- Email: [email protected]
Modulatory influences of exogenous estrogen on MCA-induced carcinogenesis in the uterine cervix of mouse
73
Cancer Letters, 43 (1988) 73-77 Elsevier Scientific Publishers Ireland Ltd.
Modulatory influences of exogenous estrogen on MCA-induced carcinogenesis in the uterine cervix of mouse P. Das, Cancer
A.R.
Biology
(Received (Accepted
Rao and P.N. Srivastava
Laboratory,
School
of Life Sciences,
Jawaharlal
Nehru
University,
New Delhi 110 067
(India)
10 August, 1988) 15 August, 1988)
Summary Placement of cotton-thread impregnated with beeswax containing methylcholanthrene (approx. 600 pgj inside the canal of uterine cervix of virgin adult mice results in the emergence of precancerous and cancerous lesions in the cervical epithelium. Employing this experimental cervical carcinogenesis model system, the present study evaluates the modulatory influence of exogenous 17@-e&radio/ on the incidences of precancerous and cancerous lesions in the cervical epithelium. When 17/3estradiol was administered weekly at the dose levels of 0.01 pg, 0.1 pg, 5 pg and 50 pg for 16 weeks following carcinogen-thread insertion, the cervical carcinoma incidences, as compared to that in positive control mice (76.1 W), were 66.6%, 61.5!%, 55.5% and 42.1!%, respectively. The decline in the incidence of carcinomas at 50 &week dose level was significant (P < 0.05). Hyperplastic and dysplastic changes did not show any definite correlation with the various treatments. Keywords: cervical methylcholanthrene;
carcinogenesis 17fi-estradiol.
(mouse) ;
Introduction Epidemiological Correspondence
studies
to: A. Ramesha
implicate
several
Rao.
0 1988 Elsevier Scientific Publishers 0304-3835/88/$03.50 Published and Printed in Ireland
modulatory factors in the incidence of cervical cancer in women [9]. However, there are no substantial studies, clinical or experimental, on the influences of natural hormones on cervical carcinogenesis. A preliminary study done in our laboratory suggested that exogenous 17fiestradiol reduces the incidence of MCAinduced cervical cancer in mice [5]. The present study evaluates the nature of the modulatory influence of graded doses of exogenous 17fi-estradiof on MCA induced cervical carcinogenesis in adult Swiss albino mice. Materials and methods Randombred, 12- 13-week-old virgin female Swiss albino mice were procured from Disease Free Small Animal House, Haryana Agricultural University, Hissar, India, and were maintained in our air-conditioned animal facility providing standard food pellets (Hindustan Lever Ltd., India) and water ad libitum. Chemicals 3-Methylcholanthrene (MCA) and 17/3estradiol were obtained from Sigma Chemical Co., U.S.A. A yellow variety of pure beeswax was procured from Mysore, India and filtered in molten state (6OOC) four times in order to remove dust particles, if any. The 17/3-estradiol was dissolved in olive oil to give solutions of concentration 0.01 pg/O. 1 ml, 0.1 pg/O. 1 ml, 5 pg/O. 1 ml and 50 pg/O. 1 ml. Ireland Ltd
19
VIII
6 7 5 7 8 8
(0.1) Wax + 17/j’-estradiol
(5) Wax + 17/3-estradiol
(50) 17/3-estradiol (0.01) 17/3-estradiol(O.l) 17/~‘-estradiol (5) 17a-estradiol(50)
XI
XII
XIII XIV xv XVI
l
l
a + , mild; + + , moderate, *Significant at P < 0.05. Not significant.
