Effects of Neonatally Administered Diethylstilbestrol on Induction of Mammary Carcinomas Induced by 7,12-Dimethylbenz[A]Anthracene in Female Rats

Abstract

Various doses of diethylstilbestrol (DES) were administered to rats once at birth. Thereafter, at 50 days after birth, the rats in all groups were given 10 mg 7, 12-dimethylbenz[a]anthracene (DMBA) and undergone necropsy at 300 days after birth. The incidence of mammary carcinomas (MCs) were 50, 54, 91, 39, 19% at 175 days after birth, and 77, 87, 100, 85, 75% at necropsy in the 0, 0.1, 1, 10, 100 μg groups, respectively. The incidence of rats without corpus luteum were 0, 0, 0, 30, 100% at 50 days after birth, and 0, 40, 53, 93, 100% at necropsy in the 0, 0.1, 1, 10, 100 μg groups, respectively. Observation of the whole mount specimens showed a higher number of terminal end buds (TEBs) in the 1 μg group and a lower number in the 100 μg group compared with the control at 50 days after birth. It suggested that the administration of a relatively low dose (1 μg) of DES during neonatal period may increase TEBs, thus resulting in a stimulatory effect on the initiation of MCs.

Introduction

The incidence of breast cancer has been increasing worldwide (Martin, 2001; Wright et al., 2003), and it is essential for its prevention to accurately determine the cause. In general, the risk of developing breast cancer is mainly related to be affected by lifestyle, environment and genetics. Exposure to certain chemicals and hormone-mimicking or endocrine disruptors is suspected to contribute to increasing incidence of breast cancer as well as precocious puberty in the United States (Fenton, 2006). There is evidence that many environmental endocrine disruptors act like sex hormones, particularly during the perinatal or neonatal period, directly or indirectly affecting reproduction (Massart et al., 2005; Safe, 2005; Willoughby et al., 2005). However, it remains to be determined whether substances that act as sex hormones at perinatal or neonatal period affect the development of MCs.

In the present study, various doses of diethylstilbestrol (DES), a synthetic estrogen with strong estrogenic activity (Palmer et al., 2006), were administered to neonatal female rats at a critical period of morphogenesis and functional development of the mammary glands to examine its effect on mammary carcinogenesis induced by 7, 12-dimethylbenz[a]anthracene (DMBA), which is known to be a polycyclic hydrocarbon carcinogen forming DNA adducts (Singletary et al., 1997).

Materials and Methods

Animals

The animals were inbred Sprague-Dawley (SD) female rats, maintained in a filtered air laminar flow at the Division of Laboratory Animal Science, Research Center for Life Science Resources, Kagoshima University. The animals were given a commercial diet (CE-2, CLEA Inc., Tokyo, Japan) and tap water ad libitum. The room temperature was maintained at 25°C ± 2°C and the relative humidity at 55% ± 10%, with a 12 hr light/dark cycle. The use of animals in this research complied with all relevant guidelines set by the Japanese government and Kagoshima University.

Study Design

Rats in groups I (n = 22), II (n = 15), III (n = 21), IV (n = 13) and V (n = 16) were administered DES (Sigma Chemical Co., St. Louis, USA) at 0, 0.1, 1, 10 and 100 μg/body, respectively, dissolved in 0.05 ml sesame oil, subcutaneously once at birth. At 50 days after birth, all groups were given 10 mg DMBA (Wako Pure Chemical Industries Ltd., Osaka Japan) dissolved in 1 ml sesame oil, by gastric intubation. All DMBA-administered animals except those sacrificed during the observation period were examined by palpation to detect mammary tumors during 50–300 days after birth. The estrus cycles of all animals were examined during 21–150 days after birth by a vaginal smear check. At 300 days after birth, all survival animals were undergone necropsy. Specimens from all mammary tumors were fixed in 10% phosphate-buffered formalin, dehydrated, and embedded in paraffin. The widest cut surface was sectioned to 5 μm, stained routinely with hematoxylin and eosin (H.E.) stain and then examined histopathologically.

