Induction of different types of uterine adenocarcinomas in Donryu rats due to neonatal exposure to high-dose p-t-octylphenol for different periods
Midori Yoshida3,
Shin-ichi Katsuda1,
Tomo Tanimoto,
Sayaka Asai2,
Dai Nakae,
Yuji Kurokawa,
Kazuyoshi Taya2 and
Akihiko Maekawa
Department of Pathology, Sasaki Institute, 22 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062,
1 Department of Biological Safety Research, Japan Food and Chemical Laboratories, Tokyo 206-0025 and
2 Veterinary Physiology, Tokyo University of Technology and Agriculture, Tokyo 183-8509, Japan
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Abstract
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Inappropriate exposure to estrogens in the fetal and/or newborn period can exert irreversible influence, including carcinogenesis on the reproductive system in mammals. The present study was conducted to investigate uterine carcinogenesis in Donryu rats treated neonatally with a high-dose estrogenic compound, p-t-octylphenol (OP) for different exposure periods. Female Donryu rats were subcutaneously administered 100 mg/kg/day OP every other day for the first 5 postnatal days (PNDs 15) or the first 2 weeks (PNDs 1-15). They received a single injection of 20 mg/kg N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) into a uterine horn at 11 weeks of age and were examined until 15 months of age. PNDs 15 OP-treated rats showed normal development of the female reproductive system, including uterine gland genesis and normal estrous cycling after vaginal opening. The treatment, however, accelerated an earlier occurrence of persistent estrus and increased the number of well differentiated uterine adenocarcinomas as compared with controls. This indicated that PNDs 15 OP treatment acts as a delayed modulator of the hypothalamus-pituitary-ovarian hormonal control system and the modulation increased the serum estrogen:progesterone ratio, resulting in induction of uterine tumors. On the contrary, PNDs 115 OP treatment demonstrated immediate and irreversible influences on the control system, called androgenization, and induced abnormal uterine development manifested by prolonged persistent estrus immediately after vaginal opening and also suppression of uterine gland genesis. In addition, uterine tumor malignancy in morphological and biological property clearly increased in this group although the total number of adenocarcinomas was not increased. The present study provides evidence that neonatal exposure to a high-dose OP enhances uterine carcinogenesis in rats, and the type of uterine tumors is changed by the periods of neonatal exposure to OP, suggesting that the mechanism of uterine tumor development is dependent upon neonatal exposure periods.
Abbreviations: DES, diethylstilbestrol; ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; ER
, estrogen receptor
; FSH, follicle-stimulating hormone; LH, luteinizing hormone; OP, p-t octylphenol; PNDs, postnatal days.
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Introduction
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The uterine endometrial carcinoma is one of the most common malignant tumors in women, and it is pointed out that estrogens may play essential roles in its development, although some epidemiological aspects remain unclear. Menoxenia, polycystic ovary syndrome, chronic anovulation, estrogen replacement therapy and the nulliparous state have been listed as risk factors (14). In experimental studies, however, there is only limited evidence due to the very low incidence of spontaneous uterine cancers in rodents. We have documented that the Donryu rat is a high-incidence strain for spontaneous endometrial adenocarcinomas with aging and the tumors have morphological and biological similarities to those found in humans (5,6). Tumor development in this strain is thought to be associated with an age-related imbalance of ovarian hormone, i.e. elevated estrogen:progesterone ratio (7). A single intrauterine administration of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) elevated the incidence and early occurrence of the tumor in this rat strain; therefore, we have established a two-stage rat uterine carcinogenesis model and shown that it is helpful in the clarification of tumor promotion effects of various chemicals (8,9).
On the other hand, exposure to estrogens in the fetal or newborn period can also induce uterine tumors in mice and rats (10,11). In rodents, the reproductive tract undergoes rapid growth and differentiation within the first 2 weeks of postnatal life named a critical point or a unique window of vulnerability because this period is very sensitive with regard to exogenous estrogens and/or androgens. In female mammals, inappropriate neonatal exposure to these hormones is known to induce the serious effect on the reproductive system, called androgenization due to irreversible disruption of the hypothalamus-pituitary-gonadal control system resulting in lowering of gonadotropins at prepuberty, in addition to direct actions to the target organs (12,13). Perhaps the most striking examples of the effects are induction of vaginal and/or cervical carcinomas by DES (10,14). Abnormal gene imprinting or role of the estrogen receptor (ER) is also proposed as one of the mechanisms of tumor development (15,16). However, the neonatal effects of estrogens on the target organs or tissues are very complex and the underlying mechanism remains to be clarified in many cases. In particular, there is little documentation about the influence of exposure times in the newborn period on uterine carcinogenesis.
