Radiation-induced mammary tumors in virgin and parous rats administered contraceptive steroids, 17
-ethinylestradiol and norethisterone
Hiroshi Inano2,
Makoto Onoda,
Keiko Suzuki,
Hisae Kobayashi1 and
Katsumi Wakabayashi1
First Research Group, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555 and
1 Institute for Molecular and Cellular Regulation, Gunma University,Maebashi-shi 371-8512, Japan
 |
Abstract
|
---|
Oral contraceptives are used among women worldwide, and radiation is being used increasingly for diagnosis or therapy. We have investigated the effects of contraceptive steroids on the risk of mammary tumors initiated by radiation. Virgin rats received whole-body irradiation with 2.6 Gy
-rays 1 month after the administration of low- or high-dose pellets of contraceptive steroids, such as 17
-ethinylestradiol (EE2) combined with 19-norethisterone (NET). The high-dose pellet was removed 1 month after irradiation, but administration of the low-dose pellet was continued for up to 1 year. The incidence (33.3%) of mammary tumors initiated with radiation was not modified by the long-term administration of the low-dose pellets. However, the incidence (58.3%) was increased significantly by the irradiation during administration of the high-dose pellets, but no significant difference in the proportion of adenocarcinoma and fibroadenoma was observed. Meanwhile, parous rats were irradiated with 2.6 Gy
-rays at weaning, a period of greater susceptibility to radiation, and then were implanted with the low-dose pellets 1 month later. The highest incidence (90%) of mammary tumors was detected in the parous rats. The proportion of adenocarcinomas in the parous irradiated rats increased significantly on treatment with the low-dose pellets. The results suggest that administration of the high-dose pellets of EE2NET, but not of the low-dose pellets, enhances susceptibility to the initiation by
-rays of mammary tumors in virgin rats, and that the low-dose pellets act as a tumor promoter in the mammary glands of parous rats irradiated at weaning.
Abbreviations: AC, adenocarcinoma; EE2, 17
-ethinylestradiol or (17
)-19-norpregn-1,3,5(10)-trien-20-yne-3,17-diol; ER, estrogen receptor; FA, fibroadenoma; FSH, follicle-stimulating hormone; LH, luteinizing hormone; MBS, maximum binding sites; NET, 19-norethisterone or (17
)-17-hydroxy-19-norpregn-4-en-20-yn-3-one; PgR, progesterone receptor; R5020, 17
-methyl-17-propionylestra-4,9-dien-3-one.
 |
Introduction
|
---|
Oral contraceptives are widely used; therefore, their effect on the risk of breast cancer is important. Most epidemiological studies suggest that there is no association between the use of oral contraceptives and the risk of breast cancer (14), but some suggest that the risk of breast cancer increases with the duration of use before the first full-term pregnancy (5,6), and commencement of use at a young age (7,8). Oral contraceptives contain a synthetic estrogen, such as 17
-ethinylestradiol (EE2), in combination with a synthetic progestin, such as 19-norethisterone (NET) (Figure 1
), and are considered to stimulate growth and development of the mammary glands in addition to inhibiting ovulation. Mammary glands in rats treated with 1 mg of EE2 alone are near maximal development with extensive lobulo-alveolar and ductal growth resembling that of late pregnancy, and a high incidence (~90%) of mammary adenocarcinomas is obtained in the rats with or without X-rays (9). Furthermore, after the administration of a daily dose of 4 mg of NET for 41 days, the number of prolactin cells in the rat pituitary glands and the DNA content in the mammary glands are significantly increased (10), and tubulo-alveolar growth of mammary glands is stimulated (11). Radiation-induced tumorigenesis of the mammary glands in rats is considered to be influenced by the developmental stage of the glands at the time of exposure. Our previous findings suggest that the mammary cells in the differentiated glands of pregnant (12,13) and lactating (12,14) rats are particularly susceptible to tumor initiation by irradiation and that more developed mammary glands, e.g. those treated with ovarian hormones, estradiol and progesterone, have a high incidence of tumors induced by irradiation (15,16). The question of whether a contraceptive mixed with estrogen and progestin increases the risk of mammary tumors induced by radiation remains unanswered. In the present study, we examined the effects of contraceptive steroids, a combination of EE2 and NET, on the sensitivities to the initiation of mammary tumors by irradiation in virgin and parous rats. Also, we discuss the promotional activities of the oral contraceptive steroids for tumorigenesis of primordial cells or stem cells in mammary glands of rats irradiated while virgins or lactating.