X
0 0 3 1
0
0
0
1
0
0
0
10 7 4 0 0
3 5 5 7
6
3
5
3
4
3
3
0 3 0 0 1
Hyperptasia
+ + + , marked; CIS, carcinoma
7
(50) Wax + 17/3-estradiol (0.01) Wax + 17/j-estradiol
IX 9
18
(5) MCA + 17/3-estradiol
VII
VI 26
10 10 4 21 24
Normal epithelium
0 0 0 0
1
1
0
1
0
1
4
0 0 0 2 6
0 0 0 0
0
0
0
0
0
1
0
0 0 0 0 2
0 0 0 0
0
0
0
0
2
5
6
in situ; MI, microinvasive;
2 2 0 0
0
2
2
4
4
1
2
0 0 0 0 0
0 0 0 0
0
0
0
0
0
5
7
0 0 0 10 9
INV
0 0 0 0
0
0
0 0 0 0
0
0
0
42.1
55.5
0
0 0
0
0
0
0
0
2
2
1
0 0 0 1 1
0 0 0 0
0
0
0
0
1
0
0
0 0 0 2 0
AC SC
of mice”.
SC, sarcoma.
0 0 0 0
0
0
0
0
44.7
27.0
19.1
61.5
0
l
l
l
0
l
0 0 0 0 12.4
% of carcinoma inhibition
0 0 0 76.1 66.6
0
8/19*
10/18*
16/26*
0 o/10 o/4 16/21 16/24
% of mice with carcinoma
INV, invasive; AC, adenocarcinoma;
MI
CIS
+ +
+
cell
Squamous cell carcinoma
changes induced by MCA in the cervical epithelium
Squamous + + +
and neoplastic
Dysplasia
of preneoplastic
No. of mice with
on the incidence
Total no. of effective mice
(0.1) MCA + 17/3-estradiol
(pg/week)
Treatments
Influence of 17fl-estradiol
Normal Wax Olive oil MCA MCA + 17/3-estradiol (0.01) MCA + 17/3-estradiol
1 II III IV V
Groups
Table 1.
75
Tumor induction technique Murphy’s string method [6,7] as described by Manoharan and Rao [4] was used for the induction of cervical cancer. Briefly, sterile double cotton thread impregnated with beeswax containing approx. 600 pg of MCA (3 : 1, w/w) was inserted into the canal of the uterine cervix of mice by means of laparotomy under mild ether anaesthesia. The thread remained in the cervical canal till the end of the observation period. Experimental design The animals were assorted into 16 control and experimental groups as follows (also see Table 1). Animals of group 1 (10) did not receive any treatment and served as normal controls. Animals of group 2 (10) were impregnated inserted with beeswax-only threads. Animals of group 3 (4) received olive oil (0.1 ml/week). Animals of group 4 (21), group 5 (24)) group 6 (26)) group 7 (18) and group 8 (19) had the intracervical insertion of carcinogen-plus-beeswax impregnated threads whereas animals of group 9 (9)) group 10 (7)) group 11 (6) and group 12 (7) had the insertion of beeswax-only impregnated threads. Then the animals of group 13 (5), group 14 (7), group 15 (8) and group 16 (8) did not have thread insertion. The animals of groups 5-8 received weekly injections of 0.01 pg, 0.1 pg, 5 pg and
Table 2. parameters
50 c(g of 17/3-estradiol, respectively. Likewise, animals of groups 9-12 also received weekly injections of 0.01 pg, 0.1 pg, 5 pg and 50 pg of 17/%estradiol, respectively, during the entire period of observation. The animals of groups 13-16 also received 0.01 pg, 0.1 pg, 5 pg and 50 pg of 17/3-estradiol, respectively, during the study periods. Moribund mice were killled routinely and cervices were fixed for studying the histopathological changes, developed during the observation period. Animals were killed 16 weeks after the initiation of the experiment. The uterine cervices were fixed in Bouin’s fluid and paraffin sections cut serially (7 pm) were stained with haematoxylin and eosin. The histopathological lesions and their criteria used for assessing the influence of hormones on the process of carcinogenesis are given in Table 2. Statistical analysis of the data Tumor incidences in control and experimental groups were compared by a Chi-square method with Yates’ correction. Results The findings of the present study are depicted in Table 1. Normal mice (group 1) as well as mice inserted intracervically with beeswax-only impregnated threads (group 2)
MCA-induced precancerous and cancerous lesions in the epithelium for the assessment of modulatory influences of 17/3-estradiol.
