Radiation-induced tumorigenesis of mammary glands in pituitary transplanted rats ovariectomized before onset of estrous cycle

Cancer Letters 138 (1999) 93±100

Radiation-induced tumorigenesis of mammary glands in pituitary transplanted rats ovariectomized before onset of estrous cycle Hiroshi Inano a,*, Keiko Suzuki a, Makoto Onoda a, Hisae Kobayashi b, Katsumi Wakabayashi b a

The First Research Group, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555, Japan b Institute for Molecular and Cellular Regulation, Gunma University, Showa-machi, Maebashi-shi 371-8512, Japan Received 26 October 1998; received in revised form 27 November 1998; accepted 27 November 1998

Abstract The role of prolactin in the initiation of mammary tumorigenesis by radiation was evaluated in ovarian hormone-free rats. Rats were bilaterally ovariectomized at 23 days of age, and then, at 2.5 months of age, two pituitaries obtained from mature rats of the same strain were transplanted underneath the kidney capsule as a means of increasing the serum prolactin level to provide stimulation of development of mammary glands. After 2 weeks, the ovariectomized rats with ectopic pituitary glands were exposed to whole body irradiation of 2.6 Gy of g-rays from a 60Co source and then treated with diethylstilbestrol as a tumor promoter. For the control, ovariectomized rats without ectopic pituitary glands were exposed and treated in the same way as the experimental group. A signi®cant increase of serum prolactin level was observed at the time of irradiation by the pituitary transplanted rats, and intense immunohistochemical reaction with a speci®c anti-prolactin antiserum was detected in the ectopic pituitary glands. Also, mammary glands in the pituitary transplanted rats, ovariectomized before puberty, showed lactiferous ducts without alveolar buds at the time of tumor initiation. The pituitary transplanted rats showed a signi®cantly increased incidence of adenocarcinoma and ®broadenoma compared with the control. Many of the mammary tumors induced in the pituitary transplanted rats given radiation were estrogen receptor (ER) (1) progesterone receptor (PgR) (1) and ER(1)PgR(2) tumors, whereas ER(2)PgR(2) tumors were mainly obtained in the control rats. In the experimental group, many of the ®broadenomas had low concentrations of ER and no PgR, while the adenocarcinomas had moderate concentrations of ER and high PgR. These results suggest that hypersecretion of prolactin from the pituitary transplants developed lactiferous ducts and accelerated the tumorigenesis of mammary glands initiated by radiation in the absence of synergism with ovarian hormones. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tumorigenesis; Radiation-induced; Prolactin; Pituaries

1. Introduction Whole-body irradiation of pregnant or lactating rats results in a higher incidence of mammary tumors than that observed in irradiated virgin rats [1,2]. We have * Corresponding author. Fax: 1 81-43-2556819. E-mail address: [email protected] (H. Inano)

reported that irradiation of lactating rats with g-rays induced a signi®cantly higher incidence of adenocarcinomas compared with rats irradiated during pregnancy [3]. The histological change in the tumors developed in rats of both these groups might be due to differences of hormonal regulation of mammary glands during pregnancy and lactation. Estradiol17b and progesterone regulate the differentiation of

0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(98)00378-4

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mammary glands of pregnant rats [4,5], and prolactin controls lactogenic activity [6,7]. Therefore, hormones clearly play an essential role in mammary carcinogenesis, but the precise role of each hormone remains to be elucidated. Husely et al. [8] reported that ectopic pituitary glands transplanted under the kidney capsule increased the circulating level of prolactin. Clifton et al. [9,10], and Kamiya et al. [11] reported that mammary tumorigenesis was increased by the transplantation of pituitary glands, or of prolactin secreting pituitary tumors after irradiation. In the present study, we transplanted pituitary glands to ovariectomized rats before irradiation to evaluate the role of prolactin, under estrogendepleting conditions, in the occurrence of mammary adenocarcinoma by radiation.

2. Materials and methods 2.1. Animals and treatment The rats used in the present study were treated and handled according to the Recommendations for the Handling of Laboratory Animals for Biomedical Research compiled by the Committee for the Safety and Handling of Laboratory Animals in our Institute. Wistar±MS rats were kept in a controlled environment (14 h light±10 h dark) at 23 ^ 18C. They received food and water ad libitum. Under the protocol, 65 rats were ovariectomized bilaterally at 23 days of age and then divided into two groups. At 2.5 months of age, 23 rats received two pituitary implants obtained from mature rats of the same strain under the left kidney capsule (Group II). Sham-operated rats (n ˆ 42) served as a control (Group I). After 14 days, ®ve rats in both the control and the transplanted groups were sacri®ced for determination of hormone concentrations, preparation of whole mounts of mammary glands, and immunohistochemical detection of prolactin in the ectopic and eutopic pituitary glands. All the remaining rats, 37 sham-operated and 18 pituitary transplanted rats, were exposed to wholebody irradiation of 2.6 Gy g-rays (0.17 Gy/min) from a 60Co source. One month after the irradiation, all rats were implanted s.c. with a diethylstilbestrol (DES) pellet ®lled with 3 mg of DES mixed with 27 mg of cholesterol in a medical grade Silastic tube (Dow

