Inhibitory effects of 17ß-estradiol and 4-n-octylphenol on 7,12-dimethylbenz[a]anthracene-induced mammary tumor development in human c-Ha-ras proto-oncogene transgenic rats
Beom Seok Han1,2,*,
Katsumi Fukamachi1,*,
Nobuo Takasuka1,
Takamasa Ohnishi1,
Mitsuaki Maeda1,
Tomomi Yamasaki3 and
Hiroyuki Tsuda1,4
1 Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
2 Division of General Toxicology, National Institute of Toxicological Research, 5 Nokbun-dong, Eunpyung-ku, Seoul 122-704, South Korea
3 Department of Nutrition and Biochemistry, National Institute of Public Health, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8638, Japan
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Abstract
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Experiments were conducted to determine whether the natural estrogen and an environmental compound with estrogenic action, 4-n-octylphenol (4nOP), could modify tumor development in human c-Ha-ras proto-oncogene transgenic (Tg) rats which are highly susceptible to mammary and skin carcinogens. Female and male Tg and non-transgenic (non-Tg) rats were given a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) (25 mg/kg body weight) at 50 days of age and thereafter subcutaneously implanted with cholesterol pellets containing 0.01, 0.1 or 1.0 mg ß-estradiol 3-benzoate (E2) per rat or received diets containing 1000 or 100 p.p.m. 4nOP for 12 weeks in females or for 20 weeks in males. E2 reduced the mammary tumor incidence and multiplicity in a dose dependent manner, especially in female Tg rats. In contrast, E2 increased mammary tumor incidence and multiplicity at the lowest dose (0.01 mg), however it reduced skin tumor induction in male Tg rats. 4nOP at a dose of 100 p.p.m. decreased mammary tumor multiplicity in female Tg rats (P < 0.001). No effects were observed in males. In separate in vitro studies, E2 at low doses (10-1110-8 M) enhanced the growth of both MCF-7 and T47D cells and this was similarly the case for 4nOP at high doses (10-710-5 M) in T47D cells. The finding that E2 and 4nOP at high doses caused reduction in mammary tumor development in female Tg and possibly non-Tg rats, may indicate that excess estrogen can exert a paradoxical inhibitory influence. E2 also appears to have bipotential effects in males, promoting mammary, but inhibiting skin carcinogenesis. These contrasting observations may be caused by differences in background physiological estrogen levels. In addition, the results suggest that Tg rats can be used in medium-term bioassay models to test for the modifying effects of estrogenic environmental compounds on mammary tumor development.
Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; E2, ß-estradiol 3-benzoate; MNU, N-methyl-N-nitrosourea; 4nOP, 4-n-octylphenol; Tg, transgenic; non-Tg, non-transgenic
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Introduction
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Mammary cancer is the second most frequent cause of cancer death in women (1). In epidemiological studies, the high incidences in women undergoing early menarche and late menopause point to a relationship with ovarian hormone exposure (2,3). Estrogen in particular, has long been associated with the risk of mammary cancer and there is now a large body of evidence suggesting that it is a crucial factor in mammary cancer etiology (4,5). In animal models, estrogen also promotes development of mammary tumors (68), while playing a key role in cell growth and proliferation of reproductive tissues such as mammary glands and uterus epithelium (4). However, the proposed links between estrogens and mammary cancer run counter to the facts that pregnancy, with long-lasting increase of estrogenic levels, is associated with protection against mammary cancer, and that the highest incidence of mammary cancer is observed in older women with very low estrogenic levels (9,10).
Rats and mice undergoing pregnancies also exhibit a greatly reduced susceptibility to chemically induced mammary carcinogenesis (11,12). In fact, it has been reported that treatment with estrogen at high doses can exhibit inhibitory effects on N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis (13,14). Therefore, the role of estrogen in mammary tumor development is equivocal.
Octylphenols, compounds derived from nonylphenol, are an estrogenic endocrine disrupting agent. They have been detected in the soils of rivers and may cause hazardous effects on certain fish and shellfish species, being passed along the biological food chain (15,16). They are reported to stimulate the growth of estrogen receptor-positive human mammary carcinoma cells (17), and also cause increase in the uterus weight and height of the endometrial epithelium in ovariectomized rats (18). However, due to the lack of an appropriate animal model to examine compounds with weak estrogenic potential, there has been no report of their influence on carcinogenesis in the mammary gland or other organs.
