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Chemopreventive effects of tamoxifen in ethyl methanesulphonateinduced rat mammary carcinogenesis
Cancer Letters. 71 (1993) 19-24 Elsevier Scientific Publishers Ireland
19 Ltd.
Chemopreventive effects of tamoxifen induced rat mammary carcinogenesis Hiroaki Ueoa, Hideo Matsuokaa, Ryosaburo Takakib and Tsuyoshi
in ethyl methanesulphonate-
Masayuki Hondaa, Akiyoshi”
Hiroshi
Inouea,
“Department of Surgery. Medical Insiitute of Bioregulation, Kyushu University, 4546, Tsurumihara. Beppu and hThe First Depurtmenl of Inrernal Medicine, Oita Medical College, Oita (Japan) (Received 31 August 1992) (Revision received 21 December (Accepted 22 March 1993)
1992)
Summary Ethyl methanesulphonate (EMS), an alkylating agent and a potent mutagen, has been shown to be an effective carcinogen for the induction of mammary carcinoma in female Wistar King A rats. We therefore utilized this new system to assess the effects of tamoxifen (TAM) on mammary carcinogenesis. In Group A rats, given EMS orally for a period of 12 weeks, mammary carcinomas were first detected at the 13th week and were found in all surviving rats at the 20th week. The concomitant administration of TAM for 4 weeks, in Group B rats, retarded the development of the tumors significantly. There was a significant reduction in the incidence of estrogen receptor (ER)-positive tumors in the rats previously exposed to TAM; 100% in Group A versus 50% in Group B. Neither the progesterone receptor (PgR) nor androgen receptor (AR) status of the tumors were significantly different between these two groups. The inhibitory effects of TAM on tumor induction was also observed when TAM treatment started after EMS administration, though the intensity was smaller than that in Group B. These findings suggest the preventive action of TAM on Correspondence to: Hiroaki Ueo. M.D.. gery, Medical Institue of Bioregulation. 4546 Tsurumihara. Beppu 874, Japan.
Department of SurKyushu University,
EMS-induced mammary carcinogenesis, and indicate that this tumor system may provide a feasible model for research on chemoprevention and hormone therapy using an antiestrogen for human mammary carcinoma.
Keywords: tamoxifen; ethyl methanesulphonate
rat mammary
carcinoma;
Introduction Chemical carcinogen-induced mammary carcinomas in rats have been extensively used as animal models for human breast carcinoma [l-4]. We previously found a high incidence of mammary carcinoma in female Wistar King A rats when ethyl methanesulphonate (EMS), a mutagenic alkylating agent, was given orally [7] and characterized this system as a new experimental model for mammary carcinogenesis [8-91. It has been shown that the EMS-induced mammary tumors contained ER and that tumor induction was prevented by ablative oophorectomy prior to EMS administration [9]. Such previous data therefore suggested that the hormonal status, especially the action of the ovarian hormones, was closely linked to the EMS-induced mammary carcinogenesis. Tamoxifen (TAM), an anti-estrogenic agent, has
been widely used for postoperative adjuvant therapy to prevent the recurrence of clinical mammary carcinoma as well as for the palliative treatment of advanced carcinoma [ 10,111. TAM is also expected as a chemopreventive agent for human mammary carcinoma [ 121 and a trial of TAM prophylaxis has been conducted in women at high risk for the development of mammary carcinoma [13]. In the present study, therefore, we examined the effects of TAM on the tumor incidence and hormone receptor status in EMS-induced rat mammary carcinogenesis and thus evaluated the efficacy of this system for research on chemoprevention in mammary carcinoma.
The rats were examined every 2 weeks for palpable tumors and the occurrence of tumors was dated from the initiation of EMS administration. All tumors were fixed in 10% formalin solution for histological examination. The tumors in Groups A and B, surgically extirpated from the 18th to the 30th week, were examined for contents of ER, PgR and AR in the cytosol by using a dextran coated charcol assay [ 161. The statistical significance of tumor incidence and the mean number of tumors per rat were determined by the x2-test and Wilcoxon test, respectively. P-values less than 0.05 were considered to be statistically significant.