of uterine
cervix of mice, used as
Lesions in the uterine cervix
Histopathological
Hyperplasia
Increase in the thickness of basal layer due to increased and orderly proliferative activity; keratinization and flattening of epithelial cells persist at the surface Crowds of cells showing atypia and loss of polarity in the hyperplastic epithelium. Dysplasia confined to lower one third of the epithelium Dysplasia confined to lower two thirds of the epithelium Dysplasia stretching nearly the entire thickness of the epithelium Carcinoma in the squamous epithelium of the uterine cervix Intraepithelial localization of carcinoma Focal, microscopic peg-like invasions of carcinoma Invasion by sheets or islands of carcinoma cells into the stroma
Dysplasia (a) Mild dysplasia (b) Moderate dysplasia (c) Marked dysplasia Squamous cell carcinoma (a) Carcinoma in situ (b) Microinvasion (c) Invasive carcinoma
criteria
76
did not develop any tumor during the observation period; so also the animals of group 3. Local exposure of the uterine cervix to MCA (group 4) resulted in the incidence of tumors in 76 % of mice. Administration of graded doses of 17/3-estradiol to the mice having MCAimpregnated threads in their uterine cervices inhibited the incidence of tumors (group 5, 66.6%; group 6, 61.5%; group 7, 55.5%; group 8, 42.1%). However, the inhibitory action was significant only in the group treated with 50 pg/week of estrogen (group 8; P < 0.05). When normal animals were treated with graded doses of 17/3-estradiol alone (groups 13-15) no tumors developed in the uterine cervices during the study period. The incidence of hyperplasia induced by wax-only impregnated thread alone (group 2) were less compared to those induced by the treatments with different doses of estradiol alone (groups 13-16). However, there were no signs of combined action of the wax and hormone on the cervical epithelium (groups 9-12). The incidences of dysplastic changes did not show any definite correlation with various treatments. Discussion
The particular strain of mouse employed in this study is not known to show spontaneous tumor incidence in the uterine cervix. Placement of cotton-thread impregnated with beeswax containing MCA inside the cervical canal in the present study resulted in long-term exposure of cervical epithelum to the carcinogen and subsequently the development of precancerous and cancerous lesions therein. Administration of graded doses of 17fi-estradiol (once a week for 16 weeks) resulted in the inhibition of MCA-induced cervical carcinogenesis but only the highest dose of estrogen used in this study (i.e. 50 pg/mouse per week) elicited significant (P < 0.05) reduction in MCA-induced tumor incidence. A preliminary study done earlier in this laboratory on 8-9week-old Swiss albino mice obtained from a different source showed that administration of
as low a dose of 17/3-estradiol as 0.5 pg twice a week for 12 weeks caused significant (P < 0.05) reduction in MCA-induced cervical cancer incidence [5]. This could be due to difference in the source of mice, the younger age at the start of the experiment and/or difference in the schedule (twice a week instead of once a week) of hormonal treatment. Forsberg [2] reported that the persistent production of ovarian estrogen in animals, which were neonatally exposed to estradiol, retarded cervical cancer growth. Pharmacological doses of estrogen have been known to inhibit DMBAinduced mammary carcinogenesis in rats [3]. Bates [l] suggested that estrogen might interfere with the tumorigenic action of DMBA on the skin in female mice since animals in the estrous phase were less susceptible than in diestrous phase. Also 17/3-estradiol is known to inhibit the induction of aryl hydrocarbon hydroxylase system in mouse tissues in vitro as well as in vivo [8] and this may result in decreased availability of ultimate carcinogenic metabolites to react with target sites and hence reduced tumor incidence. Acknowledgements
A research fellowship offered by the Council of Scientific and Industrial Research (India) to PD is gratefully acknowledged. References Bates, R.R. (1968) Sex hormones and skin tumorigenesis. I. Effect of the estrus cycle and castration on tumorigenesis by 7,12-dimethylbenz(a)anthracene. J. Natl. Cancer Inst., 41, 559-563. Forsberg, J.G. (1972) Carcinogenesis with MCA in uterine cervix of mice treated neonatally with estrogen. J. Nat]. Cancer Inst., 40, 155-172. Heuson, J.C., Legros, N., HeusonSteirnon, J.A., Leelerq, G. and Pasteels, J.L. (1976) Hormone dependency of rat mammary tumors. In: Breast Cancer: Trends in Research and Treatment, pp. 81-93. Editors. J.C. Heuson, W.H. Mattheiem and M. Rozencweig. Raven Press, New York. Manoharan, K. and Rao. A.R. (1984) Inhibitory actions of retinoic acid and butylated hydroxyanisole on cervical carcinogenesis induced by 3-methylcholanthrene in mouse. Ind. J. Exp. Biol., 22, 195-198.
