Carcinogenesis, Vol. 23, No. 9, 1549-1555,
September 2002
© 2002 Oxford University Press
Promotion, but not progression, effects of tamoxifen on uterine carcinogenesis in mice initiated with N-ethyl-N'-nitro-N-nitrosoguanidine
Masakazu Takahashi,1,
Takasumi Shimomoto,
Katsuhiro Miyajima,
Seiichi Iizuka,
Takao Watanabe,
Midori Yoshida,
Yuji Kurokawa and
Akihiko Maekawa
Department of Pathology, Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
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Abstract
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Effects of tamoxifen (TAM) on development of uterine endometrial
carcinogenesis were studied in intact and ovariectomized (OVX)
mice initiated with
N-ethyl-
N'-nitro-
N-nitrosoguanidine (ENNG).
In experiment I, animals were implanted with cholesterol (ChL,
controls) or TAM (5% w/w) and/or 17ß-oestradiol (E
2,
0.5% w/w) pellets s.c. from 9 to 25 weeks of age, until the
termination of the experiment, and all received a single intra-uterine
administration of ENNG (12.5 mg/kg) at 10 weeks of age. They
were divided into four groups: ENNG + ChL (control), ENNG +
TAM, ENNG + E
2 and ENNG + TAM + E
2. Endometrial proliferative
lesions (hyperplasias and/or carcinomas) were observed in all
groups, the incidences in the TAM- and/or E
2-treated groups
being two times higher than in the ChL-treated control animals.
High induction (11/20, 55%) of adenocarcinomas was observed
in the E
2 group but this was significantly decreased in combination
with TAM (2/20, 10%), no carcinomas being found in the TAM group.
In experiment II, animals pre-treated with TAM (10 weeks) and
receiving E
2 post-treated (4 weeks) developed adenocarcinomas,
although no cancers were observed in mice treated by ChL instead
of TAM. In animals pre-treated with TAM and post-treated with
ChL or TAM, no adenocarcinomas were also developed. In OVX mice
(experiment III), proliferative lesions were observed in the
TAM- and/or E
2-treated groups, at incidences significantly higher
than in ChL-treated animals, in which these lesions were completely
absent. However, no adenocarcinomas were found, only slight
hyperplasias being observed in the TAM group, although the incidence
of adenocarcinoma was highest in the E
2 alone group, and significantly
decreased in combination with TAM, as in experiment I. These
results indicate that TAM may itself exert promotion effects,
while exhibiting an anti-progression influence on uterine carcinogenesis
in adult mice initiated by ENNG and receiving E
2.
Abbreviations: ChL, cholesterol; ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; E2, 17ß-oestradiol; P, progesterone; E2: P ratio, 17ß-oestradiol: progesterone ratio; TAM, tamoxifen.
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Introduction
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An association between endocrine imbalance and uterine endometrial
adenocarcinomas in women has been documented in the literature
(
1–
3), and it is well established that oestrogenic compounds
such as diethylstilbestrol (DES) play an important role in development
of this tumour (
4–
6). Tamoxifen (TAM), a non-steroidal
anti-oestrogen, competes with oestrogen for binding to oestrogen-receptors.
It is as effective for treatment of hormone-dependent breast
cancer as any adjuvant therapies so far clinically tested, and
it has the advantage of fewer side effects (
7,
8). However, it
has been pointed out that the risk of endometrial cancer may
be increased in women exposed to TAM therapy, the agent acting
as a weak oestrogen agonist in response to oestrogen deficiency
in postmenopausal women (
9–
11), and TAM has been evaluated
as carcinogenic to humans (group 1) by IARC (
12). In experimental
studies, TAM can stimulate the growth of human endometrial tumours
implanted in athymic mice (
13–
15). However, Niwa
et al.
(
16) reported recently that induction of endometrial adenocarcinomas
in a two-stage mouse uterine carcinogenesis model initiated
by
N-methyl-
N-nitrosourea could not be promoted by TAM, although
development of benign endometrial proliferative lesions was
enhanced. Moreover, TAM has shown potent anticarcinogenic effects
in a rat model of uterine carcinogenesis (
17). Thus, the available
data point to species variation. Recently, we succeeded in inducing
a high incidence of uterine endometrial adenocarcinomas in CD-1
mice by a single intra-uterine administration of
N-ethyl-
N-nitrosourea
(ENU) or
N-ethyl-
N'-nitro-
N-nitrosoguanidine (ENNG), following
subcutaneous implantation of 17ß-oestradiol (E
2) (
18,
19).