Corning, Midland, MI). The pellet was renewed every 2 months. The rate of release of DES from the pellet was approximately 0:38 ^ 0:01 mg/day [12]. Rats were observed for 1 year to detect palpable mammary tumors. The transplanted pituitary glands remained in place for the duration of the experiments. When palpable tumors had grown to more than 2 cm in diameter, the rats bearing them were killed by CO2 asphyxiation, and tumors were removed for histological examination and analysis of steroid receptors. Tumor incidence was calculated based on the number of rats with mammary tumors within 1 year. 2.2. Whole mounts of mammary glands At ovariectomy at 23 days of age, or at the time of irradiation at 3 months of age, the entire inguinal mammary glands were dissected from the inner surface of the skin. After ®xing in 10% formalin buffered with 0.1 M phosphate buffer (pH 7.2) and defatting in acetone, the preparations were stained with alum carmine, destained in ethanol and stored in cedar oil [13]. 2.3. Histological examination The mammary tumors were routinely ®xed in 10% formalin buffer with 0.1 M phosphate buffer (pH 7.2), dehydrated and embedded in paraf®n. Each paraf®n section (4 mm thick) was deparaf®nized and stained with hematoxylin and eosin. The tumors were classi®ed as adenocarcinoma or ®broadenoma according to the criteria for the classi®cation of rat mammary tumors [14]. 2.4. Immunohistochemical detection of prolactin in pituitary glands The pituitary glands were ®xed in Bouin's solution without acetic acid for 4 h, dehydrated and embedded in paraf®n. Sections (4 mm thick) of the pituitary glands were deparaf®nized, and immunohistochemical staining was performed by streptavidin±biotin methods (Histo®ne, SAB-PO(R9) kit, Nichirei Co., Tokyo) using anti-rat prolactin S-9 antiserum (NIDDK) supplied by the National Hormone and Pituitary Program (Rockville, MD). Immunoreaction with the antiserum was visualized by horseradish peroxidase using 3,3 0 -diaminobenzidine. The speci®-

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and Pituitary Program, Rockville, MD). Estradiol17b was measured by a modi®cation of the method of Watanabe et al. [16]. The serum concentration of progesterone was assayed by a commercially available kit. Sensitivities were 0.2 ng/ml for prolactin, LH, FSH and progesterone and 1 pg/ml for estradiol-17b. 2.6. Assay of steroid receptors

Fig. 1. Cumulative incidence of radiation-induced mammary tumors in ovariectomized rats with or without ectopic pituitary glands.

city of staining was con®rmed by the use of nonimmunized normal rabbit serum [15]. 2.5. Radioimmunoassay of hormones The concentrations of prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in each serum sample were determined with NIDDK radioimmunoassay kits (National Hormone

The tumor tissues (1 g) were homogenized in 10 mM Tris±HCl buffer (pH 7.4) containing 1.5 mM EDTA-Na2 and 1 mM dithiothreitol. Homogenates were centrifuged at 105 000 £ g for 1 h at 48C, and the obtained cytosol fraction was used for assay of the receptors. Estrogen receptor (ER) and progesterone receptor (PgR) were analyzed by a dextran-coated charcoal method using [2,4,6,7- 3H]estradiol-17b and [17a-methyl- 3H]R5020 (Du Pont/NEN Research Product, Boston, MA), respectively, as radioligands [17,18]. Maximum binding sites and dissociation constant (Kd) values for the receptors were determined by a Scatchard plot analysis [19]. 2.7. Iball's index and statistical analysis Iball's index was calculated as follows: the ratio of

Table 1 Development of mammary tumors by radiation in ovariectomized rats transplanted with pituitary glands Group (No. of rats): Pretreatment

I (n ˆ 37) Ovariectomy 1 sham operation

Treatment

2.6 Gy 1 DES

II (n ˆ 18) Ovariectomy 1 pituitary transplantation 2.6 Gy 1 DES

8 (21.6%) 1.0 ^ 0.0 7.7

14 (77.8%) a 1.9 ^ 0.3 29.0

2 (5.4%) 2 9.1 ^ 0.7

9 (50.0%) b 12 8.8 ^ 0.6

6 (16.2%) 6 9.3 ^ 1.0

12 (66.7%) c 14 8.6 ^ 0.5

Total tumors Rats with tumors (%) No. of tumors/rat Iball's index Adenocarcinoma (AC) Rats with AC (%) No. of AC Latency period (months) Fibroadenoma (FA) Rats with FA (%) No. of FA Latency period (months) a

Signi®cantly different from Group I, P , 0:0001. Signi®cantly different from Group I, P , 0:05. c Signi®cantly different from Group I, P , 0:001. b

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per tumor-bearing rat. Probability values less than 5% were considered signi®cant.

