Yonago Acta medica 2000;43:121–130

Effects of Testosterone on Cell Proliferation and Apoptosis in BBNInduced Mouse Urinary Bladder Carcinogenesis
Hirofumi Ohno
Department of Urology, Faculty of Medicine, Tottori University, Yonago 683-0826 Japan
Human bladder cancer is nearly 3 times more common in men than in women. In
general, the sex difference in incidence of human bladder carcinoma is considered to be
due to industrial and environmental carcinogens or other factors, though there is noclear evidence supporting this. We suspected that the sex difference in incidence of
bladder carcinoma might be due to the effects of testosterone. We investigated theeffects of testosterone on mouse bladder carcinogenesis by using immunohistochemical
staining and terminal-deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nickend labeling (TUNEL). A total of 94 four-week-old male mice were divided into 3
groups. In Group I, castration was carried out, then 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was administered until the end of the experiment. In GroupII, 0.05% BBN was administered similarly, without castration. In Group III, 0.05%
BBN was administered similarly, without castration, and 3 mg/kg of testosterone wasinjected intramuscularly weekly from the beginning of the experiment. All mice werecystectomized at the end of the experimental period to investigate the incidence of bladdercancer, and immunohistochemical staining and TUNEL were performed to examine
the correlation between cell proliferation and apoptosis. Among the 3 groups, occurrenceof bladder tumor was most frequent in Group III, and tumor induction time was theshortest. The proliferation index for tumors significantly increased as the stage andgrade progressed. On the other hand, the apoptotic index for tumors significantly
decreased. The proliferation index was the highest in Group III and the lowest in GroupI. A significant difference in the proliferation index was observed among the 3 groups.
No significant difference was observed in the apoptotic index among the 3 groups. Our
results indicate that the effect of testosterone on mouse bladder carcinogenesis is moresignificantly related to cell proliferation than to suppression of apoptosis, with the resultthat testosterone promotes the occurrence of BBN-induced mouse bladder carcinomas.
Key words: apoptosis; BBN-induced mouse urinary bladder carcinogenesis; cell proliferation;
testosterone

The fact that the incidence of human urinary
bladder carcinoma is higher in males than infemales has often been reported in the literature.
It has been concretely established that the maleto-female ratio among patients is about 3:1(Laor et al., 1984; Yamada et al., 1986). However, there is no clear evidence why genderresults in different incidences of urinary bladder tumors. In general, the cause of sex differBBN, a carcinogen of urinary bladder cancer inanimal models

ence in incidence of human bladder carcinogenesis is believed to be from industrial and environmental factors. Although over the past 30
years, women have joined the male-predominant
workplace, changed their habits and been exposed to both industrial and environmental carcinogens, new cases of human bladder cancer inAbbreviations: BBN, N-butyl-N-(4-hydroxybutyl)
nitrosamine; TdT, terminal-deoxynucleotidyl-transferase; TUNEL, TdT-mediated dUTP-biotin nickend labeling; PCNA, proliferating cell nuclear antigen

