Modifying Effects of Ethinylestradiol but Not Methoxychlor on N-Ethyl-N-nitrosourea-Induced Uterine Carcinogenesis in Heterozygous p53-Deficient CBA Mice

Kunitoshi Mitsumori*,1, Takeo Shimo{dagger}, Hiroshi Onodera*, Hisayoshi Takagi*, Kazuo Yasuhara*, Toru Tamura*, Yasuji Aoki{dagger}, Osamu Nagata{dagger} and Masao Hirose*

* Division of Pathology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; and {dagger} Research Department, Research and Development Division, Hokuriku, Seiyaku Company, Ltd., 37-1-1, Inokuchi, Katsuyama, Fukui 911-8555, Japan

Received June 6, 2000; accepted July 31, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is unknown whether endocrine-disrupting chemicals (EDCs) with estrogenic activities have any modifying effects on uterine carcinogenesis. In our previous study, we established a uterine-carcinogenesis model that is useful for detecting tumor-modifying effects of EDCs by the administration of N-ethyl-N-nitrosourea (ENU) to female heterozygous p53-deficient CBA mice [p53 (+/–) mice]. To investigate the effects of ethinylestradiol (EE) and methoxychlor (MXC) on development of ENU-induced uterine tumors, female p53 (+/–) mice and their wild-type littermates [p53 (+/+) mice] received an intraperitoneal injection of 120 mg/kg body weight (bw) of ENU, followed, in Group 1, by no further treatment; in Group 2, by a diet containing 1 ppm EE; in Group 3, by a diet containing 5 ppm EE for 4 weeks and 2.5 ppm EE thereafter; and in Group 4, by a diet containing 2000 ppm MXC for 26 weeks. Uterine proliferative lesions that were induced were composed of both endometrial-stromal and epithelial-cell types. Endometrial stromal sarcomas were induced in p53 (+/–) mice of Groups 1 to 4, and the incidence (87%) in Group 3 was significantly increased compared to Group 1 (47%). Atypical hyperplasias (clear-cell type) of the endometrial gland in p53 (+/–) mice were seen at incidences of 0, 14, 60, and 0% in Groups 1, 2, 3, and 4, respectively, while their incidence in p53 (+/+) mice was 0, 7, 53, and 0%, respectively, with a significant difference between Groups 1 and 3 in both cases. One p53 (+/–) mouse in Group 3 also had an adenocarcinoma consisting of clear cells, and the PCNA labeling indices of the clear-cell atypical hyperplasias, and this endometrial adenocarcinoma, were higher than those of glandular hyperplasias. The present study suggests that 2.5 ppm EE, but not MXC, exerts tumor-promoting effects on stromal and epithelial proliferative lesions of the uteri in p53 (+/–) mice initiated with ENU.

Key Words: N-ethyl-N-nitrosourea; ethinylestradiol; methoxychlor; uterine tumorigenesis; and heterozygous p53-deficient mice.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, possible adverse effects resulting from the release of man-made, endocrine-disrupting chemicals (EDCs) with estrogenic activity into the environment have become an important social problem. Since natural and synthetic estrogens have been shown to express their biological effects mainly by binding to estrogen receptors, and have been found to be major etiological agents for uterine carcinogenesis in humans (Fox, 1984Go; Hilliard and Norris, 1979Go; Lingeman, 1979Go; Zeil, 1982Go), evaluation of the carcinogenic risk of these EDCs on the uterus is clearly a high priority. Many reports on the experimental induction of uterine adenocarcinomas in rodents using chemical carcinogens have been reported (Ando-Lu et al., 1994Go; Niwa et al., 1991Go; Ogino et al., 1989Go; Takahashi et al., 1996Go; Tanaka and Mori, 1983Go), but there seem to be no appropriate uterine-carcinogenesis models in which experimental procedures are not complicated and tumor-modifying effects to the uterus can be detected within a relatively short treatment period. In our previous study in which N-ethyl-N-nitrosourea (ENU) was intraperitoneally given to heterozygous p53-deficient mice of the CBA strain [p53 (+/–) mice] where one allele of the p53 gene is inactivated, the incidences of endometrial stromal sarcomas and atypical hyperplasia of the endometrial glands were significantly increased in p53 (+/–) mice, as compared to their wild littermates [p53 (+/+) mice] (Mitsumori et al., 2000Go). The findings obtained in our previous study—that a GCG (Ala) to GTG (Val) transition at codon 135 of exon 5 of the p53 gene was detected in these sarcomas—suggest that the uterine tumors induced by ENU in p53 (+/–) mice do not have any p53 tumor-suppressor function. Therefore, female p53 (+/–) CBA mice given ENU were considered to be useful models for detecting uterine tumor-modifying effects of EDCs with estrogenic activities.