Time-dependent promotion activity of oestrogen on uterine carcinogenesis
could subsequently be demonstrated using this two-stage model
(
20). In the present experiment, the model was further employed
to investigate the effects of TAM on development of endometrial
proliferative lesions, including adenocarcinomas, in intact
and ovariectomized (OVX) adult mice.
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Materials and methods
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Animals and treatment
Four-week-old CD-1 mice (Charles River Japan, Atsugi, Kanagawa)
were housed four animals to a plastic cage and kept in an air-conditioned
animal room at 21 ± 2°C and 55 ± 10% humidity
under a 12 h light/12 h dark cycle. At 7 weeks of age, all mice
were subjected to 24 h light conditions to induce persistent
oestrus and maintained under standard conditions throughout
the experiment. Vaginal smears from mice were checked every
morning from 7 to 9 weeks of age and only these animals exhibiting
persistent oestrus were employed for the experiment. Experiment
I: they were divided into four groups of 20 animals (Figure
1A
). ENNG, purchased from Nacalai Tesque (Kyoto), was dissolved
at a 1.5% (w/v) concentration in polyethylene glycol (PEG) just
before use. E
2 and TAM were obtained from Sigma (St Louis, MO),
and pellets containing 0.16 mg of E
2 and 31.84 mg of cholesterol
(ChL) (0.5% E
2 pellet), 1.6 mg of TAM and 30.4 mg of ChL (5%
TAM pellet), and also 0.16 mg of E
2, 1.6 mg of TAM and 30.24
mg of ChL (0.5% E
2 and 5% TAM pellet) were manufactured by the
method described previously (
18). Animals were implanted s.c.
with ChL, TAM, E
2 and TAM plus E
2 pellets at 9 weeks of age,
respectively, and thereafter the pellets were renewed after
8 weeks throughout the experiment. Values for E
2- and TAM-release
from the pellets were calculated from weight loss of the pellets,
estimated to be 3.4 ± 1.2 ng E
2/animal/day and 16.3 ±
4.5 ng TAM/animal/day, respectively. At 10 weeks of age, mice
in all groups were given a single dose of ENNG (12.5 mg/kg)
(25–35 µl/head) into one of the uterine cavities
using a 23 G needle (45 mm in length) via the vagina. The experiment
was terminated at 15 weeks after the treatment with ENNG (25
weeks of age), when all survivors were killed. In addition,
effects of several TAM doses on uterine carcinogenesis were
examined. The 20 animals in each group were implanted s.c. with
TAM pellets (0, 0.5, 1.5, 3.0 and 5.0%), combined with or without
0.5% E
2 pellets at 9 weeks of age. At 10 weeks of age, mice
in all groups were given a single intra-uterine administration
of ENNG (12.5 mg/kg). At the termination of the experiment (week
15 after the ENNG treatment), incidences of endometrial proliferative
lesions in the uterus were examined histopathologically. Experiment
II: mice in groups 1 and 2 and groups 3–5 were treated
with ChL and TAM pellets, respectively, from 9 to 20 weeks of
age (Figure 1B
). At 20 weeks of age, all groups of animals received
ChL pellets for 1 week, and then groups 1 and 3 were given ChL
pellets and groups 2 and 5 were given E
2 pellets and group 4
was given TAM from 21 weeks to the end of the experiment (25
weeks of age). Experiment III: all CD-1 mice were OVX at 7 weeks
of age and animals in groups 1–4 were treated according
to the same protocol as for experiment I. In groups 5 and 6,
animals were implanted s.c. with ChL and TAM pellets at 9 weeks
of age, respectively, and thereafter the pellets were renewed
every 8 weeks (17, 25, 33, 41 and 49 weeks of age) throughout
the experiment, and the experiment was terminated at 45 weeks
after the treatment with ENNG (55 weeks of age) (Figure 1C
).