We have recently established a Tg rat strain carrying human c-Ha-ras proto-oncogenes. These rats are highly susceptible to MNU- and DMBA-induced mammary carcinogenesis. Mammary tumors develop in females in almost all animals as short as 8 to 12 weeks after a single MNU or DMBA treatment (unless topical application) (19,20). The animals have also been found to be susceptible to N-butyl-N-(4-hydroxybutyl) nitrosamine induced urinary bladder (21) and DMBA-induced skin carcinogenesis (unpublished observation).
In the present study, for the purpose of assessing whether the natural estrogen, E2 and a suspected endocrine disrupting agent with weak estrogenic action, 4nOP, can modify mammary carcinogenesis, we conducted experiments using the c-Ha-ras proto-oncogene Tg rats.
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Materials and methods
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Animals and chemicals
The human c-Ha-ras proto-oncogene Tg rats were generated by injecting the human c-Ha-ras proto-oncogene into pro-nuclei of fertilized rat oocytes from SpragueDawley rats (Clea Japan, Tokyo, Japan) (19). A total of 265 male and female c-Ha-ras proto-oncogene Tg and non-Tg rats at 7 weeks of age were maintained in plastic cages in an air conditioned room at 22 ± 2°C and 55 ± 10% humidity. The animals were allowed free access to basal diet (Oriental MF, Oriental Yeast Co., Tokyo, Japan) and tap water. The carcinogen DMBA was purchased from Tokyo Chemical Industry (Tokyo, Japan), E2 from Wako Pure Chemical Industries (Osaka, Japan) and 4nOP from Aldrich Chem. (Milwaukee, WI). The experiments were conducted according to the `Guidelines for the care and use of laboratory animals' of the Animal Study Committee of our National Cancer Center Research Institute.
Experimental protocols
A total of 265 female and male Tg and non-Tg rats were divided into four groups, all receiving a single dose of DMBA (25 mg/kg body weight) by gavage at 50 days of age (Figure 1
). One day thereafter, rats of group 1 were subcutaneously implanted with 1.0 cm silastic tubes containing E2 at doses of 1.0, 0.1 or 0.01 mg (subgroups) in cholesterol, twice over 12 weeks for females, or three times over 20 weeks for males. Rats of group 2 received cholesterol pellets without supplement (control). Rats of group 3 were placed on powdered basal diet containing 4nOP at doses of 1000 or 100 p.p.m. (subgroups), for 12 weeks for females and for 20 weeks for males. Rats of group 4 were given the basal diet without supplement (control). Palpation and observation were regularly performed to monitor development of mammary and skin tumors after DMBA treatment. The surviving animals were killed at the end of weeks 12 or 20 for females and males, respectively. Numbers of grossly visible tumors were recorded before being processed for histological examination. Mammary tumors were weighed and the values expressed as average tumor weight of single tumors for each rat. Body, liver, kidney, ovary, uterus, and testis weights were also recorded.
Measurement of serum estrogen and 4nOP
To determine level of serum estrogen, 35 ml blood samples were collected in plastic tubes at the end of the experiment when animals were killed. After clotting at room temperature, the tubes were centrifuged and the serum was collected. Measurement of serum estrogen was performed by BML, Inc. (Tokyo, Japan) using a coated tube radioimmunoassay. The limit of detection level was 8 pg/ml. 4nOP was extracted with ethyl acetate. 4nOP concentrations were measured by LC/MS (liquid chromatography/mass spectrometry). The limit for 4nOP detection was 50 pg/ml.
Histological examination
At autopsy, visible mammary and skin tumors were excised. Mammary and skin tumors were immediately fixed in ice-cold acetone, trimmed and embedded in paraffin. Histopathological examinations of mammary tumors were then performed according to the criteria for classification of mammary tumors of Squartini and Pingitore (22), Boorman et al. (23). Skin tumors were also diagnosed with reference to the observation in mice (24) and criteria for rats (25).