Quite recently, we reported promotion effects of high-dose p-t octylphenol (OP), an environmental chemical with estrogenic activity (1725), on uterine carcinogenesis using our established model (26). However, neonatal effects on uterine carcinogenesis have not been determined to our knowledge. The compound demonstrated strong and irreversible androgenized effects in female rats, such as lower gonadotropin levels at prepuberty, inhibited uterine gland genesis, persistent estrus and polycystic ovaries when given to high-dose OP-treated rats for the first 2 weeks after birth, and the uteri exhibited luminal epithelial hyperplasias at 8 weeks of age, being similar to preneoplastic lesions observed in Donryu rats spontaneously with aging (6,18).
Furthermore, a 5-day exposure to high-dose OP in newborn animals altered estrous cyclicity in only a few animals, while persistent estrus was observed in all rats treated with the same dose of OP for the first 2 weeks of life (18). In addition, treatment at the same dose for the first 1 or 3 days did not affect any estrous cyclicity (unpublished data). The results point to the possibility that neonatal effects of estrogenic compounds might be altered by the period of exposure. In the present study, therefore, we investigated neonatal effects of high-dose OP on uterine carcinogeneses in Donryu rats, and compared the influence of different exposure periods.
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Materials and methods
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Animals, housing conditions and treatment of OP
Adult female and male Crj:Donryu rats were purchased from Charles River Japan (Kanagawa, Japan). Females were mated with males on proestrus day and plugs and sperm in the vagina were utilized to judge pregnancy.
Dams with offspring were housed in plastic cages until the pups were weaned. Dams with offspring were divided into three groups: a 0 mg/kg/day group (control group, 56 female pups), a 100 mg/kg/day OP (Wako Pure Chemicals, Osaka, Japan) group receiving OP from PND1 (within 24 h of the birth) to PND 5 (PNDs 15 OP-treated group, 46 female pups) and another treated group given the same dose of OP from PND1 to PND15 (PNDs 115 OP-treated group, 54 female pups). OP was dissolved in dimethylsulfoxide (Wako Pure Chemicals) for this purpose and injected s.c. under the back skin of pups every other day. In our previous study, the dose of 100 mg/kg/day OP from PND1 to PND 15 was effective for estrogenic activity (18,2325), although extremely high compared with the environmental concentration level (27).
The day of birth was designated postnatal day (PND) 0. Litter sizes were adjusted to 810 pups/dam at PNDs 4 or 6. All pups were weaned at PND21 and females were segregated from males. Female rats in the same treatment group were housed in cages (three or four per cage) and maintained until necropsy in an air-conditioned animal room under constant conditions of 24 ± 2°C and 55 ± 10% humidity with a 12 h light:dark cycle. Access to basal diet CRF-1 (Oriental Yeast Inc., Tokyo) and tap water was ad libitum. Animal care and use followed the NIH Guide for the Care and Use of Laboratory Animals.
Estrous cyclicity
After weaning, animals were checked daily for vaginal opening. Estrous cyclicity was manifested by examination of vaginal smears throughout the study.
Sequential observation on uterine growth and differentiation of pups up to puberty
To investigate uterine growth and gland development during the neonatal period, three or four animals per group were euthanized at PNDs 10, 14, 21 and 28, the uteri were weighed, and numbers of uterine glands per cross-section of uterine horn were histopathologically quantified.
Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels at PNDs 10, 14, 21 or 28 were measured using NIDDK radioimmunoassay kits for rat FSH and LH, for comparison between control and PNDs 15 OP-treated groups (28). Until PND14, pooled serum samples were analyzed. Data for assays of PNDs 115 OP-treated rats have already been reported (18), and hormonal assays in the PNDs 115 OP-treated group were not done in the present study. The serum was provided using the same animals examined for uterine growth and gland genesis.
