REPORT Human Chorionic Gonadotropin and Rat Mammary Cancer Prevention
The observation that mammary cancer induced by 7,12-dimethylbenz[a]anthracene (DMBA) in young, virgin, SpragueDawley rats is abolished by pregnancy led us to test the possibility of protecting the mammary gland from chemically induced carcinogenesis by using the placental hormone human chorionic gonadotropin (hCG). Fifty-day-old, outbred, virgin, Sprague-Dawley rats were utilized in two different experimental protocols. In protocol 1, four groups of virgin rats received either no hCG (group I) or a daily intraperitoneal injection of hCG at 1IU (group II), 10IU (group ffl), or 100 IU (group IV) for 21 days; group I and groups II-IV, at 21 days after the last injection, were given a single intragastric dose of 8 mg of DMBA per 100 g of body weight. In protocol 2,50-day-old rats were treated with a single intragastric dose of 8 mg of DMBA per 100 g of body weight; 21 days later, they were separated into groups V and VI. Group V rats remained undisturbed, except for palpation twice a week for detection of tumor development. Group VI rats received a daily intraperitoneal injection of 100 IU of hCG for 60 days. Tumorigenesis was evaluated 24 and 30 weeks after carcinogen administration in animals in protocols 1 and 2, respectively. In protocol 1, in which animals (with the exception of the control group) were treated with hCG prior to carcinogen administration, the incidence of adenocarcinomas decreased in a dose-dependent manner, from 43.8% in the controls (group I) to 1286
bear in mind important differences between the experimental animal and the human population (8). In experimental animals, the researcher determines the time of initiation of mammary lesions (8). In the human population, the time of initiation of neoplasms is not known nor is it known whether the human breast has been exposed to a carcinogenic stimulus at any given time in the life span of a woman. Therefore, a preventive hormone agent must efficiently protect the breast both from tumor initiation and from progression of lesions already initiated.
The observation that pregnancy protects the mammary gland from chemically induced carcinogenesis (1-3) led us to determine whether this protection was exerted through the influence of placental hormones and whether exogenous hormonal treatment could mimic the gestational process and induce the same level of refractoriness obtained after full-term pregnancy (5-5). This postulate was based on the observation that administration of the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to young, virgin, Sprague-Dawley rats during the period in which the mammary gland contains numerous undifferentiated terminal ductal structures or terminal end buds results in an incidence of carcinomas that is directly proportional to the number of these structures (6-10). Pregnancy induces full differentiation of the gland, with elimination of terminal end buds, resulting in refractoriness of the gland to carcinogenesis. A placental hormone such as human chorionic gonadotropin (hCG) administered exogenously produces in the rat mammary gland a degree of differentiation similar to that induced by pregnancy (77). This observation indicates that if gland differentiation is pivotal to carcinogenesis, it can be controlled by means other than pregnancy as a reasonable approach for mammary cancer prevention (3,5).
The objective of this work was twofold: (a) to determine whether hCG treatment before carcinogen exposure protects the mammary gland from malignant transformation and (b) to determine what effect hCG has on tumor development when the hormone is administered after exposure of the mammary gland to a carcinogen.