77
5
6 7
Manoharan, K. and Rao, A.R. (1985) Influence of exogenous hormones on experimental cervical carcinogenesis in mice. Ind. J. Exp. Biol., 23,566-568. Murphy, E.D. (1953) Studies in carcinogen induced carcinoma of the cervix in mice. Am. J. Pathol., 29,608. Murphy, E.D. (1961) Carcinogenesis of the uterine cervix in mice. Effect of diethylstilbestrol after limited application of 3methylcholanthrene. J. Natl. Cancer Inst., 27,611-653.
8
9
Nebert, D.W., Bausserman, L.L. and Bates, R.R. (1970) Effect of 17.B-e&radio] and testosterone on aryl hydrocarbon hydroxylase activity in mouse tissues in vitro and in cell culture. Int. J. Cancer, 6,470-480. Rotkin, I.D. (1981) Etiology and epidemiology of cervical cancer. In: Current Topics in Pathology of Cervical Cancer, pp. 81-110. Editor: G. Dallenbach-Hellweg. SpringerVerlag, Berlin.
Cancer Letters, 43 (1988) 73-77 Elsevier Scientific Publishers Ireland Ltd.
Modulatory influences of exogenous estrogen on MCA-induced carcinogenesis in the uterine cervix of mouse P. Das, Cancer
A.R.
Biology
(Received (Accepted
Rao and P.N. Srivastava
Laboratory,
School
of Life Sciences,
Jawaharlal
Nehru
University,
New Delhi 110 067
(India)
10 August, 1988) 15 August, 1988)
Summary Placement of cotton-thread impregnated with beeswax containing methylcholanthrene (approx. 600 pgj inside the canal of uterine cervix of virgin adult mice results in the emergence of precancerous and cancerous lesions in the cervical epithelium. Employing this experimental cervical carcinogenesis model system, the present study evaluates the modulatory influence of exogenous 17@-e&radio/ on the incidences of precancerous and cancerous lesions in the cervical epithelium. When 17/3estradiol was administered weekly at the dose levels of 0.01 pg, 0.1 pg, 5 pg and 50 pg for 16 weeks following carcinogen-thread insertion, the cervical carcinoma incidences, as compared to that in positive control mice (76.1 W), were 66.6%, 61.5!%, 55.5% and 42.1!%, respectively. The decline in the incidence of carcinomas at 50 &week dose level was significant (P < 0.05). Hyperplastic and dysplastic changes did not show any definite correlation with the various treatments. Keywords: cervical methylcholanthrene;
carcinogenesis 17fi-estradiol.
(mouse) ;
Introduction Epidemiological Correspondence
studies
to: A. Ramesha
implicate
several
Rao.
