi-
cals for carcinogenic potential, classification of animal
tumors should agree with the nomenclature used for
human neoplasms [�1]. However, the differences in their
histological appearance, growth pattern and biological
behaviour need to be emphasized [7�].
induced Tumors
Yamagiwa and Ichikawa in 1918 where the first to
proof that cancer could be induced in experimental
animals by chemical means [98]. The induction of
bladder cancer in dogs by �-naphtylamine, reported
by Hueper in 1938, established the experimental basis
of bladder carcinogenesis [40]. Early attempts to in-
duce tumors in mice bladders by means of chemicals
were unsuccessful until Armstrong and Bronser (1944)
4 Experimental Oncology �8, �–11, �006 (March)
induced papillomas and carcinomas through the oral
administration of �-acetylaminoflourene (AAF) in CBA
strain mice [4].
In the 1960s and early 1970s, organospecific
chemically-defined bladder carcinogens were discov-
ered for rodents. These chemicals and their application
provided the readily-available reproducible models
necessary for detailed studies of the biochemical,
pathobiological and immunological mechanisms in-
volved in the pathogenesis of bladder cancer [18].
There are several methods of inducing urothelial
tumors: oral administration of a chemical carcinogen
in diet, water or gastric intubation (gavage), by injec-
tion or intravesical instillation; the application of the
stimulus directly by implanting in the bladder a pellet
either by itself or with a chemical incorporated in it;
and exposure to X-rays.
Chemical carcinogens. As mentioned before,
the first compound applied in experimental bladder
carcinogenesis was AAF. Although of considerable
usefulness in experimental bladder carcinogenesis
research, the AAF is a pluripotential carcinogen, in-
ducing tumors of several tissues in addition to the
urothelium particularly the liver, pancreas, breast, skin,
forestomach and ear duct [4, 18, �1]. Hepatic tumors
may cause early death of the animals and confusion in
the interpretation of the experimental results [79].
Over the past few decades, research efforts have
focused on the development of rodent models that
permit the reproducible induction of bladder cancer
with minimal or no induction of tumors in other organs.
Three chemicals have been proved to be particularly
effective, in that, when administered via the appropri-
ate route, at the appropriate dose and in the appro-
priate strain of animal, all produce 100% incidence of
bladder tumors; these chemicals are N-[4-(5-nitro-�-
furyl)-�-thiazolyl] formamide (FANFT), N-butyl-N-(4-
hydroxybutyl) nitrosamine (BBN) and N-Methyl-N-ni-
trosurea (MNU) [30]. These compounds are complete
carcinogens, the total dose has a greater effect when
administered as several fractions, i.e. the effect of the
fractions is synergistic rather than additive. The grade
of cellular atypia and the extent of invasion increase as
the dose of carcinogen increases as well as when the
experimental period is extended [31, 51].
The nitrofuran FANFT is highly specific to the uri-
nary bladder in the rat, mouse, hamster and dog. It is
a genotoxic compound and can act as an initiator or
as a promoter. It is metabolically activated into reactive
electrophiles, produces DNA adducts and ultimately
produces mutation [6, �1, 51]. FANFT is deformylated
in �-amino-4-(5-nitro-�-furyl) thiazole by liver and
kidney enzymes before excretion [89]. Tumors induced
by this compound are predominantly transitional cell
carcinoma (TCC), with a large proportion exhibiting
squamous cell differentiation. However, hyperplasia,
dysplasia and CIS have also been observed [6, 51,
77]. FANFT is incorporated in the diet and induction
of bladder cancer requires 8 to 11 months [6, �9].
Transplantable cell lines have been developed from
these FANFT-induced tumors, the most commonly
used are labelled MBT-� and AY-�7 [96]. However, the
use of this compound presents safety concerns for the
researchers involved and the environment.
BBN is one of the most suitable urinary bladder car-
cinogens for animal models, since its carcinogenic po-
tential is essentially limited to this organ and is probably
the most commonly-referenced experimental bladder
carcinogen [55, 70]. Bladder tumors induced by BBN in
rats and mice resemble their human counterparts [55].