At 50 days after birth (before the administration of DMBA), 23, 19, 18, 20 and 22 rats in groups I, II, III, IV and V, respectively, were necropsied and corpus luteum (CL)in the ovaries were examined grossly and histopathologically.

Whole Mount Specimens

The right abdominal mammary glands (R4-6) were collected from 10, 7, 10, 7 and 10 rats in groups I, II, III, IV and V, respectively, at 50 days after birth (before the administration of DMBA). To prepare whole mount specimens, the mammary glands were fixed with 10% phosphate-buffered formalin for 24 hours, stained with alum carmine for 24 hours to prepare whole mount specimens, and finally stored in cedar oil. The terminal end buds (TEBs) were counted from the distal portions of the mammary gland in the whole mount specimens examined under a stereoscopic microscope (Yoshida et al., 1980b; Funato et al., 2006).

Immunohistochemistry

The left abdominal mammary glands (L4-6) were collected from each 7 rats in the all groups at 50 days after birth (before the administration of DMBA), fixed with 10% phosphate-buffered formalin for 24 hours, embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, and examined histopathologically. After the endogenous peroxidase activity had been blocked, deparaffinized sections were pretreated with 10 mM citrate buffer (pH 6.0) by microwaves. After rinsing with PBS, the sections were blocked with Block-Ace (Dainippon Sumitomo Pharma Co., Ltd. Osaka Japan) for 30 minutes. The sections were incubated overnight at 4°C with a diluted primary antibody, Ki-67 (Novocastra Laboratories Ltd., 1:200). After rinsing with PBS, the sections were incubated with biotinylated anti-mouse immunoglobulin for 30 minutes, rinsed again with PBS and incubated with VECTASTAIN Elite ABC KIT (Vector Laboratories, USA) for 30 minutes. To visualize the immunoreactivity, 3,3′-diaminobenzidine tetrachloride (DAB)-containing 0.02% hydrogen peroxide was used. The sections were then washed, counter-stained, dehydrated, cleared in xylene, and mounted (Kawaguchi et al., 2000; Okasaki et al., 1998; Omachi et al., 2000). For Ki-67, nuclear stained cells were interpreted as positive cells. Ki-67 positive cells in a TEB of a rat were counted. Each TEB contained approximately one hundred to three hundred epithelial cells.

Statistics

The mean differences were evaluated by Student’s t-test. The mean percentages of Ki-67 positive cells were analyzed by the nonparametric Mann–Whitney test (Shoker et al., 1999). The data are shown as the mean ± standard deviation (SD). The other percentages were tested using a 4-fold contingency table.

Results

At 50 days after birth, the percentage of rats without CL in the ovaries was significantly higher in groups IV (10 μg) (p < 0.01) and V (100 μg) (p < 0.01) in comparison to the control group (Table 1).

In groups I (control) and II (0.1 μg), no rats with PE were seen at 50, 100, and 150 days after birth. Compared with the control group, the percentage of rats with PE was significantly higher in groups III (1 μg) at 100 (p < 0.05) and 150 (p < 0.01) days after birth, IV (10 μg) at 50 (p < 0.01), 100 (p < 0.01), and 150 (p < 0.01) days after birth, and V (100 μg) at 50 (p < 0.01), 100 (p < 0.01), and 150 (p < 0.01) days after birth. At necropsy, all rats in the control had CL in ovaries. In groups II (0.1 μg) (p < 0.01), III (1 μg) (p < 0.01), IV (10 μg) (p < 0.01), and V (100 μg) (p < 0.01), the percentage of rats without CL in the ovaries was significantly higher than the control group (Table 2).

