Association Between Ovarian Tumors and Uterine Endometrial Lesions in Rats*

J Toxicol Pathol 5: 215•`222, 1992

ASSOCIATION BETWEEN OVARIAN TUMORS AND UTERINE ENDOMETRIAL LESIONS IN RATS*

Hiroshi Onodera, Yuko Matsushima, and Kunitoshi Mitsumori Division of Pathology, Biological Safety Research Center, National Institute of Health Sciences

Takaharu Nagaoka

Research Laboratories, Yoshitomi Pharmaceutical Industries Ltd.

Tsuyoshi Kitamura, Jin Lu, and Akihiko Maekawa

Department of Pathology, Sasaki Institute

Abstract: Granulosa cell tumors/luteomas of the ovary were induced at very high incidence in the offspring of F344 rats receiving N-nitrosobis (2-oxopropyl)amine (BOP) transplacentally (Jpn. J. Cancer Res., 81: 1077-1080, 1990). In addition to ovarian tumors, atrophic and hyperplastic lesions were also frequently observed in the ovary and uterus of BOP-treated animals, respectively. In order to investigate the relation between granulosa cell tumors and uterine endometrial lesions, the lesions in BOP-treated rats with ovarian tumors were compared to those in the rats without tumors. While no endometrial adenocarcinoma was found in both rat groups, the incidence and degree of atypical, and adenomatous endometrial hyperplasias were slightly high in rats with ovarian tumors, as compared to rats without tumors, while being not significant. In the assay of plasma gonad steroids, hormonal imbalance was clearly detected in BOP-treated animals and the 17ƒÀ-estradiol: progesterone (E2: P) ratio was higher in BOP-treated rats with ovarian tumors than control rats or treated rats without tumors. These results suggest that a slight association between ovarian tumors and endometrial proliferative lesions exists in rats and hormonal imbalance, particularly an increased E2: P ratio, might play an important role in the association. (J Toxicol Pathol 5: 215•`222, 1992) Key words: Ovarian tumor, Granulosa cell tumor, Hormonal imbalance, Endometrial hyperplasia/ carcinoma, F344 rat

important role in the development of endometrial Introduction carcinomas1.2. Granulosa cell tumor of the ovary An association between endocrine imbalance has been well known as one of the high risk factors and uterine endometrial adenocarcinoma in for the tumors3-5. In mice, it has been pointed women has been documented in the literature, and out that granulosa cell tumors are accompanied by it has been pointed out that estrogen may play an cystic and/or hyperplastic changes in the endometrium6, although there is no report con 小野寺博志松島裕子三森国敏永岡隆晴北村毅 cerning the relation between granulosa cell tumors 路進前川昭彦 and endometrial lesions in rats. Previously Accepted for publication: July 8, 1992 , we Mailing address: Akihiko Maekawa, Department of reported high yields of granulosa cell tumors and/ Pathology, Sasaki Institute, 2-2 Kanda-Surugadai, or luteomas in F344 rat ovaries after transplacental Chiyoda-ku, Tokyo 101, Japan. administration of N-nitrosobis (2-oxopropyl) * Part of this work was presented at the 6th Annual Meeting of the Japanese Society of Toxicological Pathol amine (BOP)7. In addition to ovarian tumors , ogy in Sapporo, February, 1990. some endometrial lesions were also frequently 216 OVARIAN TUMORS AND ENDOMETRIAL LESIONS IN RATS found in BOP-treated animals. Ovarian tumors and all reproductive organs were In the present study, we describe the detailed fixed in buffered 10% formalin, and sections were histological findings of female sex organs including routinely prepared and stained with hematoxylin the uterine endometrium observed in BOP-treated and eosin, and examined microscopically. In a rats and discuss the association between granulosa few animals of both groups, the plasma values of cell tumors/luteomas and endometrial proliferative gonad steroids such as 17ƒÀ-estradiol (E2) and lesions in rats. progesterone (P) were assayed. The precise exper imental design was described in a previous paper7.