However, in devising strategies for hormonal prevention of mammary carcinogenesis in the human population, one must
Materials and Methods Outbred, virgin, Sprague-Dawley rats were purchased from Harlan SpragueDawley (Indianapolis, Ind). The animals were housed four to a cage and were maintained at a temperature of 24 ± 1 °C with a 12-hour darkness and 12-hour light cycle. The animals received water and food ad libitum. When they reached the age of 50 days, they were divided into groups according to one of two experimental protocols (fig. 1). In protocol 1, 254 animals were divided into four groups, a control group not
Received March 19, 1990; revised May 16, 1990; accepted May 24, 1990. Supported by American Cancer Society grant BC-621 and an institutional grant from the United Foundation of Greater Detroit. Department of Pathology, Michigan Cancer Foundation, Detroit, Mich. Correspondence to: Irma H. Russo, M.D., Department of Pathology, Michigan Cancer Foundation, 110 E. Warren Ave., Detroit, MI 48201.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of Arizona on May 31, 2015
/. H. Russo,* M. Koszalka, J. Russo
34.4%, 18.2%, and 6.15% in groups II-IV treated with 1, 10, or 100 IU of hCG, respectively. Among animals in protocol 2, hCG treatment significantly reduced the incidence of adenocarcinomas, from 100% in DMBA-treated rats (group V) to 45.5% in rats treated with DMBA plus hCG (group VI). These data indicate that hCG can prevent both initiation and progression of mammary carcinogenesis. [J Natl Cancer Inst 82:1286-1289,1990]
0
2 5 8 days
71
92
start
stop
t
t
DMBA
end
50
71
50
protocol 1 — —
birth
A hCG end
0 protocol
i
birth
t
DMBA
2 5 8 days
131 •
hCG end
stop
start
Figure 1. Design of protocol 1: rats were administered hCG beginning at age 50 days (start) daily for 21 days, until they reached age 71 days (stop); DMBA was administered 21 days later. Design of protocol 2: DMBA was administered to 50-day-old animals; hCG treatment was initiated 21 days later (start) and given daily for 60 days (stop). Tumorigenic responses were evaluated 24 and 30 wk after DMBA administration for protocols 1 and 2, respectively.
protocol 1. The tumorigenic response was evaluated 30 weeks after DMBA administration (table 1, fig. 1). All tumors and the mammary glands were dissected from the skin and processed as described elsewhere (9,10). Sections of tumors were stained with hematoxylin and eosin. We classified the tumors by applying criteria published elsewhere (9,70). The proportions of DMBAinduced tumors and DMBA-induced adenocarcinomas were analyzed with the use of the chi-square test and Fisher's exact probability two-tail test (75).
Results
treated with hCG (group I) and three groups treated with an intraperitoneal injection of hCG at 1 IU (group II), 10 IU (group III), or 100 IU (group IV) daily for 21 days (table 1). The hCG was supplied by Sigma Chemical Co. (St. Louis, Mo). After the last injection, the rats were allowed to rest for an additional 21 days, after which all animals received a single intragastric dose of 8 mg of DMBA (Eastman Organic Chemicals, Rochester, NY) per 100 g of body weight. DMBA was dissolved in corn oil and then heated in a water bath at 100 °C for 15 minutes. The animals were palpated twice a week for detection of tumor development. The date of tumor appearance and the tumor location were recorded. The final tumori-
genic response was evaluated 24 weeks after DMBA administration (fig. 1). In protocol 2, 63 virgin rats received at the age of 50 days a single intragastric dose of 8 mg of DMBA per 100 g of body weight. After carcinogen administration, the animals were divided into two groups, designated groups V and VI (table 1). Animals of group V remained undisturbed, except for palpation twice a week for detection of tumor development. Starting 21 days after carcinogen administration, the time at which intraductal proliferations have been reported to be already present (8,72), group VI rats received one daily intraperitoneal injection of 100 IU of hCG for 60 days. They were palpated twice a week as previously described for
By 24 weeks after carcinogen administration, 61.3% of group I animals had developed tumors. Treatment with hCG significantly decreased the incidence of DMBA-induced mammary tumors and the number of tumors per animal; the effect seemed to be dose dependent (table 1). Tumor incidence was 53.1%, 36.7%, and 29.2% in animals of groups II, III, and IV, respectively (fig. 2). Tumors appeared earlier in control than in treated animals, but the differences were not significant (table 1). When we classified the tumors histologically, we found that two major classes of neoplasms had developed, adenocarcinomas and fibroadenomas. In group I animals, 43.8% of the tumors were adenocarcinomas, whereas in animals of groups
Table 1. Effect of hCG on DMBA-induced mammary carcinogenesis Tumors classified as adenocarcinomas
All tumors Protocol Group No. No.