It is known that ethinylestradiol (EE) has strong estrogenic activity with tumor target organs on long-term treatment in experimental animals, including the uterus, ovary, liver, and kidney (Banerjee et al., 1994Go, Vickers and Lucier, 1991Go). Methoxychlor (MXC) has been reported to be a weak estrogenic pesticide that can alter fertility in male and female rats (Bulger et al., 1978Go), but no clear positive results have been obtained in the majority of the studies on its potential carcinogenicity (Deichmann et al., 1967Go, Radomski et al., 1965Go). Therefore, our present 6-month study using a p53 (+/–) mouse short-term model initiated with ENU was performed to clarify whether EE or MXC has any modifying effects on uterine carcinogenesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and housing.
The mice used in the present study were heterozygous female, p53-deficient CBA mice [p53 (+/–)] in which exon 2 of the lateral p53 allele was inactivated. They were the F1 offspring of heterozygous p53-deficient C57 BL/6J male mice backcrossed with CBA female mice (Tsukada et al., 1993Go). Sixty female p53 (+/–) mice and 60 wild-type littermates [p53 (+/+)], 6 weeks of age, were purchased from Oriental Yeast Co., Ltd (Tokyo, Japan). Through the acclimatization and experimental periods, animals were housed at a maximum of 5 per plastic cage with absorbent hardwood bedding (White Flakes, Charles River Inc., Tokyo, Japan) in an air-conditioned animal room (room temperature, 24 ± 2°C; relative humidity of 60 ± 10%; lighting cycle, 12 light/12 dark). All animals were transferred to clean cages with fresh bedding twice weekly. The mice were quarantined for 4 weeks in the animal room assigned for the study and only those without any abnormal findings at the end of this acclimatization period were selected for experimentation. CRF-1 pellet diet (Oriental Yeast Co., Ltd.) and tap water via automatic stainless steel nozzles were freely available throughout the study. In addition, CRF-1 powder diet (Oriental Yeast Co., Ltd.) was used for treatments with EE or MXC.

Test materials.
ENU and EE were both purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and MXC from Sigma Chemical Co. (St. Louis, MO).

Experimental design.
Female p53 (+/–) and p53 (+/+) mice were each divided into 4 groups of 15 animals. Since endometrial stromal sarcomas and atypical hyperplasia of the endometrial glands were induced with a high incidence by a single intraperitoneal injection of ENU in our previous study, the same administration route was adopted in the present study. All received an intraperitoneal injection of 120 mg/kg body weight of ENU in physiological saline, followed by no further treatment (Group 1) or diets containing 1 ppm EE (Group 2: ENU + 1 ppm EE2), or 2.5 ppm EE (5 ppm EE for the first 4 weeks reduced to 2.5 ppm EE thereafter because of marked body weight depression; Group 3: ENU + 2.5 ppm EE), or 2000 ppm MXC (Group 4: ENU + MXC) for 26 weeks. Filtration of the carcinogen through a Millipore filter (MILEX-GV, Japan Millipore, Ltd., Tokyo, Japan) was performed before the injection. An intraperitoneal dose of 120 mg/kg ENU was considered to be appropriate for uterine tumorigenesis in mice, based on the results of our previous study (Mitsumori et al., 2000Go). EE and MXC were mixed into powdered diets for ad libitum consumption. Individual body weights in each group were measured every week.