Histological examination
At the termination all surviving animals were autopsied, and
the uterus, ovaries, vagina, adrenal glands, pituitary gland,
lungs, kidneys, liver and spleen were taken for histological
examination. The tissues were fixed in buffered 10% formalin,
and sections were routinely prepared and stained with hematoxylin
and eosin (H&E) for microscopic examination. Each uterus
was cut into five to seven specimens, those from the uterine
horns and the corpus uteri being sectioned transversely. In
this study, uterine endometrial proliferative lesions were histologically
classified into adenocarcinoma and hyperplasia categories. Adenocarcinomas
were composed of irregularly proliferating atypical cells forming
glands with one or more columnar layers, with clear evidence
of invasion into the muscularis. Hyperplasias were classified
into three degrees on the basis of atypia and size: slight (+),
moderate (++) and severe (+++), according to the criteria described
in our previous reports (
18,
19).
Examination of serum sex steroids
In three experiments, blood samples were obtained from the abdominal aorta at 10:00 h. After 1 h, serum was separated by centrifugation at 10 000 g for 2 min and five assay samples in each group were prepared, an assay sample being collected from three to four mice sera in the same group (total 1 ml). Serum E2 and progesterone (P) concentrations were assayed by the methods reported previously (21) with a specific sheep E2 antiserum (GDN 244) kindly supplied by Dr G.D.Niswender (Department of Physiology and Biophysics, Colorado State University, CO) and a Progesterone kit `Daiichi II' (Daiichi Radioisotope Lab., Tokyo).
Statistical methods
Differences in body, uterus and ovary weights and serum sex-steroid concentrations were analysed by Student's t-test. Incidences of endometrial proliferative lesions, including adenocarcinomas, were conducted using the Fisher's exact probability test.
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Results
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General condition
Experiment I: the mean body weights did not differ among four
groups throughout the experimental period (data not shown),
except for the decrease observed in group 4 at terminal death.
Final body, uterus and ovary weights are listed in Table I
.
Uterus weights in groups 3 and 4 were significantly higher than
in group 1 (
P < 0.05 or
P < 0.01) (E
2 effect). Those in
groups 2 and 4 were slightly decreased compared with those in
groups 1 and 3, respectively, but not significantly (TAM effect).
In contrast, ovary weights in groups 2–4 were significantly
less than in group 1 (
P < 0.01) (E
2 and/or TAM effects).
Experiment II: uterus weights in groups 2 and 5 were significantly
higher than in groups 1, 3 and 4 (
P < 0.05 or
P < 0.01)
(E
2 effect) (Table II
). In contrast, ovary weights in groups
2–5 were significantly less than in group 1 (
P < 0.01),
but group 3 was not significantly (E
2 and/or TAM effects). Experiment
III: body and uterus weights at terminal death are shown in
Table III
. Body weights in groups 3 and 4 were significantly
decreased as compared with those in groups 1 and 2 (
P < 0.01)
(E
2 effect). Uterus weights in groups 2–4 were significantly
higher than in group 1 (
P < 0.01), and in group 3 than in
groups 2 and 4 (
P < 0.01), although the value for group 4
was significantly higher than that for group 2 (
P < 0.01)
(E
2 and/or TAM effects). In long-term observed groups (groups
5 and 6), body and uterus weights showed similar tendency to
those in groups 1 and 2.
Histological characteristics and incidences of endometrial proliferative lesions in the uterus
In the present experiment, ENNG was given into one of the uterine
cavities using a 23 G needle via the vagina. Although it could
not be checked into which cavity the ENNG was given, almost
all severe endometrial hyperplasias and/or adenocarcinomas were
observed in one uterine cavity, the degree and incidence of
lesions being less in the other one of the pair. Experiment
I: data for all endometrial proliferative lesions observed in
the uterus at terminal death are summarized in Table IV
. Proliferative
lesions (hyperplasias and/or carcinomas) were observed in all
groups, and the total incidences in groups 2–4 were significantly
higher than that in group 1 (
P < 0.05 or
P < 0.01) (E
2 and/or TAM effects). The incidence of adenocarcinomas in group
3 was significantly higher than those in groups 1, 2 and 4 (
P < 0.01) (E
2 effect), the values in these groups being approximately
equal, no adenocarcinomas developing in group 2. Effects of
several TAM doses on uterine carcinogenesis in mice given ENNG
combined with or without E
2 are shown in Figure 2
. Although
endometrial adenocarcinomas were not observed in any TAM-treated
groups without E
2, total incidences of all proliferative lesions
were significantly higher than in untreated animals, with dose-dependence
(Figure 2A
) (TAM effect). In contrast, the incidences of adenocarcinomas
in animals given E
2 demonstrated a clear decrease with TAM-doses
from 0 to 5% (55, 40, 21, 15 and 10%, respectively), although
the total incidences of all proliferative lesions were the same
in all groups (Figure 2B
) (E
2 and/or TAM effects). In addition
to uterine lesions, ovarian atrophy with cystic follicles was
prominent dose dependently in all TAM-treated groups, with or
without E
2. In other organs of all groups, there were no characteristic
lesions. Experiment II: incidences of endometrial proliferative
lesions in the uterus at terminal death are shown in Table V
.