BBN is a metabolite of the symmetric dibutylnitrosamine
(DBN) [46]. In rats, both were demonstrated to be uri-
nary bladder carcinogens, with BBN being specific to
the urinary bladder, because DBN also induced tumors
of the liver, lung, kidney and oesophagus [17, 39]. In
mice, similar aspects have been found [9, �1]. The dif-
ference in lesions histopathology identified between
different rodents exposed to BBN can be attributed
to dosage, lag time until tumor development, species,
and strain. BBN is bladder specific not only in rats but
also in mice and dogs. However, it is a weak bladder
carcinogen in hamsters and pigs [51, 69]. A 100% in-
cidence of tumors can be induced by continuous and
prolonged administration of BBN in drinking water. This
compound can also be administered by oral gavage
[51]. Subcutaneous injections generally result in a lower
incidence of tumors [�1]. BBN can also be introduced
into the bladder by intravesical instillation [95]. BBN is
a yellow oily liquid not very volatile but is soluble due
to terminal hydroxylation. When dissolved in water
BBN is easy to apply. However, it is photosensitive and
therefore an opaque feeding-bottle is necessary [55].
BBN is a genotoxic compound. The alcoholic group
of administered BBN is rapidly oxidized to a carboxyl
group by the liver enzymatic system alcohol/aldehyde
dehydrogenase; the metabolite formed by N-butyl-N-
(3-carboxybutyl) nitrosamine (BCPN) is also a bladder
carcinogen and comes in contact with the urothelium
via the urine. BCPN is a stable compound that binds
covalently to cellular macromolecules and is ultimately
responsible for the initiation of the carcinogenic process
[1]. BCPN produces neoplasic transformation of rat uro-
thelial cells cultured in vitro [9]. In dogs, the dosage of
BBN is related to the grade of bladder cancer [�]. In low
dosages, BBN causes low-grade papillary carcinoma
after long periods of exposure [77].
N-Ethyl-N-(4-hydroxybutyl) nitrosamine (EHBN) is
a genotoxic compound even more potent than BBN.
It targets the urothelium of mice, rats and dogs [93].
Hamsters are however less sensitive to this carcinogen
and guinea pigs appear to be resistant [�1, 39]. EHBN
is metabolized by hepatic enzymes in N-ethyl-N-(3-
carboxypropyl) nitrosamine and excreted in urine.
In mice this compound increased the incidence of
squamous cell carcinoma and is a good model for
the investigation of nonpapillary invasive bladder
carcinoma. The carcinomas that develop demonstrate
invasion of the surrounding tissues and are associated
with hematogenous metastic spread to distant organs
[93]. Other nitroso compounds such as N, N-dibutil-
Experimental Oncology �8, �–11, �006 (March) 5
nitrosamine and N-methyl-N-dodecylnitrosamine are
carcinogenic to the urinary bladder in rats [51].
MNU is the only carcinogen known to act directly on
the urothelium following spontaneous pH-dependent
decomposition without requiring metabolic activation.
At present MNU is the only urothelial carcinogen known
to produce bladder cancer at a single dose [51]. MNU
is a fine yellowish crystalline powder stabilized by addi-
tion of 5% acetic acid [5�]. Because MNU is intrinsically
unstable, variations in carcinogenic potency can arise
unless care is taken during its storage, preparation and
use [51]. It is a genotoxic compound that can act as an ini-
tiator or as a promoter and cause persistent methylation
of the DNA [53, 57]. The MNU model of bladder cancer
has particular advantages for the experimental analysis
of complete carcinogenesis, since the carcinogen can
be administered directly in quantifiable pulse doses, via
intravesical instillation [76]. The disadvantage of this
procedure is that in some animals bladder concretions
and/or urocystitis may develop [53, 86]. Bladders treated
with intravesical MNU develop progressive neoplastic
changes, and the tumors become progressively less
differentiated with time. These lesions progress from
hyperplasia, atypia, CIS, and papillary carcinoma to
large bulky muscle invasive tumors that completely fill the
bladder lumen, obstruct the ureteres and kill the animal
[53, 91]. These tumors have a relatively low potential
for metastasis with deposits being found in abdominal
lymph nodes and within the abdominal cavity [7]. MNU
also causes local massive inflammation of the bladder
mucosa and submucosa and infiltration of the bladder
wall with acute inflammatory cells and exudates [4�].
4-Ethylsulfonylnaphthalene-1-sulfonamide (ENS)
is a carbonic anhydrase inhibitor that produces alka-
line, hypoosmolar urine with crystalluria and calculi
formation. Urinary tract hyperplasia occurred in mice
fed ENS at a dose of 0.1% of their diet [�1].