 |
Materials and methods
|
---|
Materials
High-dose pellets of EE2 (0.2 mg)NET (30 mg) (high-dose pellet), low-dose pellets of EE2 (0.02 mg)NET (1 mg) (low-dose pellet) and cholesterol pellets were purchased from Innovative Research of America (Toledo, OH). The pellets had a 60-day hormone release period. [2,4,6,7-3H]estradiol-17ß (sp. act. 4 TBq/mmol), [17
-methyl-3H]R5020 (sp. act. 3 TBq/mmol) (where R5020 is17
-methyl-17-propionylestra-4,9-dien-3-one) and non-radioactive R5020 were obtained from NEN Life Science Products (Boston, MA).
Animals and treatment
The rats used in the present study were treated and handled according to the Recommendations for Handling of Laboratory Animals for Biomedical Research compiled by the Committee for the Safety and Handling Regulations for Laboratory Animal Experiments in our institute. Wistar-MS rats were kept at 23 ± 1°C in a controlled environment (14 h light10 h dark) and received water and food ad libitum. For the first experiment, 24 virgin female rats, 2.5 months of age, were implanted subcutaneously with the high-dose pellets for 1 month, and then were irradiated with 2.6 Gy
-rays (0.15 Gy/min) from a 60Co source. Cholesterol pellets were implanted in 23 virgin rats as the control. The pellets implanted were removed from the rats 1 month after irradiation. The irradiated rats were observed for 1 year for the development of palpable mammary tumors (Figure 2
).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2. Experimental protocol used in this study. Open and shaded arrowheads pointing up, implantations of the pellets of cholesterol and EE2NET, respectively; open and shaded arrowheads pointing down, withdrawal of the cholesterol pellets and the high-dose pellets of EE2NET, respectively; closed arrow, whole-body irradiation with 2.6 Gy -rays; open bar, period of cholesterol treatment; shaded bar, period of treatment with EE2NET; number, months of age; Preg., pregnancy; Lact., lactation.
|
|
For the treated group in the second experiment, 18 virgin female rats (2.5 months old) were implanted subcutaneously with the low-dose pellet of EE2NET in the interscapular area. The hormone-treated rats were then irradiated with 2.6 Gy
-rays 1 month after implantation. The estrous cyclicity of the rats in this group was monitored by examination of vaginal smears prepared between 09:30 and 10:30 h daily from the beginning of implantation of the low-dose pellets until irradiation. A plastic tube containing 0.9% saline was inserted a few millimeters into the vagina. The saline was flushed gently into the vagina, collected and placed onto a slide glass. Cornified cells, epithelial cells and leukocytes were stained with Giemsa's solution, and identified through a light microscope. Twenty-seven age-matched control rats received cholesterol pellets of equivalent weight.
Fifty-two parous rats were used in the third experiment. They were divided into two groups 1 month after irradiation with 2.6 Gy
-rays at weaning. The low-dose pellets were implanted in 20 rats for the experimental group. For the control group, 32 rats were implanted subcutaneously with the cholesterol pellets. In the second and third experiments, the hormone and cholesterol pellets were replaced every 2 months until the end of the experiments. The rats were observed for 1 year for palpable mammary tumors. When palpable tumors reached >2 cm in diameter, the rats bearing them were killed by CO2 asphyxiation and the tumors were removed for histological examination and analysis of steroid receptors. Tumor incidence was calculated from the number of rats with mammary tumors within 1 year. Iball's index of mammary tumors was calculated as follows: the ratio of incidence (%) to the average latency period in daysx100 (17). For the determination of biological effects and morphological change by pre-treatment with the hormone pellets before irradiation, five virgin rats per group, 2.5 months of age, were implanted with the cholesterol pellets or the low- or high-dose pellets of EE2NET. One month later, corresponding to the time of irradiation, all rats were killed for examination.
Histological examination
At the time of irradiation and at the end of the experiments in virgin rats treated with high- or low-dose pellets, and in parous rats, whole mounts of mammary glands were prepared for evaluation of development according to the methods of Rothschild et al. (18) with modification (14). Briefly, the entire inguinal mammary glands were dissected and spread on filter paper. The specimens were fixed for 1 day in 10% formalin buffered with 0.1 M phosphate buffer pH 7.2, stained with alum carmine for 3 h, destained in ethanol and stored in cedar oil. The mammary tumors removed were fixed immediately in 10% neutral buffered formalin and embedded in paraffin. Paraffin sections (4 µm thick) were prepared and stained with hematoxylin and eosin. The tumors were classified as fibroadenoma (FA) or adenocarcinoma (AC) according to the criteria for the classification of rat mammary tumors (19).