3. Results 3.1. Development of mammary tumors

Fig. 2. Effects of pituitary transplantation on ovarian and pituitary hormones. aConcentration (ng/ml); bConcentration (pg/ml); *P , 0:0001. PRL, prolactin; LH, luteinizing hormone; FSH, follicle-stimulating hormone; E2, estradiol-17b; Prog, progesterone.

incidence (%) to the average latency period in days was multiplied by 100 [20]. Statistical analyses were conducted by Fisher's exact probability test for tumor incidence, and by Student's t-test for hormone concentration, latency period, and number of tumors

Of the 37 ovariectomized rats that received wholebody irradiation with 2.6 Gy g-rays at 3 months of age and were then implanted with DES (Group I), only eight (21.6%) developed mammary tumors (Fig. 1). Mammary tumors obtained from this group were classi®ed as adenocarcinomas in two cases (5.4%) and ®broadenomas in 6 (16.2%). Compared with Group I, transplantation of pituitary glands to rats ovariectomized before puberty (Group II) signi®cantly increased the incidence (77.8%) of mammary tumors (P , 0:0001) and the number (1:9 ^ 0:3) of tumors per tumor-bearing rat (P , 0:05, Table 1). Nine (50%) of the 18 pituitary transplanted rats developed mammary adenocarcinoma during the 1-year period of DES implantation. The difference of adenocarcinoma development between the rats non-transplanted and transplanted with pituitary glands was signi®cant

Fig. 3. Immunohistochemical staining of prolactin in the eutopic pituitary glands (a±c), and in the ectopic pituitary gland transplanted under the kidney capsule (d,e) at the time of initiation by radiation. Pituitary glands of ovariectomized rats without ectopic pituitary glands were immunostained with either anti-prolactin antiserum, £23 (a), or non-immunized rabbit serum, £23 (b). Eutopic pituitary gland of ovariectomized rat transplanted with ectopic pituitary glands was immunostained with anti-prolactin antiserum, £23 (c). Ectopic pituitary glands were immunostained with either anti-prolactin antiserum, £37 (d), or non-immunized rabbit serum, £37 (e).

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transplantation. As shown in Fig. 2, the prolactin concentration (49:4 ^ 1:9 ng/ml) in the transplanted rats was 9.3-fold (P , 0:0001) higher than that (5:3 ^ 1:0 ng/ml) in the non-transplanted rats. No signi®cant difference was observed in the other hormone concentrations such as LH, FSH, estradiol17b and progesterone between rats in the two groups. 3.3. Prolactin in ectopic and eutopic pituitary glands at initiation by g -ray irradiation There was no positive immunoreactive staining of prolactin in eutopic pituitary glands of ovariectomized rats with ectopic pituitary glands (Fig. 3c), in spite of intense immunoreactive staining in the pituitary glands of non-pituitary transplanted, intact rats (Fig. 3a). A higher intensity of immunohistochemical reaction with anti-prolactin antiserum was detected in the ectopic pituitary glands transplanted underneath the kidney capsule of ovariectomized rats (Fig. 3d). 3.4. Whole mounts of mammary glands at initiation by g -ray irradiation Mammary glands of 23-day-old rats, at the time of ovariectomy, demonstrated undifferentiated small ducts (Fig. 4a). Mammary glands of the 3-month-old rats ovariectomized at 23 days of age indicated thin ducts without terminal end buds branched-off in the adipose tissues (Fig. 4b); this was also the case in the pituitary transplanted rats ovariectomized before puberty (Fig. 4c).

Fig. 4. Whole mount preparations of mammary glands. (a) Rats at 23 days of age, corresponding to the time of ovariectomy, £ 5.2; (b) 3-month-old rats previously ovariectomized at 23 days of age, £ 4.5; (c) 3-month-old rats ovariectomized at 23 days of age and then transplanted with pituitary glands under the kidney capsule, £ 4.5.

(P , 0:05). Also, the ®broadenoma incidence (66.7%) in transplanted rats was signi®cantly higher than (P , 0:001) the value (16.2%) of the non-transplanted rats.