Material and Methods
Results
Mammary carcinomas were induced by the oral administration of EMS (Eastman Kodak Co., Rochester, New York, USA) in a similar manner as described previously [7-91. Briefly, lo-* M of EMS solution in tap water, in which the concentration was relatively higher than that in our previous studies, was orally given to rats for 12 weeks with a renewal of the solution every 2 days. Tamoxifen, supplied by I.C.I. Pharma Co Ltd., was first diluted in ethanol and then serially diluted to obtain the required concentrations in 0.5 ml solution. Group B, C and D rats received 50 mg/kg of TAM subcutaneously once a week for a period of 4 weeks. Single administrations of such a large dose of TAM (5-10 mg/rat) had been known to produce prolonged anti-estrogenic effects in rats [ 14,151. Equal volumes of the diluent were administered to the control group. The rats were given tap water after EMS administration and were freely fed with commercial pellet food throughout the experiments. Ninety-six female Wistar-King A rats, 4 weeks old, were divided into 6 groups according to the following treatments: Group A, EMS administration alone, beginning at 4 weeks of age; Group B, a concomitant treatment of TAM for 4 weeks with EMS administration; Group C, TAM treatment for 4 weeks subsequent to EMS administration at 16 weeks of age; Group D, TAM treatment alone for 4 weeks simultaneously with Group A; Group E, administered diluted-ethanol solution as a control and Group F, no treatment control.
The incidences of mammary carcinoma in each group are shown in Fig. 1. In Group A rats, given EMS alone, the mammary tumors were first detected at the 13th week and tumor incidence rapidly increased as the observation period proceeded, i.e. 35.3% at the 14th week, 64.7% at the 16th week and 100°/o at the 20th week. In Group B rats, concomitantly given TAM and EMS at 4 weeks of age, the development of mammary carcinomas was significantly retarded, i.e. no tumor was found up to the 16th week and the tumor inci-
EMS
treatment P
E g
80
i ,z
60
g F
40
z ?
20
-z 5u
0
0
4
8
12
Observation
16 period
20
24
28
32
(weeks)
Fig. 1. Effects of TAM on the tumor incidence of EMSinduced mammary carcinomas. EMS was orally given for 12 weeks and TAM (10 mg/kg) was injected twice a week for 4 weeks. Symbols are: 0, Group A (EMS alone): 0, Group B (TAM concomitant with EMS): A, Group C (TAM subsequent to EMS); 0, Group D (TAM alone); A. Group E (control).
EMS -
TAM alone
ethanol solution no treatment
C
D
E F
“Mean & S.D. bArrow
EMS + TAM
B
indicates
TAMb
EMS alone
A
0.13 *0.35 0 0 0
0
0.24 ZtO.44
14
16
by EMS.
0 0 0
at the following
induced
18
0 0 0
periods
by’. *P < 0.01. **P < 0.05.
Carcinomas/rat
carcinoma
‘followed
of mammary
Treatment
Multiplicity
Groups
Table I. (weeks)a
**
0 0 0
0 0 0
;I;]* y;;]*
22
2.69 f 1.30 0 0 0
0.43 izO.65
26
l ;z;]* ***;tj]*-**
20
I
* 4.00 *0.86 0 0 0
0.85 f 1.40
30
* 1
22 Table II.
Hormone
Groups
A B
receptor
status
Treaatment
EMS alone EMS + TAM
aTumors were extirpated *P < 0.05; significantly
of EMS-induced
rat mammary
carcinoma.
No. of tumors examined
No. of receptor-positive ER
PgR
AR
10 10”
10 (100) 5 (50)*
7 (70) 6 (60)
0 (0) 0 (0)
tumors
(‘!h)
22-26 weeks after TAM treatment. different from Group A.