Materials and Methods Results Rats: Female F344/DuCrj rats were pur chased from Charles River Japan Inc. (Kanagawa) Ovarian tumors were induced at very high and maintained on basal diet CRF-1 (Oriental incidence in the offspring of rats receiving BOP Yeast Ind. Co., Tokyo) and tap water. transplacentally, as shown in Table 1. His N-Nitrosobis (2-oxopropyl) amine: N-Nitro tologically, all ovarian tumors were of the sobis (2-oxopropyl) amine (BOP) was purchased granulosa cell tumor and/or luteoma type (Figs. from Ash Stevens Co. (Detroit, MI). 1-3). Data for sites, types, and incidences of all Experimental design: The experiment was tumors observed in the various organs/tissues of performed under the same design as reported by BOP-treated animals, and also for histological Pour8. Pregnant rats were given 3 subcutaneous findings of ovarian tumors were described in a injections of 10 mg/kg of BOP on the 14th, 18th, previous report7. and 20th days of gestation. Control animals All neoplastic and non-neoplastic lesions received saline only in the same manner. Animals observed in the female reproductive organs are were weaned on the 4th week, and then female rats summarized in Table 1. In the ovary, follicular were maintained on basal diet and observed for cysts and atrophic changes such as absence of the 87-93 weeks. when all survivors were killed. follicle and/or corpus luteum, in addition to

Table 1. Neoplastic and Non-neoplastic Lesions of the Female Genital Organs in Offspring Receiving BOP Transplacentally Onodera, Matsushima, Mitsumori, el a1. 217

Fig. 1. Granulosa cell tumor observed in a 93-wk-old BOP-treated F344 rat. H. E. stain, •~120

Fig. 2. Typical luteoma from an 89-wk-old BOP-treated rat. H. E. stain, •~150 granulosa cell tumors/luteomas, were observed findings in the uterine endometrium between BOP more frequently in BOP-treated animals than in treated animals with and without ovarian tumors. controls. Especially, ovarian atrophy (Fig. 4) The incidence of atypical and/or adenomatous was found in all BOP-treated rats without ovarian endometrial hyperplasias was slightly higher and tumors or in all ovaries without tumor. In the also the degree of hyperplasias was more severe in uterus, tall columnar epithelium, squamous meta rats with ovarian tumors than rats without tumors, plasia, and atypical and/or adenomatous hyper while being not significant. The incidences of plasia (Figs. 5-8) of the endometrium were also other endometrial lesions were also slightly, but more frequently found in the treated group, as not significantly, increased in BOP-treated rats compared to the control group (Fig. 9), although with ovarian tumors, as compared to rats without no endometrial adenocarcinoma was detected in tumors. both rat groups. In the vagina, cornification of Figure 10 shows the E2: P ratio in the con the epithelium was detected more commonly in the trol and BOP-treated groups. The plasma values treated animals, while the incidence of columnar of gonad steroids, especially E2, were markedly epithelium was higher in the control rats. decreased in BOP-treated animals without ovarian Table 2 shows the comparison of histological tumors, as compared to control animals, while 218 OVARIAN TUMORS AND ENDOMETRIAL LESIONS IN RATS

Fig. 3. Granulosa cell tumor mixed with luteoma from an 89-wk-old BOP-treated rat. H-E. •~120

Fig. 4. Atrophy of the ovary from a 93-wk-old BOP-treated rat. In the ovary, absence of follicle and corpus luteum, and diffuse hyperplasia of granulosa cells are observed. No ovarian tumor was found. H-E. •~40

there was wide variation in their values. The of successful induction, as reviewed previously9. decrease (especially for E2), however, was much Therefore, the experimental system reported less pronounced in rats with ovarian tumors previously7 may serve as a good animal model for although the values were still lower than those in granulosa cell tumor/luteoma in women. Atro control animals. The E2: P ratio in BOP-treated phic changes observed to occur in the ovaries of rats with ovarian tumors, especially granulosa cell the BOP-treated group may be of key importance tumors, was higher than that in the controls and in for induction of ovarian tumors. The influence of BOP-treated rats without tumors. plasma FSH (follicular stimulating hormone), LH (luteinizing hormone), and other related hormones is still uncertain and is the subject of ongoing Discussion research in our laboratory. While a number of methods have been used On the other hand, it is well known that with various degrees of success in attempts to spontaneous occurrence of uterine tumors in rats is induce ovarian tumors in rats, there are few reports generally common, but there are strain-differences Onodera, Matsushima, Mitsumori, et at 219

Fig. 5. Endometrial hyperplasia from a 92-wk-old BOP-treated rat. In this rat, a typical luteoma of the left ovary was detected. H. E. stain, •~50.