Histopathology and immunohistochemistry.
After the end of the 26-week experimental period, the surviving animals were killed by exsanguination from the posterior vena cava under ether anesthesia, and were subjected to a full autopsy. After measuring the uterine weights, a wide variety of organs and tissues, including the uterus, were fixed in 10% neutral buffered formalin. The uterine tissues were processed routinely, embedded in paraffin, sectioned at 4–5 µm, and stained with hematoxylin and eosin (H&E) for microscopic examination. Immunohistochemical staining, using a monoclonal antibody to proliferating cellular nuclear antigen (PCNA) (DAKO, Glostrup, Denmark), was performed at a dilution of 1:100. Avidin-biotin peroxidase complex kits (DAKO) were applied for the performance of immunohistochemistry with 3, 3`-diaminobenzidine as the chromogen and hematoxylin for counterstaining. The numbers of PCNA-positive cells per 100 cells in each proliferative lesion were counted in 5 different areas. The PCNA labeling index was calculated as the percentage of positive cells in each proliferative lesion.

Statistical analysis.
The incidences of uterine proliferative lesions observed were analyzed by the Fisher's exact test. Significant differences between the ENU group and the ENU + EE or ENU + MXC groups for p53 (+/–) or p53 (+/+) mice were assessed. Variation in the PCNA labeling indices for each type of proliferative lesion in the ENU + 2.5 ppm EE group was also analyzed by the Student's t test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of 15 p53 (+/–) mice of the ENU + 1 ppm EE2 group died during the early period of the experiment. p53 (+/–) and p53 (+/–) mice of the ENU + 2.5 ppm EE and ENU + MXC groups showed depression of body-weight gain compared to those in the ENU-alone group during the course of the study. The final body weights of p53 (+/–) and p53 (+/+) mice in the ENU + 2.5 ppm EE and ENU + MXC groups were significantly lower than those in the ENU-alone group (Fig. 1Go).



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FIG. 1. Final body and uterine weights for p53 (+/–) and p53 (+/+) mice treated with EE or MXC after ENU initiation. Asterisks indicate significant differences from the values of the ENU-alone group (p < 0.05).

 
On autopsy at the terminal sacrifice, multiple nodules (5 to 20 mm in diameter) of the uterine horn suggestive of uterine tumors were observed in 7, 9, 12, and 7 p53 (+/–) mice treated with ENU, ENU + 1 ppm EE, ENU + 2.5 ppm EE, and ENU + MXC, respectively. The absolute uterine weights and uterine weight/body weight ratios in p53 (+/–) and p53 (+/+) mice in the ENU + 1 ppm EE, ENU + 2.5 ppm EE, and ENU + MXC groups were significantly higher than those in the ENU-alone group (Fig. 1Go). The uterine weights of p53 (+/–) mice, especially in the ENU + 2.5 ppm EE group were markedly increased because of marked growth of uterine tumors.

Histopathologically, uterine proliferative lesions were classified into endometrial stromal polyps, endometrial stromal sarcomas, an adenocarcinoma, atypical hyperplasias, and endometrial glandular hyperplasias. Endometrial stromal polyps were characterized by pedunculated masses protruding into the uterine lumen consisting of loosely organized endometrial stromal cells with scattered cystic endometrial glands and well-differentiated cuboidal epithelial cells on the surface. Endometrial stromal sarcomas were composed of sheets, bundles, and occasionally whorls of spindle-shaped cells with pale and large chromatin-rich nuclei, abnormal mitoses, and marked cellular atypia (Fig. 2AGo). The adenocarcinoma was characterized by diffusely glandular proliferation of large, atypical epithelial cells with clear cytoplasm, and invasion into the surrounding tissues being apparent (Fig. 2BGo). Atypical hyperplasias were classified into 2 cell types, clear (Fig. 2CGo) and basophilic (Fig. 2DGo), characterized by small proliferative foci of endometrial glandular epithelia with atypia. Non-atypical endometrial glandular hyperplasias were composed of increased numbers of endometrial glands with occasional cysts (Fig. 2EGo).