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H. Ohno

United States still show no18th wk
28th–33rd wk
Castration
statistical change, and bladderGroupcancer still remains about 3
0.05% BBN
I
times more common in men
18th wk
23rd–27th wk
than in women (Messing and
Catalona, 1998). We considered that sex difference in
0.05% BBN
II
bladder carcinogenesis might
18th wk 21st–24th wk
be related to sex hormones,
Cystectomized inand specifically to testosterExperiment I
0.05% BBN + testosteroneIIIone, and decided to investiCystectomized ingate the influence of testosterExperiment II
one on bladder carcinogenesis.
Fig. 1. Experimental protocol. In Group I, castration is carried out
One of the most importantand 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) isadministered. In Group II, 0.05% BBN is similarly administered. In
molecular mechanisms in theGroup III, 0.05% BBN is administered and 3 mg/kg of testosterone isprocess of carcinogenesis is
injected intramuscularly weekly. Mice are cystectomized at the end
apoptosis. Apoptosis is aof each experimental period. wk, weeks.
morphologically and biochemically distinct form ofcell death, and its majormechanism is the maintenance of homeostasis 0.05% BBN was supplied to each group beginand the balance of cell proliferation in normal ning at 5 weeks of age.
and malignant cells (Kerr et al., 1972, 1994).
Terminal-deoxynucleotidyl-transferase (TdT)Experiment I
mediated dUTP-biotin nick end labeling(TUNEL) has been successfully used to detect A total of 44 male mice were investigated in
apoptotic cells in routine paraffin-embedded Experiment I. In Group I, castration was carried out under ether anesthesia at 4 weeks, andsections (Gavrieli et al., 1992).
Thus, we investigated whether testosterone from 5 weeks of age, mice were administeredinfluenced mouse bladder carcinogenesis and 0.05% BBN orally for a period of 18 weeks. In
analyzed the relationship between cell prolif- Group II, mice were administered 0.05% BBN
eration and apoptosis by using immunohisto- as described above, without castration, from 5
weeks of age, for a period of 18 weeks. In
chemical staining and TUNEL.
Group III, castration was not performed, andfrom 5 weeks of age, 0.05% BBN was given as
Materials and Methods
described, and 3 mg/kg (body weight) of testosterone propionate (Enarmon-oil, Teikokuzoki,
A total of 94 four-week-old male C57BL/6 mice Tokyo) was injected intramuscularly once
(Shimizu, Kyoto, Japan) were housed 5 to 6 per weekly for a period of 18 weeks. All mice wereplastic cage with hardwood chips for bedding, cystectomized on the 18th week (Fig. 1).
in an air-conditioned room at 23 ± 2˚C and 60 ±10% humidity with a 12-h light-dark cycle.
Experiment II
They were divided at random into 3 groups of
30 to 33 mice each. N-butyl-N-(4-hydroxy- A total of 50 male mice were investigated inbutyl) nitrosamine (BBN) (Tokyo Kasei, Experiment II. Mice were divided into 3
Tokyo, Japan) was dissolved in tap water to groups and housed as described in Experimentconcentrations of 0.05%, and was added to a I. All mice were cystectomized at the end offew drops of Tween 80 (Wako, Osaka, Japan). the different experimental period because we122Effects of testosterone in mouse urinary bladder carcinogenesis

believed that the difference in cystectomized
period results in no significant difference intumor stage and tumor grade among the 3groups. Thus in Group I, mice were cystectomized in groups of 3 to 5 from the 28th to the33rd week, in Group II, from the 23rd to the27th week and in Group III, from the 21st to the24th week (Fig. 1).
In Experiments I and II, all mice were cystectomized under ether anesthesia to investigate
the incidence of bladder cancer. All tissue
specimens were fixed by an injection of about
0.10 to 0.15 mL of 10% formalin into the lumen, and immersed in the same solution. After
fixation, each specimen was cut into 4 sections
and embedded in paraffin, and a series of 3µm thick sections was prepared for hematoxylin and eosin staining, immunohistochemical
staining and TUNEL.

Immunohistochemical staining of proliferating cell nuclear antigen (PCNA)
Immunohistochemical staining was performed
with a Histofine SAB-PO kit (Nichirei, Tokyo).
Paraffin-embedded sections were placed on
silane-coated glass slides and air-dried at room
temperature. Deparaffinized and rehydrated
sections were heated by microwave oven at
92˚C for 10 min, and then cooled for 60 min atroom temperature. Endogenous peroxidase
activity was inhibited by incubation with 3%hydrogen peroxidase in methanol for 20 min.
After blocking nonspecific reactions with 10%
normal rabbit serum, the sections were incubated with a monoclonal antibody against PCNA(DAKO, Glostrup, Denmark) with 1:200 dilution at 4˚C overnight. The sections were thenincubated with biotinylated rabbit anti-mouse
immunoglobulin for 30 min, followed by incubation with streptavidin-peroxidase complex
for 30 min and rinsed with several changes ofphosphate-buffered saline between steps. The
color was developed with diaminobenzidine
solution. Finally, they were lightly counterstained with hematoxylin.

