* Division of Pathology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; and
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
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ABSTRACT |
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Key Words: N-ethyl-N-nitrosourea; ethinylestradiol; methoxychlor; uterine tumorigenesis; and heterozygous p53-deficient mice.
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INTRODUCTION |
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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., 1994, Vickers and Lucier, 1991
). Methoxychlor (MXC) has been reported to be a weak estrogenic pesticide that can alter fertility in male and female rats (Bulger et al., 1978
), but no clear positive results have been obtained in the majority of the studies on its potential carcinogenicity (Deichmann et al., 1967
, Radomski et al., 1965
). 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.
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MATERIALS AND METHODS |
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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., 2000). 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 45 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.
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RESULTS |
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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. 2A). 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. 2B
). Atypical hyperplasias were classified into 2 cell types, clear (Fig. 2C
) and basophilic (Fig. 2D
), 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. 2E
).
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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. 4).
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DISCUSSION |
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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., 1991). 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., 1994a
). 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., 1994b). The strains of mice that are most susceptible are reported to be CBA and C3H (Trukhanova et al., 1998
, Turusov et al., 1994b
). The p53 (+/) mice used in our previous (Mitsumori et al., 2000
) and present studies are F1 offspring of heterozygous p53-deficient C57BL/6J male mice backcrossed with CBA female mice (Tsukada et al., 1993
), 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., 1999; Niwa et al., 1991
; Takahashi et al., 1996
). 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, 1938). 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-
(TGF
), which are responsible for cell proliferation (Gardner et al., 1989
; Nelson et al., 1992
; Sumpter et al. 1996
). 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., 1978, Gray et al., 1989
), 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.
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ACKNOWLEDGMENTS |
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NOTES |
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