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FIG. 2. Uterine endometrial tumors from p53 (+/–) mice given 2.5 ppm EE after initiation of ENU, and sacrificed at week 26 (H&E stained). (A) Endometrial stromal sarcoma, showing many mitoses in spindle-shaped or pleomorphic cells with atypical nuclei, associated with mitoses. The incidence of this tumor in this group was significantly increased as compared to the ENU-alone group; magnification x100. (B) Uterine adenocarcinoma, showing diffusely glandular proliferation of large, atypical epithelial cells with clear cytoplasm; magnification x200. (C) Uterine atypical hyperplasia of the endometrial gland with clear cytoplasm, showing histologic features characterized by proliferative foci of atypical endometrial glandular epithelial cells. The incidence of this atypical hyperplasia in this group was significantly higher than that of the ENU-alone group; magnification x200. (D) Uterine atypical hyperplasia of the endometrial gland with basophilic cytoplasm; magnification x200. (E) Uterine endometrial glandular hyperplasia, showing histologic features characterized by loosely organized, endometrial stromal cells and increased numbers of endometrial glands; magnification x200.

 
With regard to uterine stromal tumors, the incidences in p53 (+/–) mice were 87% (47% stromal sarcomas, 40% polyps), 85% (64% stromal sarcomas, 21% polyps), 87% (stromal sarcomas), and 53% (stromal sarcomas) in the ENU, ENU + 1 ppm EE, ENU + 2.5 ppm EE, and ENU + MXC groups, respectively, there being a significant difference for stromal sarcomas between the ENU and ENU + 2.5 ppm EE groups (Fig. 3Go). In p53 (+/+) mice, only stromal polyps were seen, at incidences of 20, 13, 0, and 0% in the ENU, ENU + 1 ppm EE, ENU + 2.5 ppm EE, and ENU + MXC groups, respectively, their values displaying clear decreases compared to those of stromal tumors in their counterpart groups of p53 (+/–) mice. With regard to epithelial proliferative lesions, the incidences of atypical hyperplasias of clear-cell type in p53 (+/–) mice were 0, 14, 60, and 0% in the ENU, ENU + 1 ppm EE, ENU + 2.5 ppm EE, and ENU + MXC groups, respectively. Those in p53 (+/+) mice were 0, 7, 53, and 0%, with a significant difference between the ENU and ENU + 2.5 ppm EE groups in both cases. For atypical hyperplasias of basophilic cell type, there were no significant differences among the groups. One p53 (+/–) mouse of the ENU + 2.5 ppm EE group developed an adenocarcinoma consisting of clear cells. The incidences of glandular hyperplasias for p53 (+/–) mice were 60, 79, 60, and 27% in the ENU, ENU + 1 ppm EE, ENU + 2.5 ppm EE, and ENU + MXC groups, respectively, whereas those for p53 (+/+) mice were 60, 80, 100, and 100%, the incidences in the ENU + 2.5 ppm EE and ENU + MXC groups of p53 (+/+) mice showing significant differences from the ENU-alone group value.



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FIG. 3. Incidences of uterine proliferative lesions in p53 (+/–) and p53 (+/+) mice treated with EE or MXC for 26 weeks after ENU initiation. Uterine proliferative lesions were classified into endometrial stromal polyps (ESP), endometrial stromal sarcomas (ESS), one adenocarcinoma (Adc), atypical hyperplasias of clear-cell type (AhC), atypical hyperplasias of basophilic-cell type (AhB), and endometrial glandular hyperplasias (Gh). Asterisks indicate significant differences from the values of the ENU-alone group (p < 0.05).