Benzidine, 3, 3´-dichlorobenzidine, �-naphthyl-
amine, 4-aminobiphenyl, �-acetylaminofluorene,
phenacetin, and sodium ο-phenylphenate are ad-
ditional compounds which are carcinogenic for the
urinary bladder [86].
Bracken fern (Pteridium aquilinum) induces upper ali-
mentary tract and bladder cancer in a number of species
including rats, guinea pigs and cows [�1, 81]. The major
carcinogenic compound of bracken fern is ptaquiloside,
a non-sesquiterpene that alkylates DNA when metabo-
lized into an unstable dienone under alkaline conditions
[87]. Ptaquiloside accumulates in the body of rats arti-
ficially fed bracken fern and is eliminated in urine where
its carcinogenic activity is preserved [75].
In Table �, we show the results of several studies
with different carcinogens, treatments, animals used
and lesions identified.
Using bladder cancer models, several compounds
have been evaluated as suitable for intravesical thera-
py (chemotherapy and immunotherapy) and systemic
chemotherapy [64, 66, 88, 91]. More recently photo-
dynamic therapy with hypericin and protoporphyrin IX
was also investigated [97].
Pellet implantation and urinary calculi. The pres-
ence of foreign bodies within the lumen of the bladder
can cause irritation or trauma to the urothelium, both
stimulate mitotic activity, thereby causing nodular and
papillary hyperplasia [�4, �7]. Mitotic activity predis-
poses cells to the action of both initiating and promoting
substances [�4]. If there is extensive ulceration of the
bladder surface, diffuse papillomatosis is produced
[�0]. Although direct implantation of pellets contain-
ing suspected carcinogens was attempted in 19�4, it
wasn’t until 1951 when Jull successfully induced blad-
der tumors by this method [47]. This technique involves
surgical implantation of pellets into the lumen of the
rodent bladder. The materials used as the basis for the
pellets were paraffin or cholesterol and it was thought
that these materials remained biologically inert in the
bladder lumen. Various chemicals were incorporated
into these pellets for implantation into the bladder lu-
men. It was assumed that the bladder epithelium was
incapable of metabolizing these chemicals, so that the
introduced chemical represented the ultimate form of
the carcinogen [15]. It was also assumed that the pellet
itself did not pose a carcinogenic risk to the urothelium
[47]. In 1979 Jull demonstrated in an experiment that
the pellets inserted into the mouse bladder itself repre-
sented a carcinogenic risk [48]. In addition, it became
apparent that the urothelium is metabolically active,
through evidence of the activities of enzymes involved
in the metabolism of exogenous and endogenous
chemicals [�3]. Urine was required as a co-factor for the
carcinogenic effect of the pellet [13]. The rapidity with
which the chemical was leached from the pellet varied
considerably, depending on the solubility. For highly
water-soluble compounds such as saccharin and cycla-
mate, the chemical was rapidly leached from the pellet
[1�]. In addition, the surgical procedure, to implant the
pellet, produced nodular and papillary hyperplasia [3�].
This method has since been abandoned, because these
problems made the interpretation of the results utilizing
the pellet implantation technique difficult [�0].
During the past three decades, numerous chemicals
have been identified as producing bladder tumors in ro-
dents and are associated with the appearance of urinary
calculi [16]. Such chemicals include uric acid, calcium
oxalate, uracil, thymine, melamine, and others [34, 85]. Of
these compounds, uracil is the most widely applied [67]. In
the rat urinary bladder, uracil is reported to induce calculi
and papillomatosis when administered in the diet [56, 84].
The proliferative effects of calculi are commonly sustained
in rodents since these species are normally horizontally
positioned allowing the object to remain within the lumen
of the urinary bladder, with less chance of elimination [�4].
If the calculus is removed before a neoplasm is produced,
the proliferative changes are rapidly reversed. Urinary tract
calculi represent foreign bodies, similar to pellets, but do
not require surgical implantation into the bladder lumen
[16, �0, �3]. The calculi can arise from the administered
chemical itself, from one of its metabolites or from an
endogenous metabolic product that is caused by the ad-
ministration of the chemical. In general, calculi form more
6 Experimental Oncology �8, �–11, �006 (March)
quickly in rats than in mice. Calculi-induced proliferation
appears to be considerably greater in rats than in mice.