Assays of hormones
At the time of irradiation and at the end of the experiments, blood samples collected from five rats of each group by cardiocentesis under Nembutal anesthesia were allowed to clot and then centrifuged to obtain sera. Concentrations of prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in each serum sample were determined with NIDDK radioimmunoassay kits (National Hormone and Pituitary Program). Sensitivities were 0.2 ng/ml serum for LH, FSH and prolactin.
Assay of steroid receptor in mammary tumors
Each tumor tissue (1 g) dissected was homogenized in ice-cold 10 mM TrisHCl 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 4°C, and the cytosol fraction was used for assay of the receptors. Both estrogen receptor (ER) and progesterone receptor (PgR) in the cytosol fraction were analyzed by a dextran-coated charcoal method using [2,4,6,7-3H]estradiol-17ß and [17
-methyl-3H]R5020, respectively, as radioligands (20,21). Dissociation constant (Kd) values and maximum binding sites (MBS) for the receptors were determined by a Scatchard plot analysis (22).
Statistical analysis
All statistical analyses were performed using StatView-4.5J (Abacus Concepts, Berkeley, CA). Statistical analyses were conducted by
2 test for tumor incidence and for the proportion of AC and FA, and by Student's t-test for body and organ weight, latency period and hormone concentrations. The cumulative proportions of rats with tumors (incidence curves) were calculated by the product-limit method where rats that died or were killed without mammary tumors were included, and the difference between groups was tested for statistical significance by the MantelCox test. Probability values <5% were considered significant.
 |
Results
|
---|
Modification of radiation-induced mammary tumors in virgin rats by administration of the high-dose pellets
In the control experiments, 23 virgin rats were implanted with the cholesterol pellets at the age of 2.5 months and irradiated with 2.6 Gy at 3.5 months. The pellets were removed 1 month after the irradiation. Five rats (21.7%) developed mammary tumors over the 1 year period (Table I
). The implantation of the high-dose pellets of EE2NET instead of the cholesterol pellets in 24 virgin rats significantly increased the incidence (58.3%) of mammary tumors (P < 0.01). The cumulative incidence curve of mammary tumors in the irradiated virgin rats implanted with the high-dose pellets increased significantly (P < 0.05) within the 1 year period compared with the control group (Figure 3a
). No significant differences in the proportion of ACs and FAs (P = 0.315), in the number of tumors per tumor-bearing rat (P = 0.446) or in latency periods (P = 0.205) were observed between the cholesterol-treated group and the high-dose hormone pellet-treated group (Table I
). Iball's index for the development of mammary tumors in the irradiated rats implanted with the high-dose pellets was 2.4-fold that in the rats treated with the cholesterol pellets.
View this table:
[in this window]
[in a new window]
|
Table I. Induction of radiation-induced mammary tumors in virgin rats during the administration of high- or low-dose pellets
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3. Cumulative incidence of mammary tumors in irradiated rats treated with cholesterol (dotted lines) and EE2NET (solid lines). (a) Virgin rats previously treated with the high-dose pellets of EE2NET for 2 months. (b) Virgin rats treated with the low-dose pellets of EE2NET for 14 months. (c) Parous rats treated with the low-dose pellets of EE2NET for 12 months. Statistical evaluation of the cumulative proportion data (incidence curves) by MantelCox test yielded P < 0.05 in (a), no significant difference in (b) and P < 0.0001 in (c).
|
|
Modification of radiation-induced mammary tumors in virgin rats by administration of the low-dose pellets
In the control experiments, 27 virgin rats were implanted with the cholesterol pellets from the age of 2.5 to 16.5 months and administered whole-body irradiation with 2.6 Gy at the age of 3.5 months, 1 month after the first implantation. Nine rats (33.3%) developed mammary tumors during the experimental periods (Table I
). The implantation of the low-dose pellets in 18 experimental rats did not change the incidence (33.3%) of the mammary tumors induced by radiation. No significant difference (P = 0.910) in the cumulative incidence curves was observed between the control rats and the rats treated with the low-dose pellets (Figure 3b
). No AC developed in the virgin rats administered the low-dose pellets before and after irradiation (Table I
). No significant difference in the proportion of ACs and FAs (P = 0.142), in the numbers of tumors per tumor-bearing rat (P = 0.752) or in latency periods (P = 0.278) was observed between the control and the hormone-treated groups in the virgin rats. Iball's index for the overall development of mammary tumors in the hormone-treated rats was almost the same as that in the control group.