dence during the observation period was significantly lower than that in Group A (P < 0.01). TAM treatment subsequent to EMS administration at the age of 16 weeks also retarded tumor induction in Group C rats as compared with Group A (P < 0.05), though the inhibitory effects were smaller than that in Group B. No mammary tumor was induced in either the Group D rats given TAM alone or the control Groups E and F. The multiplicity of the mammary carcinomas, expressed as the mean number/rat, was also significantly reduced by either the concomitant or consecutive administration of TAM in Group B or Group C, respectively (Table I). The inhibitory effect of TAM was more evident in Group B than in Group C, i.e. the mean number of tumors in Group B was significantly lower than that in Group C (P < 0.01). The hormone receptor status of the mammary carcinomas extirpated at week- 30-40 in Groups A and B are summarized in Table Il. There was a significant reduction in the incidence of ER-positive tumors in Group B, i.e. 5 of 10 tumors (50%) in Group B were ER-positive, whereas all 10 tumors in Group A possessed ER. On the other hand, the incidences of PgR positive-tumors were not significantly different between these two groups. There was no AR-positive tumor either in Group A or B. Discussion A system of rat mammary carcinogenesis using EMS seemed to have the following characteristics or advantages: (i) the procedure for tumor induc-
tion is simple, that is. EMS can be dissolved and given in drinking water; (ii) the incidence of tumor induction by 3 x 10m3 M of EMS solution were consistent in several independent experiments and all surviving rats have the mammary carcinomas within 40 weeks 17-91; (iii) ER-positive tumors develop at a high rate [9]; and (iv) tumor induction can obviously be prevented by endocrine ablative surgery such as oophorectomy [9]. Moreover, the present study demonstrated additional characteristics of EMS tumor induction: (v) a higher concentration of EMS solution (10-I M) was able to induce tumors in all rats within a shorter period (20 weeks); (vi) the EMS-induced tumors frequently possessed PgR but not AR; and (vii) tumor induction was significantly inhibited by TAM. In designing the present experiment, the rats were given EMS solution for 12 weeks according to our routine protocol and we attempted to evaluate the effects of TAM on mammary carcinogenesis at either an early stage (during the initial 4 weeks of EMS administration) or a later stage (immediately after EMS administration and during week 13-16) of the tumor induction. We previously found that the mammary carcinomas were first detected at around the 16th week and the tumor incidences reached 100% at week 32-40 when the rats were given 3 x 10m3 M of EMS solution [7-91. Since tumor incidence was dependent on the concentration of EMS solution [8], a relatively higher concentration of EMS solution (10-I M) was used in the present experiments with an attempt to shorten the latent period of mammary carcinogenesis. As expected, in the Group A rats given EMS alone, the tumors began to appear at the 13th week and tumor incidence reached
23
100% at the 20th week. Consequently, in the present study, the effects of TAM on both the initiation at an early stage (Group B) and tumor appearance at a later stage (Group C) of the tumor induction were examined. The present results provided an evidence for the chemopreventive effects of TAM on both early and later stages of mammary carcinogenesis induced by EMS. In Group B rats, concomitantly given EMS and TAM, tumor appearance was significantly retarded and the final tumor incidence at the 30th week was SO.O%, while in Group A rats given EMS alone, tumor incidence reached 100% at the 20th week. TAM treatment subsequent to EMS administration in Group C rats also delayed tumor appearance significantly. Similar findings were obtained in the analysis of the multiplicity of the tumors. These inhibitory effects of TAM were compatible with those in previous reports using other carcinogen-induced rat tumor systems, such as those induced by 7,12dimethylbenzanthracene (DMBA) [15,17,18] or Nmethyl-N-nitrosourea (MNU) [19,20]. Most of these rat mammary carcinomas possess ER and both the tumor induction and the growth were significantly inhibited by TAM. Jordan reported that TAM blocked estrogen binding to ER in the mammary tumors and consequently inhibited both the initiation and growth of DMBA tumors [ 151. It has also been suggested that TAM may have a direct action at the ovarian level and reduce the circulating estrogen levels [21]. In the present study, the inhibitory effects of TAM on EMSinduced mammary carcinogenesis were similar to those of ablative oophorectomy in our previous study in which the tumor development was completely prevented by oophorectomy prior to EMS administration, and partially, by oophorectomy after EMS administration [9]. As EMS-induced mammary tumors are thus dependent on ovarian hormones and all the tumors possess ER, the possible mechanism for the preventive effect of TAM in the present study seems to involve the impairment of the effects of ovarian hormones on tumor development, by either a blockade of estrogen binding to ER at the tumor level [15] or a reduction of the circulating estrogen level through some action at the ovarian level [21].