This difference is particularly due to a primary papilloma-
tous reaction in rats, whereas in mice it is predominantly a
nodular response [�3]. There is also a difference between
sexes; male rodents are more frequently affected than
females [�4]. The process involved in urothelial carcino-
genesis related to calculus formation may be influenced
by factors such as pH, volume, osmolality, cationic and
anionic concentration and quantitative and qualitative
differences in the presence of urinary protein [16].
Calculi can be surgically produced in rats by per-
forming a portacaval shunt, which leads to significant
alteration of uric acid metabolism and eventual pro-
duction of uric acid calculi, ulceration, regenerative
proliferation, and carcinoma [�6, 45].
Promoting agents. Multistage models of carci-
nogenesis proposed to explain the patterns of tumor
development observed in the urinary bladder involve the
initiation of neoplastic change in a few cells by a threshold
dose of carcinogen followed by conversion of these latent
tumor cells into an autonomous cancer by further doses
of the same and/other carcinogens, and/or promoting
agents. In the urinary bladder of mice and rats, neoplastic
change can be initiated by a few weeks treatment with
low doses of chemical carcinogens above described.
Animals exposed subsequently to promoter compounds
will develop bladder cancer [33, 73].
Urinary bladder promoters can be classified into
at least seven categories as follows: 1) sodium and
potassium salts associated with increased concen-
tration of urinary levels of sodium and potassium ions
and alkaline urine; �) urolithiasis inducing agents;
3) antioxidants; 4) anticancer drugs; 5) amino acids;
6) drugs and others [31, 43]. Table 3 shows the clas-
sification of bladder cancer promoters.
Irradiation. Treatments with ionizing radiation
have been shown to cause bladder cancer in humans.
Irradiation with X-rays is capable of inducing preneo-
plastic urothelial hyperplasia and development of
urothelial carcinoma in rodents. The rate of bladder
tumors achieved with irradiation depends on the dos-
age time of exposure and time of follow up. A single
dose of irradiation generated from a linear accelerator
induced hyperplasia of the urothelium when mice were
examined 3–19 months later [50, 9�].
Table 3. Classification of urinary bladder cancer promoters
Type Cancer promotor
Sodium or potassium
salts
Sodium saccharin
Sodium cyclamate
Sodium L-ascorbate
Sodium citrate
Sodium erithorbate
Sodium phenobarbital
Sodium barbital
Sodium chloride
Potassium carbonate with or without ascorbic acid
Urolithiasis-inducing
chemicals
Uracil
Diphenyl
Antioxidants Butylated hydroxyanisole
Butylated hydroxytoluene
Ethoxyquin
t-Butylhydroxyquinone
2-t-Butyl-4-methylphenol
Anticancer drugs Adriamycin
Mitomycin C
Amino acids DL-Tryptophan
L-Leucine
L-Isoleucine
Drugs Phenacetin
Others Components of urine
Allopurinol
Dimethylarsinic acid
Table 2. Chemically induced bladder tumors
Agent Treatment Host Tumor type
Bracken
Fern
Dietary [75] Albino rats (♂ and ♀) Hyperplasia, TCC, invasion.
BBN 0.05% in water, 40 w (weeks) [61] ACI rats ♂ (6w) 1 w no lesions, 2 w hyperplasia, 4 and 12 w papilloma, 20 w TCC
0.05%; 0.01% and 0.005% in water,
4, 8, 12, 16, 20, 26 w [68]
C3H/He ♀ mice (5w) 0.05% BBN 4 w and 8 w simple hyperplasia and dysplasia; 12, 16, 20 and 26 w
simple hyperplasia, dysplasia, CIS, invasive carcinoma.
0.01%BBN 4 w simple hyperplasia; 8, 12, 16 w simple hyperplasia and dysplasia;
20 and 26w simple hyperplasia, dysplasia, CIS and invasive carcinoma.
0.005% BBN 4 w no lesions; 8 and 12 w simple hyperplasia, 16 and 20 w simple
hyperplasia and dysplasia; 26 w simple hyperplasia, dysplasia, CIS and invasive
carcinoma.