0 1988 Elsevier Scientific Publishers 0304-3835/88/$03.50 Published and Printed in Ireland
modulatory factors in the incidence of cervical cancer in women [9]. However, there are no substantial studies, clinical or experimental, on the influences of natural hormones on cervical carcinogenesis. A preliminary study done in our laboratory suggested that exogenous 17fiestradiol reduces the incidence of MCAinduced cervical cancer in mice [5]. The present study evaluates the nature of the modulatory influence of graded doses of exogenous 17fi-estradiof on MCA induced cervical carcinogenesis in adult Swiss albino mice. Materials and methods Randombred, 12- 13-week-old virgin female Swiss albino mice were procured from Disease Free Small Animal House, Haryana Agricultural University, Hissar, India, and were maintained in our air-conditioned animal facility providing standard food pellets (Hindustan Lever Ltd., India) and water ad libitum. Chemicals 3-Methylcholanthrene (MCA) and 17/3estradiol were obtained from Sigma Chemical Co., U.S.A. A yellow variety of pure beeswax was procured from Mysore, India and filtered in molten state (6OOC) four times in order to remove dust particles, if any. The 17/3-estradiol was dissolved in olive oil to give solutions of concentration 0.01 pg/O. 1 ml, 0.1 pg/O. 1 ml, 5 pg/O. 1 ml and 50 pg/O. 1 ml. Ireland Ltd
19
VIII
6 7 5 7 8 8
(0.1) Wax + 17/j’-estradiol
(5) Wax + 17/3-estradiol
(50) 17/3-estradiol (0.01) 17/3-estradiol(O.l) 17/~‘-estradiol (5) 17a-estradiol(50)
XI
XII
XIII XIV xv XVI
l
l
a + , mild; + + , moderate, *Significant at P < 0.05. Not significant.
X
0 0 3 1
0
0
0
1
0
0
0
10 7 4 0 0
3 5 5 7
6
3
5
3
4
3
3
0 3 0 0 1
Hyperptasia
+ + + , marked; CIS, carcinoma
7
(50) Wax + 17/3-estradiol (0.01) Wax + 17/j-estradiol
IX 9
18
(5) MCA + 17/3-estradiol
VII
VI 26
10 10 4 21 24
Normal epithelium
0 0 0 0
1
1
0
1
0
1
4
0 0 0 2 6
0 0 0 0
0
0
0
0
0
1
0
0 0 0 0 2
0 0 0 0
0
0
0
0
2
5
6
in situ; MI, microinvasive;
2 2 0 0
0
2
2
4
4
1
2
0 0 0 0 0
0 0 0 0
0
0
0
0
0
5
7
0 0 0 10 9
INV
0 0 0 0
0
0
0 0 0 0
0
0
0
42.1
55.5
0
0 0
0
0
0
0
0
2
2
1
0 0 0 1 1
0 0 0 0
0
0
0
0
1
0
0
0 0 0 2 0
AC SC
of mice”.
SC, sarcoma.
0 0 0 0
0
0
0
0
44.7
27.0
19.1
61.5
0
l
l
l
0
l
0 0 0 0 12.4
% of carcinoma inhibition
0 0 0 76.1 66.6
0
8/19*
10/18*
16/26*
0 o/10 o/4 16/21 16/24
% of mice with carcinoma
INV, invasive; AC, adenocarcinoma;
MI
CIS
+ +
+
cell
Squamous cell carcinoma
changes induced by MCA in the cervical epithelium
Squamous + + +
and neoplastic
Dysplasia
of preneoplastic
No. of mice with
on the incidence
Total no. of effective mice
(0.1) MCA + 17/3-estradiol
(pg/week)
Treatments
Influence of 17fl-estradiol
Normal Wax Olive oil MCA MCA + 17/3-estradiol (0.01) MCA + 17/3-estradiol
1 II III IV V
Groups
Table 1.