Modification of radiation-induced mammary tumors in parous rats by administration of the low-dose pellets
Of the 32 rats administered whole-body irradiation with 2.6 Gy
-rays during lactation and then treated with the cholesterol pellets, 10 (31.3%) developed mammary tumors during the experimental period (Table II
). The administration of the low-dose pellets in 20 experimental rats 1 month after the irradiation significantly increased the incidence (90.0%) of total mammary tumors (P < 0.0001). The cumulative incidence curve of mammary tumors in the irradiated parous rats implanted with the low-dose pellets increased significantly (P < 0.0001) within the 1 year period compared with the control (Figure 3c
). ACs did not develop in the rats irradiated while lactating and then implanted with the cholesterol pellets. However, on treatment with the low-dose pellets, ACs developed in 27 (57.5%) of 47 tumors examined. Also, there were significant differences between the two groups in the proportion of ACs and FAs (P < 0.0001), and in the numbers of mammary tumors per tumor-bearing rat (P < 0.05). However, no significant difference in latency periods until the appearance of the first tumor (P = 0.189) was observed between the two groups. By administration of the low-dose pellets in the irradiated parous rats, Iball's index was enhanced 3-fold in comparison with that of the cholesterol-treated rats.
View this table:
[in this window]
[in a new window]
|
Table II. Induction of mammary tumors in rats irradiated during lactation and then treated with the low-dose pellets
|
|
Biological effect and hormonal change at the time of irradiation
Virgin rats implanted with the low-dose pellets showed a persistent estrous, indicated by vaginal smear. The body weight was significantly decreased (P < 0.01) in virgin rats by treatment with the low- and high-dose pellets (Table III
). Administration of the low-dose pellets induced atrophy of the ovaries with multiple small cysts and atrophy of the liver, and hypertrophy of the uterus with the presence of a substantial amount of intraluminal fluid. No change in the weight of pituitary glands and prolactin concentration was observed in the virgin rats on short-term administration of the low-dose pellets. However, the serum prolactin concentration in the virgin rats treated with the high-dose pellets for 1 month was 3-fold (P < 0.01) that in the control rats, in spite of no change in the weight of pituitary glands.
View this table:
[in this window]
[in a new window]
|
Table III. Biological effects and hormonal change by 1 month treatment with the low- and high-dose pellets containing EE2 and NET to virgin rats
|
|
Biological effect at the end of the experiments
The body weight was significantly decreased (P < 0.01) in virgin rats by long-term treatment with the low-dose pellets (Table IV
) in spite of a similar calorie intake (63.6 ± 1.1 kcal/rat/day) to the control group (64.5 ± 1.3 kcal/rat/day). The rats implanted with the low-dose hormone pellets in both virgin and parous groups had enlarged pituitary glands, which presented macroscopically friable hemorrhagic tumors. On administration of the low-dose pellets to the virgin rats, atrophy of the ovaries in comparison with the control (P < 0.05) occurred, and a tendency to hypertrophy was observed in the liver (P = 0.183) and uterus (P = 0.199). Also, a significant reduction of body weight (P < 0.01) and an increase of pituitary weight (P < 0.05) compared with the control were observed in the rats administered whole-body irradiation with
-rays during lactation and then treated with the low-dose hormone pellets. The weights of liver and uterus were increased slightly by the irradiation with
-rays during lactation and then treatment with the low-dose hormone pellets; however, no significant difference was observed (P = 0.102 for liver and P = 0.148 for uterus, respectively). When the high-dose pellets were implanted in the virgin rats 1 month before and withdrawn 1 month after the irradiation, no significant difference of body weight and organ weight was observed at the end of the experiments.