3.5. Receptors for steroid hormones in mammary tumors The mammary tumors were homogenized and ER and PgR in the cytosol fraction were analyzed by a Scatchard plot. Many of the mammary tumors induced in pituitary transplanted rats by DES (Group II) were ER(1)PgR(1), ER(1)PgR(2) and

3.2. Hormone concentrations at initiation by g -ray irradiation The serum concentrations of ovarian and pituitary hormones were measured 2 weeks after the pituitary

Table 2 Estrogen receptor and progesterone receptor in mammary tumors induced by radiation Group

No. of tumors tested

ER(1)PgR(1)

ER(1)PgR(2)

ER(2)PgR( 1 )

ER(2)PgR(2)

I II

8 18

1 8

2 8

0 2

5 0

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Fig. 5. Concentration of estrogen receptors and progesterone receptors in mammary tumors. Left, estrogen receptors (ER) and right, progesterone receptors (PgR). Open and closed circles represent data for ®broadenoma and adenocarcinoma, respectively. MBS, maximum binding sites.

ER(2)PgR(1), but no ER(2)PgR(2) tumors were detected (Table 2). Conversely, ER(2)PgR(2) tumors were mainly detected in the non-pituitary transplanted rats (Group I). As shown in Fig. 5, adenocarcinomas induced in pituitary transplanted rats by radiation and DES (Group II) had a higher concentration of both ER and PgR compared to those of ®broadenomas, respectively. 4. Discussion Recently, we reported results indicating that lobulo-alveolar development of mammary glands modulates the carcinogenic effect of radiation in rats [1,2]. Ovarian and pituitary hormones are required for the proliferation and differentiation of mammary epithelial cells that are at risk from the carcinogen. In ovariectomized rats, prolonged estrogen treatment before irradiation enhanced mammary adenocarcinoma induced by radiation, but progesterone treatment did not [21,22]. Stimulatory effects of estrogen on the growth of mammary glands of ovariectomized rats were the result of a direct action via binding to ER in mammary glands and of an indirect action to increase circulating prolactin levels. Therefore, the role of each hormone, estradiol-17b and prolactin,

as risk modi®ers of mammary tumorigenesis induced by radiation remains to be elucidated. In our previous studies to establish the role of prolactin in initiation of adenocarcinoma by radiation, ovariectomized rats were treated with either estradiol-17b or haloperidol to increase prolactin secretion and were then exposed to radiation [21,22]. However, our results regarding the role of prolactin in the initiation of mammary tumorigenesis by radiation were inconclusive for the following reasons: (i) no signi®cant increase in serum prolactin concentration by haloperidol was observed because the rats were ovariectomized, (ii) estradiol17b acted directly on mammary glands in addition to stimulating secretion of prolactin from the pituitary glands. In the present study, rats were bilaterally ovariectomized before the onset of the estrous cycle and then received pituitary transplantation. During the phase of initiation by radiation, the serum prolactin concentration in the ovariectomized rats with ectopic pituitary glands was signi®cantly higher than that of non-transplanted rats. This was well re¯ected by intense positive immunostaining in the ectopic pituitary glands using anti-prolactin antiserum. However, there was no positive staining in the eutopic pituitary gland of ovariectomized rats with ectopic pituitary glands. Prolactin secreted by the ectopic pituitary glands appears to decrease eutopic pituitary prolactin secretion. There is evidence that prolactin exerts negative feedback on its own secretion via the hypothalamus [23]. The mammary glands of the ovariectomized rats with ectopic pituitary glands showed expansion of the ducts, but did not differentiate to lobulo-alveolar structures, because of absence of synergism with estrogen. Lobulo-alveolar developments require both estrogen and prolactin, and were not induced by prolactin alone under estrogen-free conditions. When mammary glands, developed by hyperprolactinemia under estrogen-free conditions, were irradiated, a high incidence of tumors was observed after DES administration. Therefore, lobulo-alveolar structures in the differentiated mammary glands were not needed for radiation-induced tumor initiation. These results suggest that the increased incidence of adenocarcinoma and ®broadenoma is due to duct elongation by hyperprolactinemia and not ovarian hormones during the initiation with radiation. In our previous study, the incidence of development

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of ER(1)PgR(1) tumors was increased in ovariectomized rats by administration of estradiol-3-benzoate before the irradiation [22]. From those results, it was proposed that estrogen is a potent regulatory factor for hormone dependency of radiation-induced mammary tumors. However, the present results suggest that the hormone dependency on growth of mammary tumors is associated with prolactin-stimulated differentiation under estrogen-free conditions at the time of exposure. Therefore, the role of estrogen in the hormone dependency growth of mammary tumors might be to stimulate prolactin secretion from the pituitary glands. It is plausible that prolactin, which is secreted from ectopic and/or eutopic pituitary glands, might be a factor for regulation of ER expression in radiationinduced mammary tumors. In conclusion, hypersecretion of prolactin from the ectopic pituitary glands developed lactiferous ducts in the absence of synergism with ovarian hormones, and accelerated the tumorigenesis of the mammary glands by radiation.

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