I
I
n
in
2
IV V VI
Treatment*
—/DMBA hCG (1 IU) + DMBA hCG (10 IU) + DMBA hCG (100 IU) + DMBA DMBA/— DMBA + hCG (100 IU)
Total No. of No. of animals animals 80 32 77 65 30 33
49 17 28 19 30 17
%
61.3* 53.1* 36.7* 29.2* lOO.OH 51.5||
Total No. of No. of tumors No. of animals per tumors animalt 101 34 37 27 121 31
1.26 1.06 0.48 0.41 4.00 0.90
35 11 14 4 30 15
%
43.8§ 34.4§ 18.2§ 6.15§ 100.0! 45.5 U
Latent No. of Total period No. of adenocarcinoma!s (days) adenocarcinoma!5 per animalt 73 18 17 4 93 26
0.91 0.56 0.22 0.06 3.10 0.80
55-191 85-190 90-195 90-195 49-154 49-154
•DMBA was administered at 8 nig/100 g of body wt — = no hCG. t Per total No. of animals at risk. *Tumor incidence chi-square with df= 3; /> = 5.35 X 10~5. §Carcinoma incidence chi-square with df= 3; P = 6.12 X10~7. || Tumor incidence in group V vs. group VI is highly significant (Fisher's exact probability two-tail test; P = 3.18 X 10"6). II Adenocarcinoma incidence in group V vs. group VI is highly significant (Fisher's exact probability two-tail test; P = 4.34 X 10~7). Vol. 82, No. 15, August 1, 1990
REPORT 1287
Downloaded from http://jnci.oxfordjournals.org/ at University of Arizona on May 31, 2015
Effect of hCG Treatment on Tumor Initiation (Protocol 1)
10 -
Figure 2. DMBA-induced mammary tumors and adenocarcinomas in animals of protocols 1 and 2. Protocol 1: no treatment with hCG (G-l) or treatment with 1IU of hCG (G-II), 10 IU of hCG (G-III), or 100 IU of hCG (G-IV) prior to DMBA administration. Protocol 2: treatment with DMBA but no hCG (G-V) or treatment with DMBA plus 100 IU of hCG (G-VI), starting 21 days after DMBA administration. Incidence is expressed as percentage (x 10) of animals with tumors and adenocarcinomas (ordinate).
BUI Tumors 1111II Adenocarcinomas
1 I
o X
§ •" UJ
1
2 -
11H
L
G-l
nil
1 1
J4I1^L1|
— Ml*"»T* rn mm
G-II i
G-IV G-V ' '
1 G-VI
rnrni 5 Pfin 1 U v U L
The observation that mammary cancer induced by 7,12-dimethylbenz[a]anthracene (DMBA) in young, virgin, SpragueDawley rats is abolished by pregnancy led us to test the possibility of protecting the mammary gland from chemically induced carcinogenesis by using the placental hormone human chorionic gonadotropin (hCG). Fifty-day-old, outbred, virgin, Sprague-Dawley rats were utilized in two different experimental protocols. In protocol 1, four groups of virgin rats received either no hCG (group I) or a daily intraperitoneal injection of hCG at 1IU (group II), 10IU (group ffl), or 100 IU (group IV) for 21 days; group I and groups II-IV, at 21 days after the last injection, were given a single intragastric dose of 8 mg of DMBA per 100 g of body weight. In protocol 2,50-day-old rats were treated with a single intragastric dose of 8 mg of DMBA per 100 g of body weight; 21 days later, they were separated into groups V and VI. Group V rats remained undisturbed, except for palpation twice a week for detection of tumor development. Group VI rats received a daily intraperitoneal injection of 100 IU of hCG for 60 days. Tumorigenesis was evaluated 24 and 30 weeks after carcinogen administration in animals in protocols 1 and 2, respectively. In protocol 1, in which animals (with the exception of the control group) were treated with hCG prior to carcinogen administration, the incidence of adenocarcinomas decreased in a dose-dependent manner, from 43.8% in the controls (group I) to 1286
bear in mind important differences between the experimental animal and the human population (8). In experimental animals, the researcher determines the time of initiation of mammary lesions (8). In the human population, the time of initiation of neoplasms is not known nor is it known whether the human breast has been exposed to a carcinogenic stimulus at any given time in the life span of a woman. Therefore, a preventive hormone agent must efficiently protect the breast both from tumor initiation and from progression of lesions already initiated.