75
Tumor induction technique Murphy’s string method [6,7] as described by Manoharan and Rao [4] was used for the induction of cervical cancer. Briefly, sterile double cotton thread impregnated with beeswax containing approx. 600 pg of MCA (3 : 1, w/w) was inserted into the canal of the uterine cervix of mice by means of laparotomy under mild ether anaesthesia. The thread remained in the cervical canal till the end of the observation period. Experimental design The animals were assorted into 16 control and experimental groups as follows (also see Table 1). Animals of group 1 (10) did not receive any treatment and served as normal controls. Animals of group 2 (10) were impregnated inserted with beeswax-only threads. Animals of group 3 (4) received olive oil (0.1 ml/week). Animals of group 4 (21), group 5 (24)) group 6 (26)) group 7 (18) and group 8 (19) had the intracervical insertion of carcinogen-plus-beeswax impregnated threads whereas animals of group 9 (9)) group 10 (7)) group 11 (6) and group 12 (7) had the insertion of beeswax-only impregnated threads. Then the animals of group 13 (5), group 14 (7), group 15 (8) and group 16 (8) did not have thread insertion. The animals of groups 5-8 received weekly injections of 0.01 pg, 0.1 pg, 5 pg and
Table 2. parameters
50 c(g of 17/3-estradiol, respectively. Likewise, animals of groups 9-12 also received weekly injections of 0.01 pg, 0.1 pg, 5 pg and 50 pg of 17/%estradiol, respectively, during the entire period of observation. The animals of groups 13-16 also received 0.01 pg, 0.1 pg, 5 pg and 50 pg of 17/3-estradiol, respectively, during the study periods. Moribund mice were killled routinely and cervices were fixed for studying the histopathological changes, developed during the observation period. Animals were killed 16 weeks after the initiation of the experiment. The uterine cervices were fixed in Bouin’s fluid and paraffin sections cut serially (7 pm) were stained with haematoxylin and eosin. The histopathological lesions and their criteria used for assessing the influence of hormones on the process of carcinogenesis are given in Table 2. Statistical analysis of the data Tumor incidences in control and experimental groups were compared by a Chi-square method with Yates’ correction. Results The findings of the present study are depicted in Table 1. Normal mice (group 1) as well as mice inserted intracervically with beeswax-only impregnated threads (group 2)
MCA-induced precancerous and cancerous lesions in the epithelium for the assessment of modulatory influences of 17/3-estradiol.
of uterine
cervix of mice, used as
Lesions in the uterine cervix
Histopathological
Hyperplasia
Increase in the thickness of basal layer due to increased and orderly proliferative activity; keratinization and flattening of epithelial cells persist at the surface Crowds of cells showing atypia and loss of polarity in the hyperplastic epithelium. Dysplasia confined to lower one third of the epithelium Dysplasia confined to lower two thirds of the epithelium Dysplasia stretching nearly the entire thickness of the epithelium Carcinoma in the squamous epithelium of the uterine cervix Intraepithelial localization of carcinoma Focal, microscopic peg-like invasions of carcinoma Invasion by sheets or islands of carcinoma cells into the stroma
Dysplasia (a) Mild dysplasia (b) Moderate dysplasia (c) Marked dysplasia Squamous cell carcinoma (a) Carcinoma in situ (b) Microinvasion (c) Invasive carcinoma
criteria
76
did not develop any tumor during the observation period; so also the animals of group 3. Local exposure of the uterine cervix to MCA (group 4) resulted in the incidence of tumors in 76 % of mice. Administration of graded doses of 17/3-estradiol to the mice having MCAimpregnated threads in their uterine cervices inhibited the incidence of tumors (group 5, 66.6%; group 6, 61.5%; group 7, 55.5%; group 8, 42.1%). However, the inhibitory action was significant only in the group treated with 50 pg/week of estrogen (group 8; P < 0.05). When normal animals were treated with graded doses of 17/3-estradiol alone (groups 13-15) no tumors developed in the uterine cervices during the study period. The incidence of hyperplasia induced by wax-only impregnated thread alone (group 2) were less compared to those induced by the treatments with different doses of estradiol alone (groups 13-16). However, there were no signs of combined action of the wax and hormone on the cervical epithelium (groups 9-12). The incidences of dysplastic changes did not show any definite correlation with various treatments. Discussion