Fig. 6. Higher magnification of Fig. 5. H. E. stain, •~120

in their histological types10. As reported previ observation of spontaneous endometrial adenocar ously, endometrial stromal polyp was the most cinoma development in rats, it was also demon frequently observed tumor in the F344 rat strain strated that occurrence of these endometrial lesions and occurrence of endometrial adenocarcinoma was very rare until 15 months of age in F344 rats, was very rare11, while in the Donryu rat strain the as compared to Donryu rats14. These results indi most common tumor was of endometrial adenocar cate that endometrial lesions observed in the exper cinoma type 12, and it was shown that hormonal imental group are associated with BOP-treatment. imbalance might play an important role in their Ovarian atrophy induced by BOP-treatment and occurrence 13. In the present study, various lesions hormonal imbalance thereafter may be most impor of the ovary, uterus, and vagina were more fre tant for the occurrence of these uterine lesions. quently observed in BOP-treated F344 rats than in Plasma values of gonad steroids demonstrated control rats. Many of these histological findings clear hormonal imbalance and the absolute plasma observed in treated animals are similar to those values of gonad steroids decreased in the BOP observed in aged Donryu rats 13, and considered to treated group. This result coincides with ovarian be signs of estrogenization. In the sequential atrophy in this group. The E2: P ratio was 220 OVARIAN TUMORS AND ENDOMETRIAL LESIONS IN RATS

Fig. 7. Endometrial hyperplasia from an 89-wk-old BOP-treated rat. Cellular and structural atypism is prominent. This rat also had a typical luteoma shown in Fig. 2. H. E. stain, •~150

Fig. 8. Endometrial hyperplasia from an 89-wk-old BOP-treated rat. In this rat, both ovaries were atrophic and no ovarian tumor was detected. The degree of hyperplasia was very slight, compared to those in the cases of Figs. 5-7. H. E. stain, •~90

higher in rats with ovarian tumors than in control higher in rats with ovarian tumors than rats rats or rats without tumors, and increase of E2: P without tumors. There was also no difference in ratio was particularly predominant in rats with the incidence of these changes between rats with typical granulosa cell tumors. Contrary to expec typical granulosa cell tumors and rats with typical tation, however, no endometrial adenocarcinoma luteomas. The results indicate that an association was observed in BOP-treated rats with ovarian between granulosa cell tumors and endometrial tumors. No significant increase of atypical and/ proliferative lesions seems not to be strong from or adenomatous endometrial hyperplasias, which the view point of histological findings, although have been considered as precancerous changes not the endocrinological finding showed a possible only in humans15-17but also in rats14, was found association. As one of the reasons in which an in BOP-treated rats with ovarian tumors, as association was not so prominent under the present compared to rats without tumors, while the inci experimental condition, the experimental period dence of these precancerous changes was slightly might be short for occurrence and development of Onodera, Matsushima, Mitsumori, et al. 221

Fig. 9. Endometrium from an 87-wk-old rat in the control group. The endometrium is not hyperplas tic, but slightly atrophic. H. E. stain, •~150