50 or 100 mg gastric intubations,
2 x w, 9 w [6]
B6D2F1, mice ♂ TCC, SCC, Adenocarcinoma
0.05% water, 20 w [32] Wistar rats ♂ Hyperplasia, TCC, papillary tumors
0.05% water (16 w and 32 w) [60] Fisher 344 rats ♂
(6 w)
16 w and 32 w TCC
Gavage (3 fractionated doses at 24h
intervals, BBN dissolved in 1:2 pro-
pandiol 300 mg/Kg BW) [54]
Wistar rats ♀ 19 months later: Papillomas and invasive TCC
EHBN 0.025% water (4 w, 12 w, 20 w,
28 w, 36 w) [93].
B6C3F1 mice ♂ (6 w) 4w-dysplasia; 12w, 20w, 28w and 36w-dysplasia, papiloma, TCC, SCC, adenocar-
cinoma, CIS; metastasis
0.05% water 32 w [44] B6C3F1 mice ♂ (6 w) TCC (invasive), SCC
FANFT Dietary: 0.1% (45 w) [65] C3H/He mice ♀ (6 w) Dysplasia, CIS and TCC
Dietary 0.02% [22] Fisher 344 rats ♂
(4 w)
Hyperplasia (6 w)
Nodular hyperplasia (8 w)
Papillary tumours (10,12,14 w)
Dietary: 0.1% (38w) [63] C3H/He mice ♀ (6 w) Dysplasia, CIS and TCC
MNU Intravesical 5 mg/Kg BW [52] Wistar rats ♀ 15 months latter exophytic papiloma and invasive TCC
1.5 mg every 2 weeks (x 4), intra-
vesical [91]
Fisher 344 rats ♀ Hyperplasia, papillary lesions, squamous metaplasia
0.15 ml intravesical, 4 doses
over 6 weeks [76]
Fisher 344 rats ♀ 10 weeks after last treatment TCC
Notes: w — weeks; x — times; TCC — transitional cell carcinoma; SCC — squamous cell carcinoma; CIS — carcinoma in situ, BW — body weight; ♂ — male; ♀ — female
Experimental Oncology �8, �–11, �006 (March) 7
cell culTure
Cell culture represents an excellent opportunity to
study bladder cancer in vitro. The growth of normal and
neoplasic urothelial cells, obtained from rodents, or
man, in tissue culture, offers the possibility of studying
“pure” populations of cells without necessarily having
significant contamination by other tissues [77]. Differ-
ent methods for the culture of urothelial cells in vitro
are described: the propagation of primary cultures
and cell lines derived from normal urothelial cells ex-
posed to chemical carcinogens and continuous cell
lines derived from bladder tumors [59]. Such cultures
provide model systems for studying the factors that
control the growth and differentiation of both normal
and neoplastic urothelial cells, and for defining the
morphological, biochemical and genetic changes
associated with the development and progression of
bladder cancer [58, 59]. Furthermore, they provide the
opportunity to observe the evolution of cancer within
a population of cells exposed to a carcinogen and to
study the effectiveness of anticancer drugs [58, 77,
8�]. Cell cultures also offer the opportunity to study the
chromosomal changes associated with early develop-
ment of bladder cancer.
Tumor implanTaTion
As we described before, bladder tumors can be
induced in rodents using a variety of chemicals. How-
ever, these tumors required 8–1� months to develop,
they grew very slowly and many animals are required
[43, 6�, 96]. While induction of bladder tumors in
animals by chemical means has allowed the study of
carcinogenesis and, in some cases, chemotherapeutic
agents, the majority of research leading towards clini-
cal applications requires models based upon human
cancer cells [41].
Currently there are two more fundamental murine
bladder tumor models: the xenograft model (trans-
plantation of human urothelial cell carcinoma into
immunodeficient mice) [41, 80], and the syngeneic
model (transplantation of carcinogen-induced blad-
der cancer in syngeneic, i.e. immunocompetent mice)
[78, 88]. Syngeneic tumor cells can be implanted
subcutaneously (heterotopic) [4�] or intravesically
(orthotopic) [37].
For reasons of easy access, heterotopic models
(subcutaneous, intravenous and intraperitoneal)
have been preferred for human tumor implantation.
However, it is questionable if the tumor development
at these sites parallels the development of the tumor
in the organ of its origin [4�].
Orthotopic tumor implantation is more difficult.
However, the refinement of the various methods of
orthotopic implantation now allows this system to
be used routinely for a variety of applications. This
model of bladder cancer allows the growth and
spread of bladder cancer to be better observed in a
normal physiologic environment and permits novel
approaches to cancer therapy or imaging of tumor
growth. The tumor cells used for intravesical instillation
may be obtained from urothelial cell cultures exposed
to chemical carcinogens (MBT-� and AY-�7) or human
bladder neoplasic cells may be used (MGH-U3) [96].
