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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 (E2, 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 + E2 and ENNG + TAM + E2. Endometrial proliferative lesions (hyperplasias and/or carcinomas) were observed in all groups, the incidences in the TAM- and/or E2-treated groups being two times higher than in the ChL-treated control animals. High induction (11/20, 55%) of adenocarcinomas was observed in the E2 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 E2 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 E2-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 E2 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 E2.

Abbreviations: ChL, cholesterol; ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; E2, 17ß-oestradiol; P, progesterone; E2: P ratio, 17ß-oestradiol: progesterone ratio; TAM, tamoxifen.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An association between endocrine imbalance and uterine endometrial adenocarcinomas in women has been documented in the literature (13), and it is well established that oestrogenic compounds such as diethylstilbestrol (DES) play an important role in development of this tumour (46). 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 (911), 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 (1315). 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 (E2) (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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 1AGo). ENNG, purchased from Nacalai Tesque (Kyoto), was dissolved at a 1.5% (w/v) concentration in polyethylene glycol (PEG) just before use. E2 and TAM were obtained from Sigma (St Louis, MO), and pellets containing 0.16 mg of E2 and 31.84 mg of cholesterol (ChL) (0.5% E2 pellet), 1.6 mg of TAM and 30.4 mg of ChL (5% TAM pellet), and also 0.16 mg of E2, 1.6 mg of TAM and 30.24 mg of ChL (0.5% E2 and 5% TAM pellet) were manufactured by the method described previously (18). Animals were implanted s.c. with ChL, TAM, E2 and TAM plus E2 pellets at 9 weeks of age, respectively, and thereafter the pellets were renewed after 8 weeks throughout the experiment. Values for E2- and TAM-release from the pellets were calculated from weight loss of the pellets, estimated to be 3.4 ± 1.2 ng E2/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% E2 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 1BGo). 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 E2 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 1CGo).



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Fig. 1. Experimental designs for experiments I–III. Animals in each group were implanted s.c. ( ) 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, {blacktriangleup}) was given in PEG. In experiment III, all CD-1 mice were OVX at 7 weeks of age ({triangleup}).

 
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.


    Results
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 Materials and methods
 Results
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 References
 
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 IGo. Uterus weights in groups 3 and 4 were significantly higher than in group 1 (P < 0.05 or P < 0.01) (E2 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) (E2 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) (E2 effect) (Table IIGo). In contrast, ovary weights in groups 2–5 were significantly less than in group 1 (P < 0.01), but group 3 was not significantly (E2 and/or TAM effects). Experiment III: body and uterus weights at terminal death are shown in Table IIIGo. Body weights in groups 3 and 4 were significantly decreased as compared with those in groups 1 and 2 (P < 0.01) (E2 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) (E2 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.


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Table I. Body, uterus and ovary weights at terminal death in experiment I
 

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Table II. Body, uterus and ovary weights at terminal death in experiment II
 

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Table III. Body and uterus weights at terminal death in experiment III
 
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 IVGo. 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) (E2 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) (E2 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 E2 are shown in Figure 2Go. Although endometrial adenocarcinomas were not observed in any TAM-treated groups without E2, total incidences of all proliferative lesions were significantly higher than in untreated animals, with dose-dependence (Figure 2AGo) (TAM effect). In contrast, the incidences of adenocarcinomas in animals given E2 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 2BGo) (E2 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 E2. 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 VGo. 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 E2 effects) (Figure 3Go), although the incidences in both groups were the same. Endometrial adenocarcinomas were only observed in group 5 (E2 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 VIGo, 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 (E2 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) (E2 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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Fig. 2. TAM dose-dependent changes in yields of uterine proliferative lesions in mice. The incidences of all proliferative lesions (; left bar) and endometrial adenocarcinomas ( {blacksquare}; 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.

 

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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)
 
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 E2 concentrations in E2-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) (E2 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 E2-treated animals (groups 3 and 4) were significantly lower than those in groups 1 (P < 0.05) and 2 (P < 0.01), respectively (E2 effect). Experiment II: the serum E2 and P concentrations in animals post-treated with E2 (groups 2 and 5) (E2, 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 (E2, 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 (E2 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 E2 concentrations in the E2-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 E2 levels did not differ in both groups (1.1 ± 0.1 and 1.0 ± 0.2 pg/ml).


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The present experiment I study confirmed our earlier findings concerning promotion and progression effects of E2 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 E2 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 E2, they would be expected to develop into cancers following E2 treatment. Therefore, in experiment II, we examined progression effects of E2 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 (811,33), 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.


    Notes
 
1 To whom correspondence should be addressed Email: takahashi{at}sasaki.or.jp Back


    Acknowledgments
 
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.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 14, 2001; revised May 24, 2002; accepted May 27, 2002.





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