View this table:
[in this window]
[in a new window]
|
Table IV. Biological effects and hormonal change at the end of the experiments by long-term administration of the low-dose pellets of EE2 combined with NET to irradiated virgin and parous rats during the promotion stage, and by administration of the high-dose pellets during the initiation stage to irradiated virgin rat
|
|
Concentrations of pituitary hormones in serum at the end of the experiments
The concentrations of pituitary hormones were assayed using serum collected 2 months after the last implantation of pellets. The serum prolactin concentration in the virgin rats treated with the low-dose pellets was 6.3 times (P < 0.01) that in the control rats (Table IV
). No change in the concentration of LH and FSH was observed in the virgin rats treated long term with the low-dose pellets. In the rats irradiated with
-rays during lactation and then implanted with the low-dose pellet, the prolactin concentration was increased 9.5-fold (P < 0.01) compared with the cholesterol-implanted rats, but the concentrations of LH and FSH were comparable to the control value. When the virgin rats were administered the high-dose pellets from 2.5 to 4.5 months of age and irradiated at age 3.5 months, no significant difference in the serum concentration of pituitary hormones was observed at the end of the experiments.
Whole mounts of mammary glands at the time of irradiation and at the end of the experiments
Whole mounts of the mammary glands were prepared from female rats at the age corresponding to the time of irradiation. Mammary glands in the virgin rats treated with the low- or high-dose pellets for 1 month were observed to have well-developed lobulo-alveoli with expanded lactiferous ducts in the glands more than those in the rats implanted with the cholesterol pellets (Figure 4ac
). At weaning, alveolar lumina in the mammary glands were extensively dilated and milk filled. No adipose cells were visible (Figure 4d
).

View larger version (143K):
[in this window]
[in a new window]
|
Fig. 4. Whole-mount observation of inguinal mammary glands at the time of irradiation. (a) Virgin rats treated with the cholesterol pellets. (b) Virgin rats treated with the low-dose pellets of EE2NET. (c) Virgin rats treated with the high-dose pellets of EE2NET. (d) Day 21 of lactation of parous rats. Bar, 5 mm.
|
|
At the end of the experiments, mammary glands were slightly developed in the virgin rats implanted with the high-dose pellets during the initiation with
-rays (Figure 5a and b
), and proliferated in the virgin rats implanted continuously with the low-dose pellets for 1 year (Figure 5c and d
). No significant difference in the development of mammary glands was observed between the irradiated parous rats implanted with the cholesterol pellets and those implanted with the low-dose pellets (Figure 5e and f
).

View larger version (213K):
[in this window]
[in a new window]
|
Fig. 5. Whole-mount observation of inguinal mammary glands at the end of the experiments. (a and b) Virgin rats treated with the cholesterol pellets or the high-dose pellets of EE2NET for 2 months during the initiation phase and then left untreated for 1 year, respectively. (c and d) Virgin rats treated with the cholesterol pellets or the low-dose pellets of EE2NET for 14 months, respectively. (e and f) Parous rats treated with the cholesterol pellets or the low-dose pellets of EE2NET for 12 months, respectively. Bar, 5 mm.
|
|
Steroid hormone receptors in mammary tumors
In the virgin rats implanted with the high-dose pellets for 2 months at 2.5 months of age and irradiated at age 3.5 months, two ER+PgR+, three ER+PgR and nine ERPgR tumors developed within 1 year (Table V
). In the virgin rats implanted with the cholesterol pellets and irradiated with
-rays, two ER+PgR+ and five ERPgR tumors were detected. The tumors of ER+PgR+ type and ERPgR type disappeared on administration of the low-dose pellets to the irradiated virgin rats, and all tumors tested were ER+PgR. Conversely, tumors that developed in the rats irradiated during lactation and then treated with the cholesterol pellets were ER positive, and no ERPgR tumor was detected. Many (64%) of the mammary tumors obtained from the rats irradiated during lactation and then treated with the low-dose pellets were ER+PgR and only two tumors (7.1%) were ERPgR. Table VI
shows the receptor concentrations and Kd values obtained when the ER and PgR in the cytosol fraction were analyzed with a Scatchard plot. Concentrations of ER and PgR in the FAs that developed in the rats irradiated during lactation were reduced by long-term administration of the low-dose pellet, but not significantly (P = 0.086 for ER and P = 0.378 for PgR, respectively). FAs that developed in the irradiated virgin rats treated with the cholesterol or hormone pellets for 1 year were PgR negative.