Table 2. Endometrial Lesions in BOP-treated Rats with or without Ovarian Tumors

Fig. 10. Serum estrogen (E2): progesterone (P) ratio in offspring receiving BOP transplacentally. endometrial hyperplasias and/or adenocar more resistant for induction of endometrial cinomas. Another important reason may be the adenomas/carcinomas by estrogen+N-methyl-N strain difference. As mentioned above, spontane nitrosourea than Donryu rats 18. Therefore, if the ous occurrence of endometrial adenocarcinoma Donryu rat strain was used in place of the F344 rat was very rare in F344 rats, while the most common strain, the association between ovarian tumors and uterine tumor was of endometrial adenocarcinoma endometrial lesions might be more clear. This type in Donryu rats13. In fact, F344 rats were problem is now one of the subjects of ongoing 222 OVARIAN TUMORS AND ENDOMETRIAL LESIONS IN RATS research in our laboratory. 9. Maekawa, A: Tumours of the ovary. In: Pathol ogy of Tumours in Laboratory Animals. Vol. 1, From these results, it was suggested that a Tumours of the rat (second edition), Turusov, VS slight association between ovarian tumors and and Mohr, U, IARC, Lyon, pp. 473-497, 1990. endometrial proliferative lesions exists and hor 10. Maekawa, A: Neoplasms of the female reproductive monal imbalance, particularly an increased E2: P organs. In: Atlas of Tumor Pathology of the Fischer Rat. Stinson, SF, Schullar, HM and Rez ratio, might play an important role in the associa nik, G, CRC Press, Florida, pp. 431-472, 1990. tion. 11. Maekawa, A, Kurokawa, Y, Takahashi, M, Kokubo, T, Ogiu, T, Onodera, H, Tanigawa, H, Ohno, Y, Furukawa, F, and Hayashi, Y: Spontaneous tumors References in F344/DuCrj rats. Gann 74: 365-372, 1983. 12. Maekawa, A, Onodera, H, Tanigawa, H, Furuta, K, 1. Fox, H: Endometrial carcinogenesis and its relation Matsuoka, C, Kanno, J, Ogiu, T, and Hayashi, Y: to estrogens. Pathol Res Pract 179: 13-19, 1984. Spontaneous neoplastic and non-neoplastic lesions 2. Zeil, HK: Estrogen's role in endometrial cancer. in aging Donryu rats. Gann 77: 882-890, 1986. Obstet Gynecol 60: 509-515, 1982. 13. Nagaoka, T, Onodera, H, Matsushima, Y, Todate, 3. Greene, JW: Ferminizing mesenchymomas A, Shibutani, M, Ogasawara, H, and Maekawa, A: (granulosa and theca cell tumors) with associated Spontaneous uterine adenocarcinomas in aged rats endometrial carcinoma: Review of the literature and their relation to endocrine imbalance. J Cancer and a study of the material of the Ovarian Tumor Res Clin Oncol 116: 623-628, 1990. Registry. Am J Obstet Gynecol 74: 31-41, 1957. 14. Nagaoka, T, Onodera, H, Matsushima, Y, Lu, J, and 4. Gusberg, SB and Kardon, P: Proliferative en Maekawa, A: Sequential observation of spontane dometrial response to theca-granulosa cell tumors. ous endometrial adenocarcinoma development in Am J Obstet Gynecol 111: 633-643, 1971. Donryu rats. Toxicol Pathol submitted. 5. Ingram, JM and Novak, E: Endometrial carcinoma 15. Gusberg, SB and Kaplan, AL: Precursors of corpus associated with ferminizing ovarian tumors. Am J cancer. IV, Adenomatous hyperplasia as stage 0 Obstet Gynecol 61: 774-784, 1951. carcinoma of the endometrium. Am J Obstet 6. Lemon, PG and Gubareva, AV: Tumours of the Gynecol 87: 662-678, 1963. ovary. In: Pathology of Tumours in Laboratory 16. Sherman, AL and Brown, S: The precursors of Animals. Vol. II Tumours of the mouse, Turusov, endometrial carcinoma. Am J Obstet Gynecol 135: VS, IARC, Lyon, pp. 385-409, 1979. 947-956, 1979. 7. Maekawa, A, Onodera, H, Matsushima, Y, Nagaoka, 17. Kurman, RJ, Kaminski, PF, and Norris, HI: The T, Todate, A, Kanno, J, Shibutani, M, Ogasawara, behavior of endometrial hyperplasia: A long-term H, Yoshida, J, and Hayashi, Y: High yields of study of "untreated" hyperplasia in 170 patients. granulosa cell tumors/luteomas in F344 rat ovaries Cancer 56: 403-412, 1985. after transplacental administration of N 18. Nagaoka, T, Onodera, H, and Maekawa, A: Induc nitrosobis (2-oxopropyl)amine. Jpn J Cancer Res tion of endometrial adenocarcinomas in rats by 81: 1077-1080, 1990. estrogen and N-methyl-N-nitrosourea (in 8. Pour, PM: Transplacental induction of gonadal Japanese). Proc Jpn Soc Toxicol Pathol 7: 22, tumors in rats by a nitrosamine. Cancer Res 46: 1991. 4135-4138, 1986.