Cell culture
The estrogen receptor positive human mammary cancer cell line T47D was kindly provided by Dr S.Kubota, University of Tokyo and MCF-7 by Dr K.Sakabe, Kitazato University. For routine maintenance, cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose, supplemented with 10% (v/v) heat inactivated fetal-bovine serum and 25 mM HEPES. Cells were grown at 37°C in an atmosphere of 5% CO2/95% air under saturating humidity and routinely passaged at
70% confluence.
Cell proliferation assay
Cells were seeded in 96 well plates (2500 cells/well), and then incubated for 1 day (37°C, in an atmosphere of 5% CO2/95% air under saturating humidity). The seeding medium (DMEM) was removed and changed to phenol red free DMEM medium supplemented with low protein solution, `TCH' (CELOX, St Paul, MN), instead of charcoal-dextran treated fetal bovine serum. Test chemicals were then added to each well. The plates were incubated for 3 days and cell proliferation was measured by WST-1 assay (26) using a Cell Counting Kit (DOJINDO, Kumamoto, Japan). Then, absorbance (460 nm) was measured using a Bio-Rad Model 550 microplate reader. The effects of estrogen on relative cell density were assessed by dividing the optical density (OD) of cells treated with a given estrogen concentration by that of controls grown without estrogen.
Statistics
The data for body weights, organ weights, serum level of estrogen, incidence, weight and multiplicity of tumors were analyzed using the JMP software package (version 3.1) (SAS Institute, Cary, NC) on a Macintosh computer. Tumor incidence data were analyzed by Chi square test. Other data were compared with the Dunnett t-test after ANOVA analysis (27,28).
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Results
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Mortality, body weights and relative organ weights
Only five rats died before the termination of the experiment, three in E2 and two in 4nOP experimental groups, without any relation to the treatment or the Tg/non-Tg status. Data for final body weight and relative organ weights for rats given E2 or 4nOP are summarized in Tables I and II
. In females, relative liver weights in groups given E2 were increased as compared with group 2 in both Tg and non-Tg rats. Relative ovary weights of group 1 given E2 were decreased with high doses. No significant differences were noted in the weights of kidneys, ovaries and uterus in the groups fed 4nOP (data not shown). In males, final body and relative testis weights in group 1 given 1.0 mg E2 were significantly decreased as compared to group 2 (P < 0.001) in both Tg and non-Tg rats. Relative liver weights in group 3 fed 1000 p.p.m. 4nOP were significantly increased as compared to group 4 (0 p.p.m.) (P < 0.05).
Serum estrogen and 4nOP levels
Data for serum estrogen levels are summarized in Table III
. In both Tg and non-Tg rats, the estrogen levels of group 1 given 1.0 mg were significantly increased compared with group 2 (P < 0.001). Serum 4nOP was below detectable levels in all groups (data not shown).
Time-sequence cumulative and final incidences and multiplicities of mammary tumor
Cumulative multiplicity data for palpable mammary and visible skin tumors are presented in Figures 2 to 4

. The first appearance of mammary tumors in groups 1 and 3 was at weeks 5 and 10 for female and male Tg rats, whereas it was at weeks 10 and 20 for female and male non-Tg rats, respectively. In the E2 experiment, tumor multiplicity increased with time, but is suppressed in relation to treatment dose (Figure 2A
). In the 4nOP experiment, the mammary tumor multiplicity was also decreased, but dose-dependence was not as clearly documented (Figure 3A
). Although values were low, a similar tendency was observed in non-Tg rats. In male Tg animals, the tumor multiplicities for group 1 given 1.0 or 0.1 mg E2 were decreased as compared with group 2. However, the tumor multiplicities for group 1 given the intermediate dose 0.01 mg E2 were increased compared with group 2. Values for group 3 fed 100 p.p.m. 4nOP were also lower than in group 4 (Figures 2B and 3B
).

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Fig. 2. Cumulative mammary tumor multiplicity in Tg and non-Tg rats treated with E2. (A) females; (B) males.
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Fig. 3. Cumulative mammary tumor multiplicity in Tg and non-Tg rats fed 4nOP (1000 or 100 p.p.m.). (A) females; (B) males.
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Fig. 4. Cumulative skin tumor multiplicity in Tg and non-Tg male rats given E2 (1.0, 0.1 or 0.01 mg) or fed 4nOP (1000 or 100 p.p.m.). (A) E2; (B) 4nOP.