Uterine carcinogenicity study
As treatment with carcinogen, all female pups at 11 weeks of age were administered a single dose of 20 mg/kg ENNG (Nacarai Tesque Inc., Tokyo, Japan) into the uterine horn using a stainless steel catheter via the vagina, as reported previously (8). At 12 months of age, five or six animals per group were killed and examined to evaluate development of uterine proliferative lesions. At the termination, at 15 months of age, all surviving animals underwent a histopathological examination. Animals found dead and killed when moribund were also examined similarly. At necropsy, the reproductive organs were fixed in 10% neutral buffered formalin, and then routinely processed. Each uterus was cut into ~12 slices in cross-section for hematoxylin and eosin staining. Proliferative endometrial lesions were classified into three degrees of hyperplasia (slight, +; moderate, ++; severe, +++) and adenocarcinomas, according to our categories described previously (6). In addition, adenocarcinomas were subdivided into well (G1), moderately (G2) and poorly differentiated (G3) types, and also classified as to the degree of invasion: I-II (limited to the uterus), III (invading into the serosa and/or surrounding adnexae) and IV (tumors with distant metastasis), in accordance with the simplified FIGO histopathological grades for human uterine cancers (29).
Statistical analysis
Values for incidence were statistically analyzed using Fishers exact probability test. Other data were analyzed using ANOVA, and post hoc comparisons between OP-treated and control groups were made with Students t-test. P values <0.05 were considered to be statistically significant.
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Results
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The repeated injection of OP did not affect the growth and clinical observation of pups in both the OP-treated groups, except for slight scar formation observed at the injection sites during the treatment period, the lesion disappearing at weaning.
Effects of OP on uterine growth and gland genesis
Uterine weights were not different among the three groups. Sequential changes in numbers of uterine glands up to puberty are shown in Figure 1
. The values for animals in the PNDs 15 OP-treated group were comparable with those in the control group, whereas those in the PNDs 115 OP-treated group were significantly depressed. No obvious morphological changes without inhibition of uterine genesis were observed in either of the OP-treated groups before puberty.
Hormone profiles
Serum gonadotropin levels for the PNDs 15 OP-treated and control groups until 28 days of age are shown in Figure 2
. In sequential changes of FSH and LH levels, no significant decrease was detected in the PNDs 15 OP-treated group compared with controls although FSH levels in this group were varied with a wide range at PNDs.

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Fig. 2. Sequential change in gonadotropin levels (A, FSH; B, LH) before puberty in the control ( ) and the PNDs 15 OP-treated () groups. Serum was pooled from pups of the same age at each stage of development during the immature period, and analyzed for individual rats at later ages. Results presented as means ± SE.
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Estrous cyclicity
In control animals, precise 4-day cycles of the estrous stages started after vaginal opening. Persistent estrus, characterized by vaginal smears exhibiting nucleated epithelial and/or cornified cells, began to appear after 4 months of age and gradually increased with age in this group, all animals showing estrus at 11 months of age, as shown in Figure 3
. In PNDs 15 OP-treated rats, the time of vaginal opening was not different from that of controls, and most of the animals showed a regular estrous cycle similar to controls after the opening, although several showed irregular cyclicity. At 6 weeks of age in this group, however, persistent estrus already began to appear, and the incidence significantly increased with age compared with that of controls, becoming 100% by 6 months. In contrast, in PNDs 115 OP-treated rats, the time of vaginal opening was shortened to ~4 days compared with controls, and all animals then showed persistent estrus until the termination.
Carcinogenicity of OP
Data for uterine endometrial proliferative lesions and subclassified adenocarcinomas are summarized in Table I
. In both control and PNDs 15 OP-treated groups, hyperplasias and their grades increased with age dependence. In the PNDs 15 OP-treated group, well-differentiated adenocarcinomas were observed in two animals (40%) at 12 months of age, when none were detected in control animals. The incidences of adenocarcinomas in the control and PNDs 15 OP-treated groups at 15 months of age were 6/23 (26%) and 18/28 (64%), respectively, the difference being significant. Most of them were of well-differentiated type and limited in the uteri (Figure 4
), although one in the PNDs 15 OP-treated group had metastasized to the lungs. In contrast, in the PNDs 115 OP-treated group, total numbers of hyperplasias were significantly decreased at termination, compared with the controls. An adenocarcinoma was observed at 12 months of age, but the incidence at termination was almost the same as in the controls. However, in the subclassification of adenocarcinomas in this group, malignancy was clearly increased in terms of both histopathological grades, most tumors being of moderately or poorly differentiated type, and degree of invasion, involvement of the serosa and/or surrounding tissue or metastasis to the lung being prominent. In moderately differentiated adenocarcinomas, tumor cells with irregularly shaped glands were observed invading through the myometrium to the serosa of the uterus. In poorly differentiated cases, scattered cuboidal tumor cells without gland formation demonstrated invasion into the myometrium (Figure 5
). The ovaries in all groups, including the controls, were atrophic with small cystic follicles and a loss or decrease of corpus lutea at 12 and 15 months of age, the changes being similar to those known for aged rats of this strain.