All proliferative lesions (hyperplasias and/or carcinomas) were
observed in all groups, and the total incidences in groups 2,
4 and 5 were significantly higher than those in groups 1 and
3 (
P < 0.05 or
P < 0.01) (TAM and/or E
2 effects) (Figure
3
), although the incidences in both groups were the same. Endometrial
adenocarcinomas were only observed in group 5 (E
2 effect). Experiment
III: effects of TAM on uterine carcinogenesis were also examined
in OVX mice by the same two-stage endometrial carcinogenesis
protocol as used in experiment I. As shown in Table VI
, endometrial
proliferative lesions (hyperplasias and/or carcinomas) were
observed in groups 2–4, and the total incidences in these
groups were significantly higher than in group 1 (
P < 0.01),
which lacked any proliferative lesions (E
2 and/or TAM effects).
In group 2, however, endometrial adenocarcinomas were not found,
and only slight hyperplasia (+) was observed (TAM effect). In
group 3, the incidence of adenocarcinoma was highest and significantly
greater than those in groups 1, 2 and 4 (
P < 0.01) (E
2 effect).
As no endometrial adenocarcinomas were observed in group 1 and
2 animals, the observation period was prolonged to 45 weeks
in groups 5 and 6. No endometrial proliferative lesions were
again evident in group 5. In group 6, not only slight (+) (60%)
but also moderate (++) (30%) endometrial hyperplasias were observed,
although no severe hyperplasias or endometrial adenocarcinomas
were encountered. In other tissues and organs of all groups
in experiments I–III, no proliferative lesions were apparent.
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Table IV. Incidences of endometrial proliferative lesions in the uterus at terminal death in experiment I (% incidences in parentheses)
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Table V. Incidences of endometrial proliferative lesions in the uterus at terminal death in experiment II (% incidences in parentheses)
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Fig. 3. Histological findings of endometrial proliferations. (A) Sever hyperplasia (+++) in group 2 (experiment II). Irregular proliferation of atypical gland is obvious in the endometrium, without an invasion into the muscle. (B) Endometrial adenocarcinoma in group 5 (experiment II). The lesion shows irregular proliferation of atypical glands in the endometrium, and invasion of tumour cells into the muscle layer. H&Ex125.
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Table VI. Incidences of endometrial proliferative lesions in the uterus at terminal death in experiment III (% incidences in parentheses)
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Serum sex-steroid hormone concentrations
All assay samples were collected from three to four mice sera
in the same group and five samples in each group were examined.