Compared with the control group, no significant changes in the body weights (BWs) were seen in the all groups administered with DES. In group V (100 μg), the absolute weight (AW) (p < 0.01) and AW/BW (relative weight) (p < 0.01) of the ovaries were significantly lower than the control group. The AW of the uterus was significantly lower than the control group in groups III (1 μg) (p < 0.01), IV (10 μg) (p < 0.01), and V (100 μg) (p < 0.01), and appears to be dose-dependent. The AW/BW of the uterus was also significantly lower than the control group in groups III (1 μg) (p < 0.01), IV (10 μg) (p < 0.01), and V (100 μg) (p < 0.01), and likewise appear to be dose-dependent (Table 3).

In group III (1 μg), the incidence of rats with MCs was significantly higher than the control group at 175 (p < 0.01), 200 (p < 0.05), 225 (p < 0.05), 250 (p < 0.01), 275 (p < 0.01) and 300 (p < 0.05) days after birth, and the number of MCs per rat was significantly higher than the control group at 175 (p < 0.05), 200 (p < 0.05), 225 (p < 0.05), 250 (p < 0.01), 275 (p < 0.01) and 300 (p < 0.01) days after birth. In group II (0.1 μg), the number of MCs per rat was significantly higher than the control group at 275 (p < 0.05) and 300 (p < 0.05) days after birth. In group IV (10 μg), the number of MCs per rat was significantly higher than the control group at 250 (p < 0.05), 275 (p < 0.05) and 300 (p < 0.05) days after birth. In group V (100 μg), the incidence of rats with MCs was significantly lower than the control group at 125 (p < 0.05), 150 (p < 0.01) and 175 (p < 0.05) days after birth, and the number of MCs per rat was significantly lower than the control group at 125 (p < 0.05), 150 (p < 0.01) and 175 (p < 0.05) days after birth (Table 4).

In group V (100 μg), the mean days of detection (latent periods) were significantly longer than the control group (p < 0.05). Compared with the control group, no significant changes in the weight and size of MCs per rat were observed in all groups administered DES (Table 5).

In the abdominal mammary glands at 50 days after birth, the number of TEBs was significantly higher in groups II (0.1 μg) (p < 0.05) and III (1 μg) (p < 0.01), while it was significantly lower in groups IV (100 μg) (p < 0.05) and V (100 μg) (p < 0.01) compared with the control group. In addition, in groups II (1 μg) (p < 0.05) and III (1 μg) (p < 0.01), the percentage of Ki67-positive cells in TEB was significantly higher than the control group (Table 6, Figures 1 and 2).

Discussion

We previously reported that a high dose (1000 μg) of 17μ-estradiol (E₂) during the neonatal period resulted in rats without CL or with PE due to the disturbance of the gonadotropin-secreting system in the hypothalamus (Funato et al., 2006). We also previously reported that the administration of 1.25 mg testosterone proprionate (TP) to female rats at neonatal periods induced loss of CL in the ovaries (a hormonal environment with markedly low levels of progesterone while estrogen exists), and that the administration of 20 mg DMBA at 50 days after birth resulted in a significant decrease in the number of induced MCs (Yoshida and Fukunishi, 1978; Yoshida et al., 1980c; Kawaguchi et al., 2000). An additional administration of progesterone to these rats showed rapid tumorigenesis of MCs (Yoshida et al., 1980a, 1980b).

From these results, it was considered that tumorigenesis of MCs in neonatally androgenized female rats induced by DMBA was suppressed by a long-term decrease in progesterone during the progression period. The present study revealed that the administration of DES, which is a synthetic estrogen, during the neonatal period, also induced a dose-dependent disturbance of the gonadotropin-secreting system, resulting in rats without CL or with PE and that female rats administered with a high dose (100 μg) of DES at the neonatal period frequently developed MCs which were able to proliferate in a hormonal environment with markedly low levels of progesterone similar to those levels observed in the TP-treated rats although the latent periods of these MCs were long. Therefore, it was hypothesized that the characteristics of established MCs could be different between TP- and DES-treated rats (e.g., the number of MCs of DES treated rats was higher and the latent period was longer compared with the MCs of TP-treated rats).