 |
Discussion
|
---|
The results of our previous study demonstrated that chlormadinone acetate, one of the progestins in oral contraceptives (23), is not a potent promoter of the tumorigenesis initiated by radiation in mammary cells, but diethylstilbestrol (DES), a synthetic estrogen, is (24). However, a promoter effect of synthetic progestins was reported by Pazos et al. (25,26), who found that medroxyprogesterone acetate (MPA), as a contraceptive (27) or an effective agent for breast cancer therapy (28), enhanced the incidence of N-methyl-N-nitrosourea-induced mammary tumors in mice. The low-dose pellets, EE2 (0.02 mg)NET (1 mg) of 60-day release type, used in this study reflect the most recent oral contraceptive pills in that they contain a low dose (<0.05 mg/pill) of EE2 combined with varying doses of the progestins, such as NET, levonorgestrel or desogestrel (29). Dilatation of acini and ducts in mammary glands and the absence of tumorigenic potential were demonstrated on the long-term administration of the oral contraceptives (0.052.55 mg combined dose of EE2 and NET acetate/kg/day) in sexually mature female rhesus monkeys over a 10 year period (30). However, the long-term administration of an oral contraceptive mixed with EE2-3-methylether and WY-4355 induced a high incidence of malignant mammary tumors in female beagle dogs (31,32). The present study indicates that long-term treatment with the low-dose pellets did not modify the risk of mammary tumors initiated with radiation in virgin rats (incidence 33.3% in control versus 33.3% by EE2NET); however, it significantly increased the risk in parous rats irradiated during lactation (incidence 31.3% in control versus 90.0% by EE2NET, P < 0.0001). At the time of irradiation, the mammary glands were weakly developed by the pre-treatment with the low-dose hormone pellets in virgin rats, and were markedly differentiated during the lactation of parous rats. The extent to which mammary tumorigenesis is initiated by radiation is dependent on the developmental stage of the mammary glands at the time of irradiation (15,16). The more advanced the stage, the higher the rate of mammary tumorigenesis. Also, irradiation of virgin rats implanted with the high-dose pellets resulted in a significantly higher incidence (58.3%, P < 0.01) of tumors than that (21.7%) of rats implanted with the cholesterol pellets. These findings suggest that the risk for radiation-induced mammary tumorigenesis depends on the dose of estrogen and progestin in the pellet, whether it is implanted before or after irradiation, and also on the developmental stage of mammary glands at irradiation.
No AC developed in the parous rats irradiated during lactation and implanted with cholesterol pellets as the control. However, in those implanted with the low-dose pellet instead of the cholesterol pellet, the proportion of ACs increased significantly to 57.5% of the total tumors. On the other hand, the proportion of ACs in the total tumors in the null-parous irradiated rats decreased from 23.1% for the cholesterol pellet as the control to 0% for the low-dose pellet of EE2NET, and from 28.6% in the control to 11.8% for the high-dose pellet. In the initiation phase with the radiation, the primordial cells or stem cells in the mammary glands that develop during pregnancy and following lactation have a tendency to become ACs in the presence of the low-dose pellet of EE2NET as a tumor promoter. However, primordial cells or stem cells in the glands developed by treatment with the low-dose pellet of EE2NET before irradiation tend to become FAs on continuous administration of the low-dose pellet after the irradiation. Primordial cells and stem cells in the glands developed by treatment with the high-dose pellet of EE2NET before irradiation also have a tendency to develop into FAs in the presence of physiological hormones secreted from the ovaries, because of elimination of the pellet used as initiation modifier after irradiation. From those results, it is suggested that the primordial cells or stem cells in the lactating mammary glands differ in their disposition to radiation from those in the glands developed with EE2NET.
Regarding the association between EE2 and radiation, Holtzman et al. (9) reported that the final incidence of mammary AC was no different between rats treated with EE2 and X-rays versus rats treated with EE2 alone. On the other hand, regarding the association between oral contraceptives and chemical carcinogens, Welsch and Meites (33) have reported that Enovid (98.5% norethynodrel and 1.5% EE2-3-methylether) significantly inhibited the development of 7,12-dimethylbenz[a]anthracene-induced mammary tumors, but enhanced the growth of those mammary tumor cells already present. Using normal, non-malignant atypical and malignant human mammary epithelial cells, it was found that the oral contraceptive mixed with EE2-3-methylether and NET promoted the growth of previously transformed cells, rather than the transforming event by carcinogen initiation (34). Also, Baggs et al. (35) reported that EE2NET did not increase the risk of developing vaginal tumors in female rat offspring treated in utero with DES.