The difference in the inhibitory effects of TAM on EMS-induced mammary carcinogenesis between Group B (TAM treatment concomitant with EMS administration at 4 weeks of age) and Group C (TAM treatment after EMS administration at 16 weeks of age) might reflect the different responsiveness of the mammary glands to TAM treatment. When the Group C rats were treated with TAM after EMS administration, the majority of the mammary glands might have already been fully initiated and sensitized for tumor production by the previous EMS administration in the presence of ovarian hormones. The other possibility was that the hormone dependency of the initiated mammary glands and the latent mammary carcinomas might have changed at a later stage of tumor development and consequently the effects of TAM were less evident in Group C rats. This possibility may be supported by our previous finding that the ER contents of the EMS induced mammary carcinomas declined during tumor progression [9]. In another experimental system, Sukumar et al. [5] and Turcot-Lamay [18] also reported that the hormone responsiveness of MNU induced rat mammary carcinomas had been reduced during the late stages of tumor development. Another purpose of the present study was to examine the hormone receptor status of the EMSinduced mammary tumors occurring in the rats which had been exposed to TAM. The data showed that concomitant TAM treatment with EMS administration reduced the incidence of ERpositive tumors. Since it has been shown that the circulating levels of ovarian hormones influence the concentration of ER in rat mammary tumors [3], a reduction of the circulating ovarian hormones by TAM treatment appears to be responsible for the reduced incidence of ER-positive tumors. Although further investigation is needed for a better understanding of the biological mechanisms in which TAM affects the tumor induction and the hormone receptor status, it is considered that EMS-induced rat mammary carcinoma may serve as a useful model for research on chemoprevention by antiestrogens as well as help elucidate a role of hormone receptor status during mammary carcinogenesis.
24
Acknowledgement We thank Dr. Brian T. Quinn ‘on the manuscript.
10
for his comments 11
References 12 Welch. C.W. (1985) Host factors affecting the growth of carcinogen-induced rat mammary carcinomas: A review and tribute to Charles Brenton Huggins. Cancer Res.. 45. 3415-3443. Sinha. D., Cooper, D. and Dao. T.L. (1973) The nature of estrogen and prolactin effects on mammary tumorigenesis. Cancer Res., 33, 41 l-415. Hawkins, R.A.. Hill, A.. Freedman, B., Killen. E.. Buchan. P., Miller. W.R. and Forrest. A.P.M. (1977) Oestrogen receptor activity and endocrine status in DMBA-induced rat mammary tumors. Eur. J. Cancer. 13, 223-228. Williams, J.C., Gusterson. B. Humphreys, J., Monaghan. P.. Coomber. R.C., Rudland, P. and Neville. A.M. (1981) N-methyl-N-nitrosourea-induced rat mammary tumors. Hormone responsiveness but lack of spontaneous metastasis. J. Natl. Cancer Inst., 66, 147-155. Sukumar, S., Carney, W.P. and Barbacid, M. (1988) Independent molecular pathways in initiation and loss of hormone responsiveness of breast carcinomas. Science, 240, 524-526. McCormick, D.L., Adamowski, C.B., Fiks, A. and Moon. R.C. (1981) Lifetime dose-response relationships for mammary tumor induction by a single administration of N-methyl-N-nitrosourea. Cancer Res., 41. 1690-1694. Ueo. H., Takaki, R., Yamagami, H., Nakano, S.. Okeda, T. and Sakakibara. K. (1979) High incidence of rat mammary carcinoma by oral administration of ethyl methanesulphonate. Cancer Lett., 79. 79-84. Ueo. H., Takaki, R.. Yamagami. H. and Sugimachi. K. (1981) Mammary carcinoma induced by oral administration of ethyl methanesulphonate. Determination of some of the parameters affecting tumor induction. Carcinogenesis, 2, 1223-1228. Ueo, H., Matsuoka. H., Akiyoshi, T., Sugimachi. K. and Takaki. R. (1990) Estrogen receptor status and effects of endocrine ablative surgery in ethyl methanesulphonateinduced rat mammary carcinoma. Cancer Lett., 51. 151-155.