 
With regard to non-proliferative lesions in the uterus, dilatation of the endometrial glands, suggestive of estrogen action, was observed in all of the ENU + MXC groups of p53 (+/–) and p53 (+/+) mice as well as the ENU + EE groups of p53 (+/+) mice. However, such a change was not consistently apparent in the ENU + EE groups of p53 (+/–) mice because of the expansive growth of endometrial stromal sarcomas.

The PCNA labeling indices for the atypical hyperplasias, consisting of clear cells and the endometrial adenocarcinoma in the ENU + 2.5 ppm EE group, were significantly and markedly higher than that for glandular hyperplasias (Fig. 4Go).



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FIG. 4. PCNA labeling indices for uterine proliferative lesions in p53 (+/–) mice of the ENU + 2.5 ppm EE group. Asterisks indicate significant differences from the values of "glandular hyperplasia" (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been postulated that genomic instability, defined as genetic changes such as deletions and point mutations at the level of signal genes, can be caused by loss of function of the p53 gene (Fukasawa et al., 1997Go). A contribution of such genomic instability to carcinogenesis might explain the fact that homozygous p53-deficient mice [p53 (–/–) mice] in which both p53 alleles are inactivated develop tumors early in their life (Donehower et al., 1992Go, Fukasawa et al., 1997Go). In our previous study, we demonstrated rapid induction of uterine tumors with a point mutation of GCG (Ala) to GTG (Val) transition at codon 135 of exon 5 of the p53 gene in p53 (+/–) CBA mice given a single intraperitoneal injection of ENU (Mitsumori et al., 2000Go). The present study was performed to examine whether EDCs such as EE and MXC have any tumor-modifying effects on the uterine tumors induced by ENU in p53 (+/–) mice that are considered not to have any p53 tumor-suppressor function.

Uterine endometrial adenocarcinomas and atypical hyperplasias have been reported to be significantly increased in ICR mice fed a diet containing 5 ppm 17ß-estradiol for 20 weeks after intra-vaginal instillation of 10 mg/kg of N-methyl-N-nitrosourea (MNU), as compared to the case with MNU treatment alone (Niwa et al., 1991Go). In addition, Maekawa et al. (1999) reported high production of uterine endometrial adenocarcinomas in CD mice given subcutaneous implantation of 17ß-estradiol for 15 weeks after a single intra-uterine application of ENU. On the other hand, in the present study, treatment with 2.5 ppm EE enhanced the development of ENU-induced uterine endometrial-stromal sarcomas, a significant increase of such sarcomas in p53 (+/–) mice of the ENU + 2.5 ppm EE group as compared to the ENU group being found. It has been reported that the uterine stromal sarcomas induced by 1,2-dimethylhydrazine (DMH) are estrogen-sensitive, and that combined DMH and estrogen treatment results in a reduced latency period and higher incidence of uterine stromal sarcomas (Turusov et al., 1994aGo). These findings strongly suggest that EE also has a tumor-promoting effect on the endometrial stromal cells of the uterus.

A question thus arises as to whether endometrial stromal sarcomas are specifically induced in p53 (+/–) mice or can be easily induced in CBA mice per se by the treatment of ENU. It has been reported that such uterine sarcomas were also induced with a high incidence in CBA mice by repeated subcutaneous administration of DMH, an alkylating agent (Turusov et al., 1994bGo). The strains of mice that are most susceptible are reported to be CBA and C3H (Trukhanova et al., 1998Go, Turusov et al., 1994bGo). The p53 (+/–) mice used in our previous (Mitsumori et al., 2000Go) and present studies are F1 offspring of heterozygous p53-deficient C57BL/6J male mice backcrossed with CBA female mice (Tsukada et al., 1993Go), and are accordingly on a CBA genetic background. The CBA strain thus appears to be susceptible to the induction of uterine endometrial stromal sarcomas by the administration of alkylating agents such as DMH and ENU, irrespective of the genetically engineering procedure to knock out the p53 gene.