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Final mammary tumor incidences and multiplicities determined by histological examination are summarized in Tables III and IV
. In female Tg rats, all mammary tumors were diagnosed as adenocarcinomas, the incidence for the 1.0 mg E2 group 1 rats being significantly decreased compared with the controls. Similarly, the multiplicity for group 1 rats given 1.0 and 0.1 mg E2 was significantly decreased (6.6% and 24.8% of the group 2 value, respectively). The value for group 3 given 100 p.p.m. 4nOP was also significantly reduced (26.9% of the group 4 value). In female non-Tg rats, mammary tumor incidences for group 1 treated with 1.0 or 0.1 mg E2 were significantly decreased, along with the tumor multiplicity. On the other hand, in male Tg rats, total mammary tumor incidence for group 1 given 0.01 mg E2 was significantly increased (180% of the group 2 value, P < 0.05). No inter-group differences were observed for either incidence or multiplicity of adenomas, adenocarcinomas (80 case) and sarcomas (four cases) in Tg and non-Tg rats in the 4nOP experiment (data not shown).
Time-sequence cumulative and final incidence and multiplicity of skin tumors
Skin tumors were induced only in males. In Tg rats, the first appearance was at week 17 for group 1 given E2, week 14 for group 2, at week 16 for group 3 given 4nOP and week 16 for group 4, whereas it was at week 20 for group 1 in non-Tg rats (Figure 4
). Cumulative multiplicity of skin tumors in group 1 Tg rats was decreased with E2 treatment as compared to group 2, but dose-dependence was not as clearly documented (Figure 4A
). Slight decrease in Tg rats of group 3 fed 100 p.p.m. 4nOP was also evident as compared with group 4 (Figure 4B
).
Data for skin papillomas and squamous cell carcinomas in male rats are summarized in Table V
. In Tg rats, total skin tumor incidences with 1.0, 0.1 and 0.01 mg E2 of group 1 were significantly decreased as compared to group 2. With 4nOP, no significant inter-group differences were observed in male Tg and non-Tg rats.
Effects of E2 and 4nOp on growth of human mammary cancer cells
Data for effects of E2 and 4nOP on growth of human mammary cancer cells are summarized in Figure 5
. E2 caused growth of both MCF and T47D cells even at low doses (10-1110-8 M), whereas 4nOP only exerted effects on T47D cells at high doses (10-710-5 M). However, E2 caused inhibitory effects on both cells at higher dose range.

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Fig. 5. Influence of E2 and 4nOP on human mammary carcinoma cell proliferation in a low protein medium. Cell proliferation activities were expressed as relative to control (untreated) values.
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Discussion
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In the present study, E2 significantly inhibited mammary tumor development in both Tg and non-Tg females. In contrast, 0.01 mg E2 caused enhancement of tumor development in male Tg rats. Inhibitory effects on mammary tumor development were also observed with 4nOP at a dose of 100 p.p.m. in female Tg animals. The inhibitory effects did not appear to be due to toxic effects of the compounds, because body weights were not reduced by the treatment. Although obvious differences in body weights, males in E2 experiment group being larger than those in 4nOP were noted, they were not within the treatment groups to be compared. Involvement of seasonal factors and inherent variation within the SpragueDawley strain is presumed. Rats used for the two experiments were not supplied at the same time and wild females from outbred colonies (CLEA Japan, Tokyo) were used to mate with Tg male rats.
The results are thus not clearly in line with animal and human studies indicating that estrogen is a major factor for proliferation of mammary epithelial cells and also a prerequisite for mammary carcinogenesis (58). However, this may be explained by the dose of estrogen. 17ß-estradiol at high dose and progesterone treatment after DMBA administration were shown to inhibit mammary tumor development in an earlier study (29) and it was recently reported that 17ß-estradiol at high dose (30 mg, 200 µg and 100 µg/rat) significantly reduced mammary tumor incidence and multiplicity induced by MNU in rats (13,14). The fact that E2 stimulated growth of MCF-7 and T47D cells at low dose, whereas it inhibited cell growth at high dose, is clearly of interest in this context. Similarly, it has been reported that genistein and other flavonoids with estrogenic action stimulate cell growth at low dose in human mammary cancer cells, whereas they inhibit cell proliferation at high doses (30).