The observation that pregnancy protects the mammary gland from chemically induced carcinogenesis (1-3) led us to determine whether this protection was exerted through the influence of placental hormones and whether exogenous hormonal treatment could mimic the gestational process and induce the same level of refractoriness obtained after full-term pregnancy (5-5). This postulate was based on the observation that administration of the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to young, virgin, Sprague-Dawley rats during the period in which the mammary gland contains numerous undifferentiated terminal ductal structures or terminal end buds results in an incidence of carcinomas that is directly proportional to the number of these structures (6-10). Pregnancy induces full differentiation of the gland, with elimination of terminal end buds, resulting in refractoriness of the gland to carcinogenesis. A placental hormone such as human chorionic gonadotropin (hCG) administered exogenously produces in the rat mammary gland a degree of differentiation similar to that induced by pregnancy (77). This observation indicates that if gland differentiation is pivotal to carcinogenesis, it can be controlled by means other than pregnancy as a reasonable approach for mammary cancer prevention (3,5).
The objective of this work was twofold: (a) to determine whether hCG treatment before carcinogen exposure protects the mammary gland from malignant transformation and (b) to determine what effect hCG has on tumor development when the hormone is administered after exposure of the mammary gland to a carcinogen.
However, in devising strategies for hormonal prevention of mammary carcinogenesis in the human population, one must
Materials and Methods Outbred, virgin, Sprague-Dawley rats were purchased from Harlan SpragueDawley (Indianapolis, Ind). The animals were housed four to a cage and were maintained at a temperature of 24 ± 1 °C with a 12-hour darkness and 12-hour light cycle. The animals received water and food ad libitum. When they reached the age of 50 days, they were divided into groups according to one of two experimental protocols (fig. 1). In protocol 1, 254 animals were divided into four groups, a control group not
Received March 19, 1990; revised May 16, 1990; accepted May 24, 1990. Supported by American Cancer Society grant BC-621 and an institutional grant from the United Foundation of Greater Detroit. Department of Pathology, Michigan Cancer Foundation, Detroit, Mich. Correspondence to: Irma H. Russo, M.D., Department of Pathology, Michigan Cancer Foundation, 110 E. Warren Ave., Detroit, MI 48201.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of Arizona on May 31, 2015
/. H. Russo,* M. Koszalka, J. Russo
34.4%, 18.2%, and 6.15% in groups II-IV treated with 1, 10, or 100 IU of hCG, respectively. Among animals in protocol 2, hCG treatment significantly reduced the incidence of adenocarcinomas, from 100% in DMBA-treated rats (group V) to 45.5% in rats treated with DMBA plus hCG (group VI). These data indicate that hCG can prevent both initiation and progression of mammary carcinogenesis. [J Natl Cancer Inst 82:1286-1289,1990]
0
2 5 8 days
71
92
start
stop
t
t
DMBA
end
50
71
50
protocol 1 — —
birth
A hCG end
0 protocol
i
birth
t
DMBA
2 5 8 days
131 •
hCG end
stop
start
Figure 1. Design of protocol 1: rats were administered hCG beginning at age 50 days (start) daily for 21 days, until they reached age 71 days (stop); DMBA was administered 21 days later. Design of protocol 2: DMBA was administered to 50-day-old animals; hCG treatment was initiated 21 days later (start) and given daily for 60 days (stop). Tumorigenic responses were evaluated 24 and 30 wk after DMBA administration for protocols 1 and 2, respectively.
protocol 1. The tumorigenic response was evaluated 30 weeks after DMBA administration (table 1, fig. 1). All tumors and the mammary glands were dissected from the skin and processed as described elsewhere (9,10). Sections of tumors were stained with hematoxylin and eosin. We classified the tumors by applying criteria published elsewhere (9,70). The proportions of DMBAinduced tumors and DMBA-induced adenocarcinomas were analyzed with the use of the chi-square test and Fisher's exact probability two-tail test (75).