13 14
15
16
17
18
19
Early Breast Cancer Trialist’s Collaborative Group (1988) Effect of adjuvant tamoxifen and of cytotoxic therapy on mortality in early breast cancer. N. Engl. J. Med., 319. 1681-1692. Cole, M.P., Jones, C.T.A. and Todd, I.D.H. (1971) A new antiestrogenic agent in late breast cancer: An early clinical appraisal of ICI 46474. Br. J. Cancer, 25, 270-275. Jordan, V.C. (1991) Chemosuppression of breast cancer with long-term tamoxifen therapy. Prev. Med. 20, 3-14. Cuzick. J.. Wang, D.Y. and Bulbrook, R.D. (1986) The prevention of breast cancer. Lancet. January I I. 83-86. Emmens, C.W. (1971) Compounds exhibiting prolonged anti-oestrogenic and anti-fertility activity in rats and mice. J. Reprod. Fertil.. 26, 175-182. Jordan, V.C. (1976) Effect of tamoxifen (ICI 46,474) on initiation and growth of DMBA-induced rat mammary carcinoma. Eur. J. Cancer, 12. 419-424. Thibodeau, S.N., Freeman, L. and Jiang, NS. ( 1981) Simultaneons measurement of estrogen and progesterone receptors in tumor cytosols with use of “51-1abeled estradiol and 3H-R5020. Clin. Chem., 27, 687-691. Welsch, C.W., Goodrich-Smith. M., Brown. C.K.. Mackie. D. and Johnson. D. (1982) ?-Bromo-oergocyryptine (CB-154) and tamoxifen (ICI 46.474) induced suppression of the genesis of mammary carcinomas in female rats treated with 7,12-dimethylbenzanthracene (DMBA): A comparison. Oncology, 39. 88-92. Turcot-Lemay, L. and Kelly, P.A. (1980) Characterization of estradiol, progesterone, and prolactin receptors in nitrosomethylurea-induced mammary tumors and effect of an antiestrogen treatment on the development and growth of these tumors. Cancer Res., 40, 3232-3240. Osborne. M.P.. Ruperto. J.F., Crowe, J.P.. Rosen, P.P. and Telang N.T. (1992) Effect of tamoxifen on preneoplastic cell proliferation in N-nitroso-Nmethylurea-induced mammary carcinogenesis. Cancer Res.. 52. 1477-1480.
20
Thompson. H.T. and Ronan, A.M. (1986) Effects of D.L2-difluoromethylornithine and endocrine manipulation on the induction of mammary carcinogenesis by I-methyll-nitrosourea. Carcinogenesis, 7. 2003-2006.
21
Watson, J.. Anderson. F.B., Alam. M.. O’Grady, J.E. and Heald, P.J. (1975) Plasma hormones and pituitary luteinizing hormone in the rat during the early stages of pregnancy and after post-coital treatment with tamoxifen (ICI 46.474). J. Endocrinol., 65. 7- 13.
19 Ltd.
Chemopreventive effects of tamoxifen induced rat mammary carcinogenesis Hiroaki Ueoa, Hideo Matsuokaa, Ryosaburo Takakib and Tsuyoshi
in ethyl methanesulphonate-
Masayuki Hondaa, Akiyoshi”
Hiroshi
Inouea,
“Department of Surgery. Medical Insiitute of Bioregulation, Kyushu University, 4546, Tsurumihara. Beppu and hThe First Depurtmenl of Inrernal Medicine, Oita Medical College, Oita (Japan) (Received 31 August 1992) (Revision received 21 December (Accepted 22 March 1993)
1992)
Summary Ethyl methanesulphonate (EMS), an alkylating agent and a potent mutagen, has been shown to be an effective carcinogen for the induction of mammary carcinoma in female Wistar King A rats. We therefore utilized this new system to assess the effects of tamoxifen (TAM) on mammary carcinogenesis. In Group A rats, given EMS orally for a period of 12 weeks, mammary carcinomas were first detected at the 13th week and were found in all surviving rats at the 20th week. The concomitant administration of TAM for 4 weeks, in Group B rats, retarded the development of the tumors significantly. There was a significant reduction in the incidence of estrogen receptor (ER)-positive tumors in the rats previously exposed to TAM; 100% in Group A versus 50% in Group B. Neither the progesterone receptor (PgR) nor androgen receptor (AR) status of the tumors were significantly different between these two groups. The inhibitory effects of TAM on tumor induction was also observed when TAM treatment started after EMS administration, though the intensity was smaller than that in Group B. These findings suggest the preventive action of TAM on Correspondence to: Hiroaki Ueo. M.D.. gery, Medical Institue of Bioregulation. 4546 Tsurumihara. Beppu 874, Japan.