Regarding inhibitory mechanisms, it has been suggested that preneoplastic cells induced by carcinogen treatment differentiate into lobulo-alveolar cells from terminal end buds (31,32) or undergo apoptosis (33). Unlike the results for female Tg rats, 0.01 mg E2 significantly increased development of mammary tumors in males. We therefore conclude that E2 may stimulate growth of mammary gland tumors when low background estrogen levels prevail.
E2 also inhibited development of skin tumors, particularly papillomas, in male Tg rats. To our knowledge, this is the first demonstration such inhibition, although it is reported that 17ß-estradiol can reduce the invasive potential of a human cutaneous melanoma cell line (34). The mechanism remains to be clarified, but again the sex difference provides a clue. In the progeny of SHR male mice exposed to X-ray irradiation (35) and the newborn SENCAR mouse (36), induction of skin papillomas by a 2-stage chemical carcinogenesis protocol was higher in males than in females. This observation may be in line with the lack of skin tumor induction in female rats observed in this study, indicating that feminization by E2 may have played a role in reducing the skin tumors.
While some epidemiological studies failed to find associations between serum hormone levels and mammary cancer (37,38), some reports featured a correlation (39,40). In the present study, the serum estrogen level at a dose of 1.0 mg E2 was similarly 5- to 8-fold the control level in female Tg and non-Tg rats (Table III
). This is clearly in line with a previous report that high 17ß-estradiol levels (168.8 pg/ml) in virgin Lewis rats treated with 30 mg 17ß-estradiol, being 9-fold the control value (18.3 pg/ml) exerted inhibitory effects on mammary tumors (13). Further studies are thus needed to elucidate the relation between serum estrogen level and tumor development in the mammary gland at lower doses of E2.
Octylphenols, environmental estrogens, may stimulate mammary cell proliferation and thereby promote mammary tumorigenesis (17). Our finding that 4nOP causes cell proliferation of T47D cells is indicative of estrogenic action and slight enhancement of lesion development was observed in male rats in which estrogen levels were low. However, the reduction of mammary tumors in female Tg rats fed 100 p.p.m. 4nOP suggests that any estrogenic action of this compound may be overwhelmed by endogenous estrogens. Since the serum level of 4nOP was below the detectable level, the mechanisms underlying the observed inhibitory effects are unclear.
In conclusion, in the present study, E2 at high dose effectively reduced the development of mammary glands in female, and skin tumors in male human c-Ha-ras proto-oncogene Tg rats. Similarly, 4nOP at high doses demonstrated inhibitory potential on mammary tumors. While further studies are now required to elucidate what influence E2 and 4nOP might exert at low-dose levels in c-Ha-ras proto-oncogene Tg rats, it should be noted that similar inhibitory effects were observed in non-Tg rats. The similar tendency observed for mammary carcinogenesis in non-Tg rats, although not significant, is also of interest in this context. The results also brought additional evidence supporting the use of Tg rats in medium-term bioassay models to study the modifying potential of environmental estrogenic compounds on mammary and skin carcinogenesis.
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Notes
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* The first two authors contributed equally to this work. 
4 To whom correspondence should be addressed Email: htsuda{at}gan2.ncc.go.jp 
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control, Grant-in-Aid for Cancer Research and Grant-in Aid (H11-Seikatsu-018) for Research on Environmental Health from the Ministry of Health, Labor and Welfare, a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology and a Grant-in-Aid for CREST (Core Research of Evolutional Science and Technology) of Japan Science and Technology Corporation (JST), Japan. Beom Seok Han was a recipient of the Foreign Research Fellowship Program for Invitation of Foreign Researchers from the Foundation for Promotion of Cancer Research, supported by the 2nd Term Comprehensive 10-Year Strategy for Cancer Control. Katsumi Fukamachi and Takamasa Ohnishi were recipients of Research Resident Fellowships from the Foundation for Promotion of Cancer Research in Japan. We thank Dr Malcolm A.Moore for his kind advice regarding the English language in the manuscript. We also would like to express our appreciation to Dr Yohichiro Matsuoka for pertinent assistance and advice on animal experimentation with 4nOP.
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Received August 2, 2001;
revised March 20, 2002;
accepted March 20, 2002.