Experiment I: the serum E
2 concentrations in E
2-treated animals
(groups 3 and 4) (30.0 ± 5.5 and 24.9 ± 3.9 pg/ml)
were significantly higher (
P < 0.01) than those in groups
1 and 2 (11.1 ± 2.4 and 11.3 ± 0.7 pg/ml) (E
2 effect) respectively. The serum P concentrations in TAM-treated
mice (groups 2 and 4) (11.7 ± 2.4 and 6.6 ± 1.7
ng/ml) were significantly higher (
P < 0.01) than those in
groups 1 and 3 (9.0 ± 1.8 and 3.7 ± 1.6 ng/ml),
respectively, but Group 1
vs 2 was not significant (TAM effect),
and E
2-treated animals (groups 3 and 4) were significantly lower
than those in groups 1 (
P < 0.05) and 2 (
P < 0.01), respectively
(E
2 effect). Experiment II: the serum E
2 and P concentrations
in animals post-treated with E
2 (groups 2 and 5) (E
2, 28.7 ±
4.2 and 28.8 ± 3.9 pg/ml; P, 4.0 ± 1.7 and 4.2
± 1.8 ng/ml) were significantly higher (
P < 0.01)
and lower (
P < 0.01) than those in groups 1, 3 and 4 (E
2,
11.1 ± 2.0, 11.2 ± 1.0 and 11.0 ± 0.9 pg/ml;
P, 8.8 ± 1.5, 10.0 ± 2.0 and 10.4 ± 2.2
ng/ml), respectively (E
2 effect). On the other hand, the serum
P concentrations in mice pre- and post-treated with TAM (group
4) was higher than in animals treated by ChL instead of TAM
(group 3), but not significant (TAM effect). Experiment III:
with the short experiment of period (groups 1–4), the
serum E
2 concentrations in the E
2-treated groups (groups 3 and
4) (26.5 ± 1.6 and 25.5 ± 1.8 pg/ml) were significantly
higher (
P < 0.01) than those in the untreated mice (groups
1 and 2) (1.0 ± 0.2 and 0.8 ± 0.2), respectively.
Serum P content in mice treated with TAM groups (groups 2 and
4) (1.3 ± 0.2 and 1.2 ± 0.1 ng/ml) was significantly
higher (
P < 0.01) than in the corresponding TAM-untreated
groups (groups 1 and 3) (0.7 ± 0.3 and 0.7 ± 0.1
ng/ml), respectively (TAM effect). In the long-experiment (groups
5 and 6), the P concentration in group 6 (1.3 ± 0.2 ng/ml)
was significantly higher (
P < 0.01) than in group 5 (0.8
± 0.2 ng/ml) (TAM effect), although the E
2 levels did
not differ in both groups (1.1 ± 0.1 and 1.0 ±
0.2 pg/ml).
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Discussion
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The present experiment I study confirmed our earlier findings
concerning promotion and progression effects of E
2 on development
of uterine endometrial adenocarcinomas in ENNG-initiated CD-1
mice (
19), while showing that TAM can increase the yield of
uterine endometrial hyperplasias but not affecting adenocarcinomas.
Conversely, when given in combination with E
2 the incidence
of malignancies was decreased dose-dependently. Previously,
Niwa
et al. (
16) reported effects of TAM on uterine carcinogenesis
in mice initiated with
N-methyl-
N-nitrosourea, high yields of
endometrial hyperplasias, but no adenocarcinomas, being observed.
Our present findings are in line with their result. It is generally
well known that the classical separation of the carcinogenic
process in the liver and other organs can be divided into three
stages: initiation, promotion (selection) and progression (
22,
23).
If endometrial cells in mice initiated by ENNG and promoted
by TAM are truly pre-neoplastic, similar to those promoted by
E
2, they would be expected to develop into cancers following
E
2 treatment. Therefore, in experiment II, we examined progression
effects of E
2 on endometrial cells in mice initiated by ENNG
and promoted by TAM, and found this to indeed be the case. From
these results, the process for uterine endometrial carcinogenesis
in mice can also be divided into three stages: initiation, promotion
and progression stages, and TAM may have promotion but not progression
effects on mouse uterine carcinogenesis, apparently inhibiting
the progression stage due to oestrogen-antagonistic influence.
In the present experiment III, uterine weights in TAM-treated
animals were significantly increased, compared with those in
TAM-untreated animals, pointing to oestrogen agonism, it is
in fact well known that TAM shows uterotropic effects on OVX
mice and rats (
24,
25). In mice given ENNG and TAM, only slight
hyperplasias were observed, the lack of severe hyperplasias
or adenocarcinomas even after 45 weeks, the result again indicating
that TAM has promotion but not progression activity in uterine
carcinogenesis in OVX mice.