In conclusion, the current investigation demonstrates that long-term administration of the low-dose pellets of EE2NET increases the risk of developing mammary tumors in parous rats irradiated at weaning, but not in rats irradiated while virgins. The risk of radiation-induced mammary tumorigenesis increased in virgin rats previously administered the high-dose pellets of EE2NET.
 |
Acknowledgments
|
---|
This work was partly supported by a project research grant for `Experimental Studies on the Radiation Health, Detriment and its Modifying Factors' and also by a grant from the Special Program for Bioregulation of the National Institute of Radiological Sciences.
 |
Notes
|
---|
2 To whom correspondence should be addressed Email: inano{at}nirs.go.jp 
 |
References
|
---|
-
Jick,S.S., Walker,A.M., Stergachis,A. and Jick,H. (1989) Oral contraceptives and breast cancer. Br. J. Cancer, 59, 619621.
-
Henderson,B.E., Powell,D., Rosario,I., Key,C., Hanisch,M., Young,M., Casagrande,J., Gerkins,V. and Pike,M.C. (1974) An epidemiologic study of breast cancer. J. Natl Cancer Inst., 53, 609614.[ISI][Medline]
-
Paffenbarger,R., Fasal,E., Simmons,M.E. and Kampert,J.B. (1977) Cancer risk as related to use of oral contraceptives during fertile years. Cancer, 39, 18871891.[ISI][Medline]
-
Romieu,I., Berlin,J.A. and Colditz,G.A. (1990) Oral contraceptives and breast cancer: review and meta-analysis. Cancer, 66, 22532263.[ISI][Medline]
-
Henderson,B.E., Ross,R. and Bernstein,L. (1988) Estrogens as a cause of human cancer: The Richard and Hinda Rosenthal Foundation Award Lecture. Cancer Res., 48, 246253.[ISI][Medline]
-
Colditz,G.A. (1993) Epidemiology of breast cancer: findings from the Nurses' Health Study. Cancer, 71 (suppl.), 14801489.[ISI][Medline]
-
Olsson,H., Landin-Olsson,M., Möller,T.R., Ranstam,J. and Holm,P. (1985) Oral contraceptive use and breast cancer in young women in Sweden. Lancet, i, 748749.
-
Wingo,P.A., Lee,N.C., Ory,H.W., Beral,V., Peterson,H.B. and Rhodes,P. (1993) Age-specific differences in the relationship between oral contraceptive use and breast cancer. Cancer, 71 (suppl.), 15061517.[ISI][Medline]
-
Holtzman,S., Stone,J.P. and Shellabarger,C.J. (1981) Synergism of estrogens and x-rays in mammary carcinogenesis in female ACI rats. J. Natl Cancer Inst., 67, 455459.[ISI][Medline]
-
Aumüller,G., Wagner,R. and Gräf,K.J. (1978) Fine structure of rat prolactin cells after treatment with a long acting depot contraceptive. Acta Endocrinol., 89, 251262.[ISI][Medline]
-
Nishino,Y. and Neumann,F. (1979) Estrogenic partial effect of norethisterone enanthate in relation to tumor induction in rat mammary gland. Arch. Toxicol., 2 (suppl.), 439443.
-
Inano,H., Suzuki,K., Onoda,M. and Yamanouchi,H. (1996) Susceptibility of fetal, virgin, pregnant and lactating rats for the induction of mammary tumors by gamma-rays. Radiat. Res., 145, 708713.[ISI][Medline]
-
Inano,H., Suzuki,K., Ishii,H., Ikeda,K. and Wakabayashi,K. (1991) Pregnancy-dependent initiation in tumorigenesis of Wistar rat mammary glands by 60Co-irradiation. Carcinogenesis, 12, 10851090.[Abstract]
-
Suzuki,K., Ishii-Ohba,H., Yamanouchi,H., Wakabayashi,K., Takahashi,M. and Inano,H. (1994) Susceptibility of lactating rat mammary glands to gamma-ray-irradiation-induced tumorigenesis. Int. J. Cancer, 56, 413417.[ISI][Medline]
-
Yamanouchi,H., Ishii-Ohba,H., Suzuki,K., Onoda,M., Wakabayashi,K. and Inano,H. (1995) Relationship between stages of mammary development and sensitivity to gamma-ray irradiation in mammary tumorigenesis in rats. Int. J. Cancer, 60, 230234.[ISI][Medline]
-
Inano,H., Yamanouchi,H., Suzuki,K., Onoda,M. and Wakabayashi,K. (1995) Estradiol-17ß as an initiation modifier for radiation-induced mammary tumorigenesis of rat ovariectomized before puberty. Carcinogenesis, 16, 18711877.[Abstract]
-
Iball,J. (1939) The relative potency of carcinogenic compounds. Am. J. Cancer, 35, 188190.