TUNEL
TUNEL was performed by using an Apop Tag
Plus in situ apoptosis detection kit (Intergen,
New York, NY). In brief, paraffin sections
were de-waxed, rehydrated through a gradedalcohol series and washed with distilled water.
Subsequently, tissues were digested with 40µL/mL proteinase K (Wako) at 37˚C for 80
min, because proteinase K is known to enhance
positive nuclear labeling in apoptotic cells.
After prehybridization treatment, the sections
were incubated with TdT and digoxigenindUTP in a moist chamber for 90 min at 37˚C.
Incubation with anti-digoxigenin-antibodyperoxidase for 30 min at room temperature was
employed for detection of digoxigenin-dUTP
labeling. The color was developed with diaminobenzidine solution and counterstainedwith hematoxylin or methyl green.

The proliferation and apoptotic indices
PCNA-positive cells were used to quantify the
proliferation index (percentage of PCNA-positive cells in 1000 cells). Similarly, TUNELpositive cells were used to quantify the apoptotic index (percentage of TUNEL-positive cells
in 1000 cells). In benign lesions and carcinomas in situ, the proliferation and apoptotic indices were measured in the mucosal layers, and ininvasive carcinomas, counted at the infiltrative
layers.

Statistics
The difference for occurrence of bladder tumor
in Experiment I was analyzed with the KruskalWallis rank test. The distribution of histological stages, tumor grades and tumor induction
times in Experiment II was analyzed with theone-way analysis of variance. The relationshipof the proliferation or apoptotic index to thestage and grade was examined with the KruskalWallis rank test. The correlation between the
proliferation index and the apoptotic index was
analyzed by using a Pearson’s correlation coefficient. A level of P < 0.05 was regarded asstatistically significant.

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H. Ohno

Table 1. Occurrence of bladder tumors in Experiment I and invasive tumor induction time inExperiment II

Occurrence of bladder tumor(%)
Invasive tumor induction time (week)† Kruskal-Wallis rank test.
‡ One-way analysis of variance.

Group IGroupIIGroup III

Statistical analysis

13.3
30.3

64.3
25.4

80.0
22.8

P < 0.001†
P < 0.001‡

3 groups. The tumor induction time was a meanof 30.3 weeks in Group I, 25.4 weeks in GroupII and 22.8 weeks in Group III. A significant
difference in tumor induction time was foundamong the 3 groups (P < 0. 001) (Table 1).

Results

Experiment I
A total of 44 mice were cystectomized in the
18th week. Occurrence of urinary bladder cancer was 13.3% (1 carcinoma in situ; 1 invasive
carcinoma) in 15 mice from Group I, 64.3% (6carcinomas in situ; 3 invasive carcinomas) in 14
mice from Group II and 80.0% (7 carcinomas insitu; 5 invasive carcinomas) in 15 mice fromGroup III. Statistically significant differenceswere found among the 3 groups (P < 0. 001)
(Table 1).

Experiment II
A cumulative total of 50 mice were cystectomized between the 28th and 33rd week inGroup I, between the 23rd and 27th week inGroup II and between the 21st and 24th week inGroup III. Occurrence of invasive bladdercancer was 72.2% in Group I, 82.4% in GroupII and 86.7% in Group III. No significant
distribution of cases was observed according to
the histological stage and grade of tumors in the

The proliferation and apoptotic indices
The relationship between the mean proliferation index and the stage of tumors is shown in
Table 2. In each group, the proliferation index
significantly increased as the tumor stage
progressed. In invasive carcinomas, a significant difference in the proliferation index was
observed among the 3 groups (P = 0. 005). The
relationship between the proliferation index andthe grade of invasive carcinomas is shown in
Table 3. In Groups I and III, the proliferationindex significantly increased as the tumor grade
increased. In Group II, no significant difference of the proliferation index was observed,
though values tended to increase as mentioned
above. In every grade of tumor, a significant
difference in the proliferation index was foundamong the 3 groups. The relationships between
the mean apoptotic index and the tumor stage or
grade are shown in Tables 4 and 5, respectively.
In each group, the apoptotic index significantly

Table 2. Relationship between the proliferation index and the stage of tumors
Benign lesion(%)
Group IGroup IIGroup III
Statistical analysis† Kruskal-Wallis rank test.
NS, not significant.