The particular strain of mouse employed in this study is not known to show spontaneous tumor incidence in the uterine cervix. Placement of cotton-thread impregnated with beeswax containing MCA inside the cervical canal in the present study resulted in long-term exposure of cervical epithelum to the carcinogen and subsequently the development of precancerous and cancerous lesions therein. Administration of graded doses of 17fi-estradiol (once a week for 16 weeks) resulted in the inhibition of MCA-induced cervical carcinogenesis but only the highest dose of estrogen used in this study (i.e. 50 pg/mouse per week) elicited significant (P < 0.05) reduction in MCA-induced tumor incidence. A preliminary study done earlier in this laboratory on 8-9week-old Swiss albino mice obtained from a different source showed that administration of
as low a dose of 17/3-estradiol as 0.5 pg twice a week for 12 weeks caused significant (P < 0.05) reduction in MCA-induced cervical cancer incidence [5]. This could be due to difference in the source of mice, the younger age at the start of the experiment and/or difference in the schedule (twice a week instead of once a week) of hormonal treatment. Forsberg [2] reported that the persistent production of ovarian estrogen in animals, which were neonatally exposed to estradiol, retarded cervical cancer growth. Pharmacological doses of estrogen have been known to inhibit DMBAinduced mammary carcinogenesis in rats [3]. Bates [l] suggested that estrogen might interfere with the tumorigenic action of DMBA on the skin in female mice since animals in the estrous phase were less susceptible than in diestrous phase. Also 17/3-estradiol is known to inhibit the induction of aryl hydrocarbon hydroxylase system in mouse tissues in vitro as well as in vivo [8] and this may result in decreased availability of ultimate carcinogenic metabolites to react with target sites and hence reduced tumor incidence. Acknowledgements
A research fellowship offered by the Council of Scientific and Industrial Research (India) to PD is gratefully acknowledged. References Bates, R.R. (1968) Sex hormones and skin tumorigenesis. I. Effect of the estrus cycle and castration on tumorigenesis by 7,12-dimethylbenz(a)anthracene. J. Natl. Cancer Inst., 41, 559-563. Forsberg, J.G. (1972) Carcinogenesis with MCA in uterine cervix of mice treated neonatally with estrogen. J. Nat]. Cancer Inst., 40, 155-172. Heuson, J.C., Legros, N., HeusonSteirnon, J.A., Leelerq, G. and Pasteels, J.L. (1976) Hormone dependency of rat mammary tumors. In: Breast Cancer: Trends in Research and Treatment, pp. 81-93. Editors. J.C. Heuson, W.H. Mattheiem and M. Rozencweig. Raven Press, New York. Manoharan, K. and Rao. A.R. (1984) Inhibitory actions of retinoic acid and butylated hydroxyanisole on cervical carcinogenesis induced by 3-methylcholanthrene in mouse. Ind. J. Exp. Biol., 22, 195-198.
77
5
6 7
Manoharan, K. and Rao, A.R. (1985) Influence of exogenous hormones on experimental cervical carcinogenesis in mice. Ind. J. Exp. Biol., 23,566-568. Murphy, E.D. (1953) Studies in carcinogen induced carcinoma of the cervix in mice. Am. J. Pathol., 29,608. Murphy, E.D. (1961) Carcinogenesis of the uterine cervix in mice. Effect of diethylstilbestrol after limited application of 3methylcholanthrene. J. Natl. Cancer Inst., 27,611-653.
8
9
Nebert, D.W., Bausserman, L.L. and Bates, R.R. (1970) Effect of 17.B-e&radio] and testosterone on aryl hydrocarbon hydroxylase activity in mouse tissues in vitro and in cell culture. Int. J. Cancer, 6,470-480. Rotkin, I.D. (1981) Etiology and epidemiology of cervical cancer. In: Current Topics in Pathology of Cervical Cancer, pp. 81-110. Editor: G. Dallenbach-Hellweg. SpringerVerlag, Berlin.