Department of SurKyushu University,
EMS-induced mammary carcinogenesis, and indicate that this tumor system may provide a feasible model for research on chemoprevention and hormone therapy using an antiestrogen for human mammary carcinoma.
Keywords: tamoxifen; ethyl methanesulphonate
rat mammary
carcinoma;
Introduction Chemical carcinogen-induced mammary carcinomas in rats have been extensively used as animal models for human breast carcinoma [l-4]. We previously found a high incidence of mammary carcinoma in female Wistar King A rats when ethyl methanesulphonate (EMS), a mutagenic alkylating agent, was given orally [7] and characterized this system as a new experimental model for mammary carcinogenesis [8-91. It has been shown that the EMS-induced mammary tumors contained ER and that tumor induction was prevented by ablative oophorectomy prior to EMS administration [9]. Such previous data therefore suggested that the hormonal status, especially the action of the ovarian hormones, was closely linked to the EMS-induced mammary carcinogenesis. Tamoxifen (TAM), an anti-estrogenic agent, has
been widely used for postoperative adjuvant therapy to prevent the recurrence of clinical mammary carcinoma as well as for the palliative treatment of advanced carcinoma [ 10,111. TAM is also expected as a chemopreventive agent for human mammary carcinoma [ 121 and a trial of TAM prophylaxis has been conducted in women at high risk for the development of mammary carcinoma [13]. In the present study, therefore, we examined the effects of TAM on the tumor incidence and hormone receptor status in EMS-induced rat mammary carcinogenesis and thus evaluated the efficacy of this system for research on chemoprevention in mammary carcinoma.
The rats were examined every 2 weeks for palpable tumors and the occurrence of tumors was dated from the initiation of EMS administration. All tumors were fixed in 10% formalin solution for histological examination. The tumors in Groups A and B, surgically extirpated from the 18th to the 30th week, were examined for contents of ER, PgR and AR in the cytosol by using a dextran coated charcol assay [ 161. The statistical significance of tumor incidence and the mean number of tumors per rat were determined by the x2-test and Wilcoxon test, respectively. P-values less than 0.05 were considered to be statistically significant.
Material and Methods
Results
Mammary carcinomas were induced by the oral administration of EMS (Eastman Kodak Co., Rochester, New York, USA) in a similar manner as described previously [7-91. Briefly, lo-* M of EMS solution in tap water, in which the concentration was relatively higher than that in our previous studies, was orally given to rats for 12 weeks with a renewal of the solution every 2 days. Tamoxifen, supplied by I.C.I. Pharma Co Ltd., was first diluted in ethanol and then serially diluted to obtain the required concentrations in 0.5 ml solution. Group B, C and D rats received 50 mg/kg of TAM subcutaneously once a week for a period of 4 weeks. Single administrations of such a large dose of TAM (5-10 mg/rat) had been known to produce prolonged anti-estrogenic effects in rats [ 14,151. Equal volumes of the diluent were administered to the control group. The rats were given tap water after EMS administration and were freely fed with commercial pellet food throughout the experiments. Ninety-six female Wistar-King A rats, 4 weeks old, were divided into 6 groups according to the following treatments: Group A, EMS administration alone, beginning at 4 weeks of age; Group B, a concomitant treatment of TAM for 4 weeks with EMS administration; Group C, TAM treatment for 4 weeks subsequent to EMS administration at 16 weeks of age; Group D, TAM treatment alone for 4 weeks simultaneously with Group A; Group E, administered diluted-ethanol solution as a control and Group F, no treatment control.
The incidences of mammary carcinoma in each group are shown in Fig. 1. In Group A rats, given EMS alone, the mammary tumors were first detected at the 13th week and tumor incidence rapidly increased as the observation period proceeded, i.e. 35.3% at the 14th week, 64.7% at the 16th week and 100°/o at the 20th week. In Group B rats, concomitantly given TAM and EMS at 4 weeks of age, the development of mammary carcinomas was significantly retarded, i.e. no tumor was found up to the 16th week and the tumor inci-
EMS
treatment P
E g
80
i ,z
60
g F
40
z ?