Cells can be introduced into the bladder cavity after
chemical urothelial denudation (leading to multifocal
lesions) or after mechanical urothelial lesion (requir-
ing cystotomy) [8, 4�]. The first method is less time-
consuming, requires no surgery and does not bring
up diffuse ulceration, oedema or urinary stones. Its
tumor implantation success rate ranges from �8 to
97%. The tumors are multifocal and their localization
is unpredictable [8]. Transplantable animal models
are not technically complicated, are well tolerated by
the animals and result in minimal morbidity associated
with occasional haematuria subsequent to tumor cell
instillation [4�, 96]. More importantly is that these
tumors are produced intravesically and arise from the
urothelium. Consequently, the tumor can be directly
exposed to intravesical anticancer drugs in its natural
environment [49]. However, this model is not perfect.
Its one disadvantage is that the tumors prove to be
invasive carcinomas from the start; this is related to
the mechanical urothelial abrasion [8].
Transgenic models For bladder
cancer
Genetically engineered animals, usually trans-
genic or gene targeted mice, allow the examination
of whether genetic changes, including oncogene over
expression/mutation or tumor suppressor gene loss,
can increase the risk of neoplasic progression; whether
genetic changes can cooperate during bladder car-
cinogenesis; and how the genetic signature of a neo-
plasm correlates with particular biological aspects of
tumor development [36]. Transgenic mice carrying the
human C-Ha-ras proto-oncogene, v-Ha-ras transgenic
mice, pim-1 transgenic mice, and several knockout
strains of mice deficient in tumor suppressor genes
such as p53 have been shown to exhibit increased
carcinogen susceptibility [35]. p53 knockout mice are
much more sensitive to BBN urinary bladder carcino-
genesis than the parental mice strain C57Bl/6 [74, 99].
For studies of chemical carcinogenesis rats are gen-
erally more frequently used than mice. However, only
limited types of transgenic rats have been developed
for studying carcinogenesis. The establishment of a
rat model with similarities may be the ideal solution. A
transgenic rat line carrying three copies of the human
c-Ha-ras proto-oncogene was developed with its own
original promotor region. This rat is highly susceptible
to BBN carcinogenesis and may be utilized as a rat
model for analysis of bladder tumor development [71].
Tsuda et al. [95] concentrated their attention on the
generation of transgenic rats with the same human
c-Ha-ras proto-oncogene used for the establishment
of transgenic mice. The rat line developed by this team
called Hras1�8 is highly susceptible to BBN bladder
carcinogenesis [94]. Table 4 shows the utility of each
model in function of the subject to be studied [77].
8 Experimental Oncology �8, �–11, �006 (March)
Table 4. Utility of different models to study bladder cancer
Subject for study
Tumors
chemically
induced
Cell cul-
tures
Tumor im-
plantation
Transgenic
models
Conventional histology yes no yes yes
Ultraestructure yes yes yes yes
Immunohistochemical yes yes yes yes
Histochemical yes no yes yes
Molecular genetics and
proteomics
yes yes yes yes
Image cytometry yes yes yes yes
Image analysis yes yes yes yes
Treatment yes? yes? yes yes?
Kinetics yes yes? yes yes
Carcinogenesis yes yes no yes
Note: ? — not fully validated.
evaluaTion oF neoplasic
developmenT in rodenTs
Urinary cytology has been useful in the diagnosis
and intermittent observation of patients with urinary
bladder carcinoma. Surprisingly, urinary cytology has
received little study in rodents. Confirmation of tumor
development and therapeutic effects in rodents is made
possible by the detection of a palpable suprapubic
mass, gross haematuria, weight loss or when lapa-
rotomy or cystectomy is performed [8]. However, these
techniques do not have reliable parameters which en-
able the tumor growth to be assessed. Only large tumors
(> �00 mg) are clearly detected by abdominal palpation
[38]. To solve this limitation non-invasive diagnosis of
superficial bladder tumors in animals can be applied.
It is possible to detect and follow the development of
induced bladder tumors using ultrasonography, a non-
invasive imaging technique that accurately estimates
tumors’ morphologic characteristics [3, 8]. Magnetic
resonance has also been reported for this purpose [96].