With regard to epithelial proliferative lesions in the uteri in our study, the induction of atypical hyperplasias of clear cell type was enhanced in p53 (+/–) and p53 (+/+) mice of the ENU + 2.5 ppm EE group and the only endometrial adenocarcinoma was found in a p53 (+/–) mouse receiving ENU + 2.5 ppm EE. In addition, the PCNA-labeling indices of the atypical hyperplasias of clear-cell type and this endometrial adenocarcinoma were significantly higher than those of glandular hyperplasias. These results are in line with a pathogenesis leading from atypical hyperplasias to adenocarcinomas in humans. The fact that atypical hyperplasias were also observed in a few ENU + 1 ppm EE mice of both p53 (+/–) and p53 (+/+) types suggests that adenocarcinomas may also be induced by treatment with 1 ppm EE over a long period. Such uterine endometrial adenocarcinomas and atypical hyperplasias have been shown to occur in ICR mice given 17ß-estradiol for 15 to 20 weeks after initiation of MNU or ENU (Maekawa et al., 1999Go; Niwa et al., 1991Go; Takahashi et al., 1996Go). These atypical hyperplasias in ICR mice had a morphological resemblance to those observed in our study. The data thus strongly indicate that EE exerts tumor-promoting effects on the endometrial epithelial cells as well as stromal cells of the uterus.

No experiments designed to clarify the potential mechanism in the enhancement of uterine carcinogenesis of EE have been done yet. Administrations of EDCs that have estrogenic action are uterotrophic to immature or ovariectomized rats, resulting in the rapid growth of uterine tissues (Astwood, 1938Go). It is known that such chemicals typically bind the estrogen receptor, that the estrogen receptor-ligand complex subsequently interacts with estrogen-response elements (ERE), and that transcription factors are recruited to the estrogen receptor-ligand/ERE complex, resulting in enhanced mRNA synthesis and gene expression of polypeptide growth factors such as epidermal growth factor (EGF) and transforming growth factor-{alpha} (TGF{alpha}), which are responsible for cell proliferation (Gardner et al., 1989Go; Nelson et al., 1992Go; Sumpter et al. 1996Go). The tumor-promoting effect of EE to the uterus, observed in the present study, is considered to be attributable to the above-mentioned biological effects of estrogenic compounds, mainly by binding to estrogen receptors. However, additional studies to clarify the potential mechanism on the tumor-promoting effects of EE are absolutely needed.

In the present study, the induction of endometrial stromal sarcomas as well as atypical hyperplasias was not enhanced in p53 (+/–) mice of the ENU + MXC group, although the increased uterine weights and cystic dilatation of the endometrial glands, consistently observed in both p53 (+/–) and p53 (+/+) mice given the chemical, indicate that MXC per se has an estrogenic action. Since MXC has been reported to be an EDC with a weak estrogenic potential (Bulger et al., 1978Go, Gray et al., 1989Go), we suspected that it might have some tumor-promoting effect on the uterus, acting via estrogen-receptor binding, but the negative result in our study suggests that MXC does not have such a promoting effect on the uterus in either p53 (+/–) or p53 (+/+) mice. However, it would be premature to conclude from the present study that endocrine disruptors with a strong estrogenic activity have tumor-promoting potential in the uterus while those with weak estrogenic activity do not. Additional studies using other endocrine disruptors with weak estrogenic activity, such as bisphenol A, nonylphenol, and genistein are now in progress to clarify this point.

In conclusion, the present study indicates that treatment with 2.5 ppm EE, but not with MXC, enhanced the development of ENU-induced uterine endometrial stromal sarcomas and atypical hyperplasias of clear-cell type in p53 (+/–) mice initiated with ENU, and suggests that only EE with high estrogenic activity exerts tumor-promoting effects on endometrial-stromal and epithelial proliferative lesions of the uteri.


    ACKNOWLEDGMENTS
 
This work was supported in part by a grant-in-aid from the Ministry of Health and Welfare of Japan for research on modifying effects of endocrine-disrupting chemicals on tumorigenesis.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +81-3-3700-1425 E-mail: mitsumor{at}nihs.go.jp. Back


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