20
-z 5u
0
0
4
8
12
Observation
16 period
20
24
28
32
(weeks)
Fig. 1. Effects of TAM on the tumor incidence of EMSinduced mammary carcinomas. EMS was orally given for 12 weeks and TAM (10 mg/kg) was injected twice a week for 4 weeks. Symbols are: 0, Group A (EMS alone): 0, Group B (TAM concomitant with EMS): A, Group C (TAM subsequent to EMS); 0, Group D (TAM alone); A. Group E (control).
EMS -
TAM alone
ethanol solution no treatment
C
D
E F
“Mean & S.D. bArrow
EMS + TAM
B
indicates
TAMb
EMS alone
A
0.13 *0.35 0 0 0
0
0.24 ZtO.44
14
16
by EMS.
0 0 0
at the following
induced
18
0 0 0
periods
by’. *P < 0.01. **P < 0.05.
Carcinomas/rat
carcinoma
‘followed
of mammary
Treatment
Multiplicity
Groups
Table I. (weeks)a
**
0 0 0
0 0 0
;I;]* y;;]*
22
2.69 f 1.30 0 0 0
0.43 izO.65
26
l ;z;]* ***;tj]*-**
20
I
* 4.00 *0.86 0 0 0
0.85 f 1.40
30
* 1
22 Table II.
Hormone
Groups
A B
receptor
status
Treaatment
EMS alone EMS + TAM
aTumors were extirpated *P < 0.05; significantly
of EMS-induced
rat mammary
carcinoma.
No. of tumors examined
No. of receptor-positive ER
PgR
AR
10 10”
10 (100) 5 (50)*
7 (70) 6 (60)
0 (0) 0 (0)
tumors
(‘!h)
22-26 weeks after TAM treatment. different from Group A.
dence during the observation period was significantly lower than that in Group A (P < 0.01). TAM treatment subsequent to EMS administration at the age of 16 weeks also retarded tumor induction in Group C rats as compared with Group A (P < 0.05), though the inhibitory effects were smaller than that in Group B. No mammary tumor was induced in either the Group D rats given TAM alone or the control Groups E and F. The multiplicity of the mammary carcinomas, expressed as the mean number/rat, was also significantly reduced by either the concomitant or consecutive administration of TAM in Group B or Group C, respectively (Table I). The inhibitory effect of TAM was more evident in Group B than in Group C, i.e. the mean number of tumors in Group B was significantly lower than that in Group C (P < 0.01). The hormone receptor status of the mammary carcinomas extirpated at week- 30-40 in Groups A and B are summarized in Table Il. There was a significant reduction in the incidence of ER-positive tumors in Group B, i.e. 5 of 10 tumors (50%) in Group B were ER-positive, whereas all 10 tumors in Group A possessed ER. On the other hand, the incidences of PgR positive-tumors were not significantly different between these two groups. There was no AR-positive tumor either in Group A or B. Discussion A system of rat mammary carcinogenesis using EMS seemed to have the following characteristics or advantages: (i) the procedure for tumor induc-
tion is simple, that is. EMS can be dissolved and given in drinking water; (ii) the incidence of tumor induction by 3 x 10m3 M of EMS solution were consistent in several independent experiments and all surviving rats have the mammary carcinomas within 40 weeks 17-91; (iii) ER-positive tumors develop at a high rate [9]; and (iv) tumor induction can obviously be prevented by endocrine ablative surgery such as oophorectomy [9]. Moreover, the present study demonstrated additional characteristics of EMS tumor induction: (v) a higher concentration of EMS solution (10-I M) was able to induce tumors in all rats within a shorter period (20 weeks); (vi) the EMS-induced tumors frequently possessed PgR but not AR; and (vii) tumor induction was significantly inhibited by TAM. In designing the present experiment, the rats were given EMS solution for 12 weeks according to our routine protocol and we attempted to evaluate the effects of TAM on mammary carcinogenesis at either an early stage (during the initial 4 weeks of EMS administration) or a later stage (immediately after EMS administration and during week 13-16) of the tumor induction. We previously found that the mammary carcinomas were first detected at around the 16th week and the tumor incidences reached 100% at week 32-40 when the rats were given 3 x 10m3 M of EMS solution [7-91. Since tumor incidence was dependent on the concentration of EMS solution [8], a relatively higher concentration of EMS solution (10-I M) was used in the present experiments with an attempt to shorten the latent period of mammary carcinogenesis. As expected, in the Group A rats given EMS alone, the tumors began to appear at the 13th week and tumor incidence reached
23