Tables 1 and 2 demonstrated that a disturbance of the gonadotropin-secreting system occurred at 50 days after birth (the period of administration of DMBA) in rats administered 10 and 100 μg DES at neonatal period. In addition, a number of rats with estrus cycle at 50 days after birth in groups administered with low (0.1 μg) or moderate (1, 10 μg) doses of DES during the neonatal period exhibited a disappearance of ovulation and CL at earlier periods in comparison to the control group.

The number of TEBs in the groups administered with DES during the neonatal period significantly decreased in the 100 μg group and increased in the 1 μg group compared with the control group at 50 days after birth. This phenomenon may be correlated with the direct effect of DES on the differentiation of mammary gland because a decrease in the number of TEBs was observed in the rats administered 100 μg DES during the neonatal period resulted from rapid differentiation of the mammary glands (unpublished data). TEBs, the most undifferentiated terminal ductal structures with a multilayer of highly proliferative epithelial cells, are highly vulnerable to chemical carcinogenesis (Russo and Russo, 1978). The incidence of carcinomas in rodents is directly associated with the density of TEBs in the mammary glands at the time of carcinogen administration (Russo and Russo, 1978; Russo et al., 1979). In response to stimulation from the mammogenic hormones, TEBs differentiate to more mature structures, namely, alveolar buds (ABs) and lobules (LOBs), which are less susceptible to carcinogens (Russo and Russo, 1978, 1987). The progressive differentiation of TEBs into ABs is accentuated by the estrous cycle, which begins at 30–42 days after birth. The development of the mammary gland in nonpregnant females is strongly controlled by the ovary.

An ovariectomy can cause regression of the end buds and cessation of growth (Russo and Russo, 1996). Since TEBs are considered to be located in the region of the mammary gland most sensitive to DMBA (Shilkaitis et al., 2000; Rowlands et al., 2002), the increased incidence of MCs in the 1μg group may be related to the increased number of TEBs. The increased number of MCs in the 0.1 μg group was also considered to have resulted from the increased number of TEBs seen in this group. In turn, the incidence of MCs in the 100 μg group until 175 days after birth was considered to have resulted from the decreased number of TEBs seen in this group. However, the incidence of MCs in the 100 μg group did not decrease at 300 days after birth. In the 10 μg group, the number of MCs increased in comparison with the control group from 250 days after birth although the number of TEBs decreased.

We previously reported that 1000 μg E₂ during the neonatal period resulted in marked suppression of MCs due to the strongly decreased number of TEBs and 10 μg E₂ resulted in promotion of MCs due to the increased number of TEBs (Funato et al., 2006). Therefore, the difference of mammary carcinogenesis may be associated with the difference of estrogenic potentials between DES (synthesized estrogen), which is inherently more estrogenic than E₂ (Andersen et al., 1999; Hendry et al., 2004), and E₂ (physical estrogen). In the present study, Ki-67 (proliferative index) expression in TEB was also examined whether DES affected the proliferative capability of the epithelial cells in TEB. From these results, it was possible that not only the number of TEBs but also proliferative activity of epithelial cells in TEB was associated with mammary carcinogenesis because of the increased Ki-67 positive cells in the 0.1 and 1 μg groups. However, the difference of various numbers of gene expressions at the TEBs should be investigated at 50 days after birth.

In conclusion, it is suggested that neonatally administered DES affects the gonadotropin-secreting system, resulting in a change in the endocrine system, which is thought to influence mammary carcinogenesis induced by DMBA. Moreover, it was suggested that the administration of a relatively low dose (1 μg) of DES during neonatal period increased the number of TEBs, thus resulting in a stimulatory effect on the initiation of MCs. Based on these results, it is necessary to further investigate the effects of other endocrine disrupting chemicals, such as dichlorodiphenyltrichloroethane (DDT) (Uppala et al., 2005), octylphenol (Han et al., 2002) and so on, during the neonatal period on the development of MCs.