-
Rothschild,T.C., Boylan,E.S., Calhoon,R.E. and Vonderhaar,B.K. (1987) Transplacental effects of diethylstilbestrol on mammary development and tumorigenesis in female ACI rats. Cancer Res., 47, 45084516.[Abstract]
-
World Health Organization (1981) Histological Typing of Breast Tumors, 2nd edn. WHO, Geneva, pp. 1525.
-
Johnson,R.B., Nakamura,R.M. and Libby,R.M. (1975) Simplified Scatchard plot assay for estrogen receptor in human breast tumor. Clin. Chem., 21, 17251730.[Abstract/Free Full Text]
-
Johnson,R.B. and Nakamura,R.M. (1978) Simplified Scatchard plot assay for progesterone receptor in breast cancer; comparison with single-point and multipoint assay. Clin. Chem., 24, 11701176.[Free Full Text]
-
Scatchard,G. (1949) The attractions of proteins for small molecules and ions. Ann. NY Acad. Sci., 51, 660672.[ISI]
-
Butler,C. and Hill,H. (1969) Chlormadinone acetate as oral contraceptive. Lancet, i, 11161119.
-
Inano,H., Suzuki,K., Onoda,M., Kobayashi,H. and Wakabayashi,K. (1999) Comparative effect of chlormadinone acetate and diethylstilbestrol as promoters in mammary tumorigenesis of rats irradiated with
-rays during lactation. Breast Cancer Res. Treat., 53, 153160.[ISI][Medline]
-
Pazos,P., Lanari,C., Meiss,R., Charreau,E.H. and Pasqualini,C.D. (1991) Mammary carcinogenesis induced by N-methyl-N-nitrosourea (MNU) and medroxyprogesterone acetate (MPA) in BALB/c mice. Breast Cancer Res. Treat., 20, 133138.[ISI]
-
Pazos,P., Lanari,C., Elizalde,P., Montecchia,F., Charreau,E.H. and Molinolo,A.A. (1998) Promoter effect of medroxyprogesterone acetate (MPA) in N-methyl-N-nitrosourea (MNU) induced mammary tumors in BALB/c mice. Carcinogenesis, 19, 529531.[Abstract]
-
Chilvers,C. (1994) Breast cancer and depot-medroxyprogesterone acetate: a review. Contraception, 49, 211222.[ISI][Medline]
-
Lundgren,S. (1992) Progestins in breast cancer treatment. Acta Oncol., 31, 709722.[ISI][Medline]
-
Harlap,S. (1991) Oral contraceptives and breast cancer. Cause and effect? J. Reprod. Med., 36, 374395.
-
Fitzgerald,J., de la Iglesia,F. and Goldenthal,E.I. (1982) Ten-year oral toxicity study with Norlestrin in rhesus monkeys. J. Toxicol. Environ. Health, 10, 879896.[ISI][Medline]
-
Giles,R.C., Kwapien,R.P., Geil,R.G. and Casey,H.W. (1978) Mammary nodules in beagle dogs administered investigational oral contraceptive steroid. J. Natl Cancer Inst., 60, 13511364.[ISI][Medline]
-
Kwapien,R.P., Giles,R.C., Geil,R.G. and Casey,H.W. (1980) Malignant mammary tumors in beagle dogs dosed with investigational oral contraceptive steroids. J. Natl Cancer Inst., 65, 137144.[ISI][Medline]
-
Welsch,C.W. and Meites,J. (1969) Effects of a norethynodrelmestranol combination (Enovid) on development and growth of carcinogen-induced mammary tumors in female rats. Cancer, 23, 601607.[ISI][Medline]
-
Longman,S.M. and Buehring,G.C. (1987) Oral contraceptives and breast cancer, in vitro effect of contraceptive steroids on human mammary cell growth. Cancer, 59, 281287.[ISI][Medline]
-
Baggs,R.B., Miller,R.K. and Odoroff,C.L. (1991) Carcinogenicity of diethylstilbestrol in the Wistar rats: effect of postnatal oral contraceptive steroids. Cancer Res., 51, 33113315.[Abstract]
Received October 18, 1999;
revised December 30, 1999;
accepted January 5, 2000.