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Fig. 4. (a) Well-differentiated endometrial adenocarcinoma limited to the uterus in a PNDs 15 OP-treated rat. x60. (b) Higher magnification of Figure 4a , x150. Hematoxylin and eosin stain.
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Fig. 5. (a) Moderatelydifferentiated endometrial adenocarcinoma disseminated into abdominal adipose tissue in a PNDs 115 OP-treated rat. x150. (b) Poorly-differentiated endometrial adenocarcinoma in a PNDs 1-15 OP-treated rat. Scattered cuboidal tumor cells losing gland formation and invading into myometrium. x200. Hematoxylin and eosin stain.
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Discussion
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The present study provides evidence that neonatal exposure to a high-dose OP enhanced uterine carcinogenesis in Donryu rats treated with ENNG. Most interestingly, different types of uterine adenocarcinomas, being different in biological and morphological properties, were noted dependent on the neonatal exposure periods.
Rats given OP for a short exposure-time (PNDs 15) showed normal uterine growth and development including gland genesis, normal serum gonadotropin levels at prepuberty, regular estrous cyclicity after vaginal opening but early occurrence of persistent estrus thereafter. Although a number of investigators reported effects of neonatal exposure to estrogens or androgens, few have revealed such a status in our present study except MacLusky and Naftolin (30). They proposed the status as delayed anovulatory syndrome characterized by a few normal estrous cycles after puberty and spontaneous anovulation observed in female rats treated neonatally with a low dose androgen, and focused on pointing to incomplete androgenization of the central nervous system although its mechanism remains to be undetermined (30). Our results are in line with the syndrome reported by MacLusky and Naftolin, and demonstrate that neonatal exposure to OP for the first 5 days of life did not disrupt completely the hypothalamus-pituitary-gonadal control system and that animals showed normal growth in early life, but affected delayed modulations of the system to enhance earlier occurrence of persistent estrus phage. Particularly, it should be emphasized that the delayed modulation results in an increase of uterine adenocarcinomas which were histologically and biologically similar to those observed spontaneously. Previously we investigated the mechanisms of spontaneous uterine adenocarcinoma development in the Donryu rat, a high-incidence strain of the tumor, and documented that earlier occurrence of modulation of ovarian hormonal control than other rat strains was a feature of Donryu rats (6). In this strain of rats, persistent estrus by vaginal cytology occurs after 4 months of age, and the incidence increases with agedependency. The most obvious histological changes in these animals are atrophy of the ovaries with polycystic follicles and decrease and/or lack of corpus lutea, resulting in prolonged increase of the serum estrogen:progesterone ratio (6,7). From the evidence, we have hypothesized that spontaneous uterine cancer development in this strain is strongly linked to age-dependent modulation of the ovarian hormonal control, similar to that in humans. From this point of view, PNDs 15 OP-treatment might promote ovarian aging in this strain of rat.