However, due to the spatial resolution of magnetic reso-
nance imaging accurate diagnosis of small early lesions
could not be obtained. The use of the non invasive ultra
thin endoscope, developed by Asanuma et al. [5] allows
the detection of bladder tumors with a minimal lesion of
1 mm. Cytoscopic examination is a reliable noninvasive
method for detecting and monitoring superficial tumors
[5]. All methods described above proved to be suitable
means of monitoring intravesical growth, but each ima-
ging procedure required anaesthesia and catheteriza-
tion. Almost �0% of the animals died prior to the end of
the study due to procedural mishaps [3].
conclusion
We have reviewed experimental data related to the
induction of bladder cancer in rodents. Our intention is
not to propose any specific model for researching blad-
der cancer but to give enough information to develop a
framework in each instance to encourage questioning
of the process. Multiple experimental approaches have
been employed to study bladder cancer, including the
use of rodents. Rodents have the advantage that they
can be manipulated to address specific hypotheses
concerning the mechanisms underlying this disease.
However, it should not be forgotten that the direct study
of human bladder cancer continues to be vitally impor-
tant in understanding the biology of bladder cancer.
In the human bladder, it is likely that the urothelium is
chronically exposed to sub-cytotoxic doses of genotox-
ins and, consequently, acute exposure may not be rep-
resentative of the process in man. Each rodent model
to study bladder carcinogenesis has strong and weak
points. The one that is most suitable for the purpose of
each study must be selected. The rodent urinary blad-
der is very similar to the human urinary bladder, which
helps to understand bladder carcinogenesis. Like in
humans, the spectrum of lesions observed in urinary
bladder in mice and rats include preneoplastic lesions,
papillary tumors and invasive carcinoma. The selection
between mice and rats may be based on the aims of the
study. For instance, the small bladder size of mice limits
both histologic characterization and the application of
intravesical therapeutics.
Chemical induction of bladder cancer in rodents
usually requires 8–1� months. However, the adminis-
tration of chemicals in water or diet although effective
present inherent risks to the safety of laboratory per-
sonnel. Moreover, it is difficult to quantify the amount
of carcinogen ingested by each animal. This disadvan-
tage of carcinogen dosing may be eliminated by the
administration of the carcinogen via gavage.
Transgenic rodent models of bladder cancer also
have both advantages and disadvantages. Since all
feature a specific transgene and the latent period of
cancer development is relatively short, they should
provide useful tools for analysing the fundamental
biology and roles of specific genes in tumor develop-
ment and progression.
By monitoring the responses to chemical carcino-
gens using experimental models, it has been possible
to identify many of the mechanisms through which
tumors developed. Animal tumors also provide an op-
portunity to study the chromosomal changes associ-
ated with the early development of bladder cancer. In
addition, such studies may establish whether different
carcinogens are associated with specific cytogenetic
abnormalities, hence defining the specific gene(s)
involved in the development of bladder cancer. Finally,
with the progress in the development of the rat and
mouse genome programs, without difficult ethical
problems and with much shorter generations, as yet
undescribed genes related to cancer development in
human cases should be successfully detected.
acknowledgemenTs
This study was supported by a Grant-in-Aid from
the Fundação para a Ciência e Tecnologia, Ministério
da Ciência e Ensino Superior, Portugal, with the num-
ber 1�453/�003.
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Экспериментальные опухоли мочевого пузыря:
модели на грызунах
Для изучения механизмов развития рака мочевого пузыря было создано несколько экспериментальных моделей на гры-
зунах. Целью обзора была сравнительная оценка различных экспериментальных моделей для изучения канцерогенеза
мочевого пузыря, профилактики и терапии. За исключением трансгенных и нокаутных животных, для получения высокого
выхода опухолей в любой экспериментальной модели требуется 8–12 мес. Модели спонтанного канцерогенеза мочевого
пузыря крайне редки. Выбор экспериментальной модели с определенными параметрами определяет значимость получен-
ных результатов. В статье описаны различные методики, используемые для индукции рака мочевого пузыря in vivo, ряд
методических подходов, таких как культура клеток, имплантация опухоли и трансгенные модели рака мочевого пузыря
и современные методы мониторинга опухолевой прогрессии.
Ключевые слова: рак мочевого пузыря, экспериментальные модели, грызуны.
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