Results
treated with hCG (group I) and three groups treated with an intraperitoneal injection of hCG at 1 IU (group II), 10 IU (group III), or 100 IU (group IV) daily for 21 days (table 1). The hCG was supplied by Sigma Chemical Co. (St. Louis, Mo). After the last injection, the rats were allowed to rest for an additional 21 days, after which all animals received a single intragastric dose of 8 mg of DMBA (Eastman Organic Chemicals, Rochester, NY) per 100 g of body weight. DMBA was dissolved in corn oil and then heated in a water bath at 100 °C for 15 minutes. The animals were palpated twice a week for detection of tumor development. The date of tumor appearance and the tumor location were recorded. The final tumori-
genic response was evaluated 24 weeks after DMBA administration (fig. 1). In protocol 2, 63 virgin rats received at the age of 50 days a single intragastric dose of 8 mg of DMBA per 100 g of body weight. After carcinogen administration, the animals were divided into two groups, designated groups V and VI (table 1). Animals of group V remained undisturbed, except for palpation twice a week for detection of tumor development. Starting 21 days after carcinogen administration, the time at which intraductal proliferations have been reported to be already present (8,72), group VI rats received one daily intraperitoneal injection of 100 IU of hCG for 60 days. They were palpated twice a week as previously described for
By 24 weeks after carcinogen administration, 61.3% of group I animals had developed tumors. Treatment with hCG significantly decreased the incidence of DMBA-induced mammary tumors and the number of tumors per animal; the effect seemed to be dose dependent (table 1). Tumor incidence was 53.1%, 36.7%, and 29.2% in animals of groups II, III, and IV, respectively (fig. 2). Tumors appeared earlier in control than in treated animals, but the differences were not significant (table 1). When we classified the tumors histologically, we found that two major classes of neoplasms had developed, adenocarcinomas and fibroadenomas. In group I animals, 43.8% of the tumors were adenocarcinomas, whereas in animals of groups
Table 1. Effect of hCG on DMBA-induced mammary carcinogenesis Tumors classified as adenocarcinomas
All tumors Protocol Group No. No.
I
I
n
in
2
IV V VI
Treatment*
—/DMBA hCG (1 IU) + DMBA hCG (10 IU) + DMBA hCG (100 IU) + DMBA DMBA/— DMBA + hCG (100 IU)
Total No. of No. of animals animals 80 32 77 65 30 33
49 17 28 19 30 17
%
61.3* 53.1* 36.7* 29.2* lOO.OH 51.5||
Total No. of No. of tumors No. of animals per tumors animalt 101 34 37 27 121 31
1.26 1.06 0.48 0.41 4.00 0.90
35 11 14 4 30 15
%
43.8§ 34.4§ 18.2§ 6.15§ 100.0! 45.5 U
Latent No. of Total period No. of adenocarcinoma!s (days) adenocarcinoma!5 per animalt 73 18 17 4 93 26
0.91 0.56 0.22 0.06 3.10 0.80
55-191 85-190 90-195 90-195 49-154 49-154
•DMBA was administered at 8 nig/100 g of body wt — = no hCG. t Per total No. of animals at risk. *Tumor incidence chi-square with df= 3; /> = 5.35 X 10~5. §Carcinoma incidence chi-square with df= 3; P = 6.12 X10~7. || Tumor incidence in group V vs. group VI is highly significant (Fisher's exact probability two-tail test; P = 3.18 X 10"6). II Adenocarcinoma incidence in group V vs. group VI is highly significant (Fisher's exact probability two-tail test; P = 4.34 X 10~7). Vol. 82, No. 15, August 1, 1990
REPORT 1287
Downloaded from http://jnci.oxfordjournals.org/ at University of Arizona on May 31, 2015
Effect of hCG Treatment on Tumor Initiation (Protocol 1)
10 -
Figure 2. DMBA-induced mammary tumors and adenocarcinomas in animals of protocols 1 and 2. Protocol 1: no treatment with hCG (G-l) or treatment with 1IU of hCG (G-II), 10 IU of hCG (G-III), or 100 IU of hCG (G-IV) prior to DMBA administration. Protocol 2: treatment with DMBA but no hCG (G-V) or treatment with DMBA plus 100 IU of hCG (G-VI), starting 21 days after DMBA administration. Incidence is expressed as percentage (x 10) of animals with tumors and adenocarcinomas (ordinate).
BUI Tumors 1111II Adenocarcinomas
1 I
o X
§ •" UJ
1
2 -
11H
L
G-l
nil
1 1
J4I1^L1|
— Ml*"»T* rn mm
G-II i
G-IV G-V ' '
1 G-VI
rnrni 5 Pfin 1 U v U L