In normal oestrous cycling animals, serum hormone levels change with the oestrous cycle and sex-steroid hormone synthesis and secretion are regulated by various factors. Thus, for the purpose of synchronizing oestrous cycles, illumination-induced persistent oestrus mice were used in the present study, so that oestrous cycles in animals with or without E2- and/or TAM-treatment were comparable. In these animals, it has been established that the serum levels of steroid hormones are almost the same as in normal mice on dioestrus, probably due to blockage of the negative feedback, although these animals show persistent oestrus in vaginal smears. Promotion effects of hormonal imbalance (persistent oestrus; an increased E2:P ratio) on uterine carcinogenesis are well known in rodents (26). Although endometrial proliferative lesions (slight and moderate hyperplasias) develop from an earlier age in illumination-induced persistent oestrus mice compared with normal 12 h light/dark cycle animals, artificially induced persistent oestrus alone is not sufficient for high induction of adenocarcinomas (19). In the present experiment, serum E2 levels in intact and OVX mice given ENNG were ~10 and 1 pg/ml, respectively. Whereas the E2:P ratios were almost the same (1.31 ± 0.41, 1.49 ± 0.52x10-3), endometrial hyperplasias and adenocarcinomas were only found in the intact case, indicating that appreciable serum oestrogen may be necessary for their development.
Newbold et al. (27) reported that TAM may act as an oestrogen agonist at low doses but as an oestrogen antagonist at higher doses in mice. In the present study, uterine weights of intact animals treated with TAM was decreased as compared with controls, although not significantly. In contrast, the weights in OVX mice treated with TAM were significantly increased. However, the weights with E2 and TAM in both intact and OVX mice were decreased relative to E2 alone. Depending on whether the serum E2 level is high or low, TAM appears to show anti-oestrogenic (oestrogen antagonistic) or oestrogenic (oestrogen agonistic) actions, respectively. Histologically, ovarian atrophy, characterized by cystic dilatation of follicles and decrease and/or lack of follicle and corpus luteum, was prominent in animals given E2 and/or TAM, similar to the polycystic ovary disease in humans, considered to be a risk factor for endometrial carcinomas (1,28,29). These results indicated that the influence of E2 or TAM on the ovary may be oestrogenic, the two compounds exhibiting mutually potentiating effects.
While it has been reported that endometrial adenocarcinomas can be induced in mice and rats by neonatal exposure to TAM (27,30), or oestrogen and DES treatment (31), as well as with postnatal exposure to E2 (19,31), TAM or DES did not cause adenocarcinomas with post-neonatal exposure (16,32). These chemicals given neonatally are potent carcinogens and act as an E2 agonist in rodents. Thus, the treatment period is clearly important, presumably related to hormonal imprinting of oestrogen receptor responses in stem cells.
In women administered TAM continuously as an ablative or additive therapy for breast cancer, the major concern has been sustained oestrogen-agonistic effects on the endometrium. In a number of studies, this has been shown to induce endometrial hyperplasias, and eventually lead to an increased incidence of endometrial carcinomas (8–11,33), although the mechanism of uterine carcinogenesis in humans by TAM is still unknown. In our animal model, TAM showed oestrogen-agonistic effects on the uterus in the promotion stage, the results being of interest in the context of influence in the human uterus. However, at the doses used in the present studies, the effects of TAM on the uterus in the progression stage appeared to be different from the oestrogen agonism reported for human beings. To understand this discrepancy between humans and mice, additional studies into interspecies differences in metabolism of TAM and localization of oestrogen-receptors expression are needed.
In conclusion, this study demonstrated that TAM may itself exert promotion but not progression effects on uterine endometrial carcinogenesis in adult mice initiated by ENNG, and TAM apparently inhibits the progression stage induced by E2 due to oestrogen-antagonistic influence.
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Notes
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1 To whom correspondence should be addressed Email:
takahashi{at}sasaki.or.jp 
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Acknowledgments
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The authors would like to express their appreciation to Miss
Hiromi Tokuda for technical assistance. This study was supported
by Grant-in-Aids from Ministry of Health and Labor for (Scientific
Research C) Japan.
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Received February 14, 2001;
revised May 24, 2002;
accepted May 27, 2002.
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Abstract
Introduction
) with ChL (
), TAM (
) E2 (
) and TAM + E2 (
symbol) pellets, and thereafter the pellets were renewed every 8 weeks throughout the experiment. The single intra-uterine application of ENNG (12.5 mg/kg, 
) was given in PEG. In experiment III, all CD-1 mice were OVX at 7 weeks of age (
).
; right bar) in mice without E2 pellets (A) and combined with 0.5% E2 pellets (B). Significant difference from the 0% TAM group (*P < 0.05 and **P < 0.01) by Fisher's exact probability test.