In the PNDs 115 OP-treated group, however, manifestations of OPs effects on the uteri before puberty have already differed from those in the PNDs 15 OP-treated group. We have already reported the effects on the female reproductive tract of rats given high-dose OP at PNDs 115, the serum FSH and LH being kept at low levels before puberty due to complete disruption of the hypothalamus-pituitary control system called androgenization and the uterine gland genesis also being inhibited (18,31). The present study partly confirmed the previous results and also added a new result, in that the large exposure of OP for 15 days clearly increased malignancy of uterine adenocarcinomas, although the treatment decreased the incidence of endometrial hyperplasias, preneoplastic lesions of uterine tumors, and did not increase the total number of uterine carcinomas. Perinatal exposure to high doses of estrogens suppresses uterine gland genesis in rats and mice (32,33). Carthew et al. (34) reported that endometrial adenocarcinomas without hyperplasias were induced in rats by neonatal treatment with tamoxifen and concluded that suppression of uterine gland genesis by the compound might be causally related to the low incidence of hyperplasias, and our present result is comparable with their result (34). In humans, it has been pointed out that the presence or absence of hyperplasia as the background is important for assessing the biological behavior of endometrial adenocarcinomas (35). Concerning the histogenesis of uterine adenocarcinomas in the Donryu rat, the tumors are considered to arise from hyperplasias of the luminal or glandular epithelium, especially the latter (6). We previously found luminal epithelial hyperplasia in the uteri at 8 weeks of age in rats exposed to high-dose OP at PNDs 115 (18). The increase of malignant uterine tumors without hyperplasias is therefore interesting for consideration of the histogenesis of uterine adenocarcinomas in rats. Employment of the present experimental model under different conditions will hopefully provide a useful insight into the case in humans.
Persistent estrus was a common result in the PNDs 15 and 115 OP-treated groups, although the manifested time was different in both groups. Persistent estrus is recognized to reflect on dysfunction of the hypothalamus-pitutary-ovarian hormonal control system, exhibiting an elevated estrogen: progesterone ratio as described above. After ovariectomy, persistent estrus and uterine hyperplasia promptly disappeared and changed to castration-like status (10,18), and no uterine tumors developed in ovariectomized mice exposed to DES neonatally (10). No tumors also developed in ovariectomized Donryu rats exposed to high-dose OP during adulthood (26). The present study also proposed that persistent estrus in existence of the ovary might be an important factor for development of uterine tumors.
On the other hand, as a direct action of estrogens on the uteri, Newbold et al. (10,36) and other investigators proposed the idea that DES acts to initiate transformation during neonatal development, causing permanent changes in the uterine target cells. Abnormal expression of estrogen receptor
(ER
) and induction of c-fos have been reported in the mouse uterus neonatally exposed to DES, supporting this conclusion (37,38). Recently, we also found neonatal exposure to OP for PNDs 1-15 altered differentiation of the developing uteri via abnormal ER
expression and subsequent alteration of cell proliferating activity (39). Therefore, we speculate that abnormal development of the uteri in rats treated neonatally with OP during PNDs 115 might contribute to tumor progression.
Previously we confirmed neonatal OP-treatment of 50 mg/kg/day every other day for PNDs 1-15 did not affect estrous cyclicity (18), the total administered dose (400 mg/kg) being higher than that (300 mg/kg) of the PNDs 15 group in the present study. In toxicokinetic studies, metabolic rates of the compound were rapid and it was no longer detectable 48 h after application, suggesting that the differences between the two groups in the present study are due to the treatment period, rather than total dosing volume (40,41).
In conclusion, neonatal exposure to high-dose OP during either the first 5 or 15 days after birth enhances uterine carcinogenesis in Donryu rats. Our hypothesis of the uterine carcinogenesis in the present study is shown in Figure 6
. The exposure for the first 5 days of life exerts an earlier appearance of persistent estrus, acts as a delayed modulator of the hypothalamus-pituitary-ovarian hormonal control system and results in increased incidence of well-differentiated uterine adenocarcinomas. On the contrary, the immediate disruption of the control system and the abnormal development of the uteri by the OP-treatment for the first 2 weeks lead to the evaluation of tumor malignancy without uterine hyperplasia, proposing an idea that mechanisms of uterine tumor development might be different between them.

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Fig. 6. Hypothesis of uterine carcinogenesis in the present study. PNDs 115, neonatal treatment of p-t octylphenol from 1 to 15 days of age. PNDs 15, neonatal treatment of p-t octylphenol from 1 to 5 days of age. E:P ratio, estrogen:progesterone ratio.
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Notes
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3 To whom correspondence should be addressed Email: pathol{at}sasaki.or.jp 
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Acknowledgments
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This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology. The expert technical assistance of Mrs Hiromi Ichihara for her histopathological examination is sincerely appreciated.
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Received February 1, 2002;
revised July 2, 2002;
accepted July 2, 2002.