1.1
0.2
1.1
NS

Carcinomain situ (%)

Invasivetumor (%)

Statistical analysis†

6.7
6.4
13.2
NS

17.1
31.0
42.7
P = 0.005

P < 0.001
P < 0.001
P < 0.001

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Effects of testosterone in mouse urinary bladder carcinogenesis

Table 3. Relationship between the proliferation index and the grade of tumors
Grade 1 (%)
Group I
9.9
Group II
24.7
Group III
14.0
Statistical analysis
P = 0.012† Kruskal-Wallis rank test.
NS, not significant.

Grade 2 (%)

Grade 3 (%)

Statistical analysis†

16.1
26.3
33.0
P = 0.039

25.3
41.2
71.4
P = 0.004

P = 0.035
NS
P = 0.001

decreased as the stage and grade progressed.
No significant difference in apoptotic index was
found among the 3 groups. Examples shown in
Figs. 2, 3 and 4 are a benign lesion in Group II,
a Grade-3 invasive bladder carcinoma in GroupI and a Grade-3 invasive bladder carcinoma inGroup III, respectively.

Correlation between the proliferation index and apoptotic index
The correlation between the proliferation index
and apoptotic index is shown in Fig. 5. A negative significant correlation was observed (r =–0.7, P < 0.001).

Discussion
Druckrey et al. (1964) first reported that oral
administration of BBN in rats induced bladdercarcinoma only organotrophically. Since
then, some investigators have reported on
histogenetic studies of urinary bladder tumors
in various animals induced by BBN (Kakizoe,
1995). In the 1970’s, several reports indicated hormonal effects on the sex differencein the incidence of bladder carcinogenesis.
Some of these studies showed that the occurrence of bladder carcinoma in animalmodels induced by BBN is higher in males
than in females (Bertram and Craig, 1972;
Iriya, 1979). Others have shown that testos-

Table 4. Relationship between the apoptotic index and the stage of tumors
Benign lesion(%)
Group IGroup IIGroup III
Statistical analysis† Kruskal-Wallis rank test.
NS, not significant.

54.0
64.5
69.0
NS

Carcinomain situ (%)

Invasivetumor (%)

Statistical analysis†

51.3
43.5
36.3
NS

26.7
21.4
18.8
NS

P = 0.001
P < 0.001
P = 0.009

Table 5. Relationship between the apoptotic index and the grade of tumors
Grade 1 (%)
Group IGroup IIGroup III
Statistical analysis† Kruskal-Wallis rank test.
NS, not significant.

35.4
28.5
36.4
NS

Grade 2 (%)

Grade 3 (%)

Statistical analysis†

39.3
28.7
21.9
NS

8.0
8.2
5.1
NS

P = 0.010
P = 0.042
P = 0.006

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H. Ohnoterone promotes the occurrenceof bladder carcinomas, and that
diethylstilbestrol suppresses it in
BBN-induced rats (Okajima etal., 1975; Kono et al., 1975).
However, at present there is noclear evidence to explain these
hormonal influences on urinary
bladder carcinogenesis. In general, the effects of human bladdercarcinogenesis are considered tobe due to industrial and environmental carcinogens (Risch et al.,1995) such as aniline dye, cigarette smoking, etc., and hormonal
influence is considered negligible. Recently, few reports haveappeared on hormonal effects inbladder carcinogenesis. It was estimated that, in 1995, among 50,500
new cases of human bladder cancer diagnosed in the United States,
the incidence was about 3 times
more common in men than in women (Wingo et al., 1995). Overthe past 30 years, women havejoined the male-predominant
workplace, changed their habits
and have subsequently been exposed to both industrial and environmental carcinogens, but surprisingly, this sex difference inthe number of new cases of human bladder cancer remains(Messing and Catalona, 1998).
We considered that hormonal influence, especially of testosterone, might relate to this contradictory sex difference in incidence, and that the hormonal influence on bladder carcinogenesis should be investigated.
Human bladder carcinomas
can be classified into papillarysuperficial bladder carcinoma
and non-papillary invasive bladder carcinoma. The former frequently recurs after treatment
such as transurethral resection or

Fig. 2. A benign bladder lesion in Group II. a: Hematoxylin andeosin staining. b: A few proliferating cell nuclear antigen-positive
cells shown by immunohistochemical staining. c: A number of
apoptotic cells shown by terminal-deoxynucleotidyl-transferase(TdT)-mediated dUTP-biotin nick end labeling, × 200.

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