100% at the 20th week. Consequently, in the present study, the effects of TAM on both the initiation at an early stage (Group B) and tumor appearance at a later stage (Group C) of the tumor induction were examined. The present results provided an evidence for the chemopreventive effects of TAM on both early and later stages of mammary carcinogenesis induced by EMS. In Group B rats, concomitantly given EMS and TAM, tumor appearance was significantly retarded and the final tumor incidence at the 30th week was SO.O%, while in Group A rats given EMS alone, tumor incidence reached 100% at the 20th week. TAM treatment subsequent to EMS administration in Group C rats also delayed tumor appearance significantly. Similar findings were obtained in the analysis of the multiplicity of the tumors. These inhibitory effects of TAM were compatible with those in previous reports using other carcinogen-induced rat tumor systems, such as those induced by 7,12dimethylbenzanthracene (DMBA) [15,17,18] or Nmethyl-N-nitrosourea (MNU) [19,20]. Most of these rat mammary carcinomas possess ER and both the tumor induction and the growth were significantly inhibited by TAM. Jordan reported that TAM blocked estrogen binding to ER in the mammary tumors and consequently inhibited both the initiation and growth of DMBA tumors [ 151. It has also been suggested that TAM may have a direct action at the ovarian level and reduce the circulating estrogen levels [21]. In the present study, the inhibitory effects of TAM on EMSinduced mammary carcinogenesis were similar to those of ablative oophorectomy in our previous study in which the tumor development was completely prevented by oophorectomy prior to EMS administration, and partially, by oophorectomy after EMS administration [9]. As EMS-induced mammary tumors are thus dependent on ovarian hormones and all the tumors possess ER, the possible mechanism for the preventive effect of TAM in the present study seems to involve the impairment of the effects of ovarian hormones on tumor development, by either a blockade of estrogen binding to ER at the tumor level [15] or a reduction of the circulating estrogen level through some action at the ovarian level [21].
The difference in the inhibitory effects of TAM on EMS-induced mammary carcinogenesis between Group B (TAM treatment concomitant with EMS administration at 4 weeks of age) and Group C (TAM treatment after EMS administration at 16 weeks of age) might reflect the different responsiveness of the mammary glands to TAM treatment. When the Group C rats were treated with TAM after EMS administration, the majority of the mammary glands might have already been fully initiated and sensitized for tumor production by the previous EMS administration in the presence of ovarian hormones. The other possibility was that the hormone dependency of the initiated mammary glands and the latent mammary carcinomas might have changed at a later stage of tumor development and consequently the effects of TAM were less evident in Group C rats. This possibility may be supported by our previous finding that the ER contents of the EMS induced mammary carcinomas declined during tumor progression [9]. In another experimental system, Sukumar et al. [5] and Turcot-Lamay [18] also reported that the hormone responsiveness of MNU induced rat mammary carcinomas had been reduced during the late stages of tumor development. Another purpose of the present study was to examine the hormone receptor status of the EMSinduced mammary tumors occurring in the rats which had been exposed to TAM. The data showed that concomitant TAM treatment with EMS administration reduced the incidence of ERpositive tumors. Since it has been shown that the circulating levels of ovarian hormones influence the concentration of ER in rat mammary tumors [3], a reduction of the circulating ovarian hormones by TAM treatment appears to be responsible for the reduced incidence of ER-positive tumors. Although further investigation is needed for a better understanding of the biological mechanisms in which TAM affects the tumor induction and the hormone receptor status, it is considered that EMS-induced rat mammary carcinoma may serve as a useful model for research on chemoprevention by antiestrogens as well as help elucidate a role of hormone receptor status during mammary carcinogenesis.
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Acknowledgement We thank Dr. Brian T. Quinn ‘on the manuscript.
10
for his comments 11
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