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Transplacental effect of diethylstilbestrol in female rats
Cancer Letters, 6 (1979) 107--114
107
© Elsevier/North-Holland ScientificPublishers Ltd.
TRANSPLACENTAL EFFECT' O F DIETHYLSTILBESTROL IN FEMALE RATS
N.P. N A P A L K O V
and V.N. A N I S I M O V
Petrov Research Institute o f Oncology, 68 Leningradskaya St., Pesoehny.2, Leningrad 188~46 (U.S.S.R.)
(Received 27 April 1978} (Revised version received 27 September 1978) (Accepted 28 Septersber 1978}
SUMMARY
The effect of diethylstilbestrol propionate (DES; 1 mg/kg body wt. was studied in the female offspring of rats exposed subcutaneously on the 19th day o f gestation. Examination of vaginal smears showed persistent estrus in adult rats treated prenatally with DES. Compensatory ovarian hypertrophy (COH) induced by hemicastration of these rats was not suppressed by estrogens. A per oral test for glucose-tolerance revealed decreased utilization of glucose in the offspring o f DES-treated r a t s Preliminary observations demonstrated that tumors developed in 14 of 18 (77.8%) progeny transpiacentally exposed to DES and in 9 o f 34 (26.5%) intact control rats. Tumors of the ovary and the endometrium were found only in the rats treated prenatally with DES; no tumors of the uterine cervix or vagina were observed in either experimental or control groups. It is suggested that a transplacental effect of DES in the female is impairment of the sex differentiation o f the hypothalamus. The resulting hormonal and metabolic shift:~ might promote tumorigenesis.
INTRODUCTION During the last decade, reports on she development of cervical and vaginal adenocarcinomas in human females exposed in utero to DES have been pub?,ished [17,18]. Hyperplastic ~esions in the uterine cervix and vagina were induced in mice treated with estrogens during: the first week of ilfe [12,15,19,23]. A single administration of DES to mice on the :~.5--19th day of pregnancy led ~o hypertrophy of the vaginal portio of the cervix and to persistent estrus in the A bbreviations: COH, compensatory ovarian hypertrophy; DES, diethyIstilbestrol propionate.
208
progeny; however, development of cervical and vaginal adenocarcinomas was n o t observed [25]. Vorherr et al, [30] obsevped genital abnormalities, squamous cell vaginal carcinomas, endometriaI and ovarian adenocarcinomas in rats transplacentally exposed to DES: g u s t i a and Shubik [27] showed the development of hyperkeratcsis and parakeratosis and o f squamous cell papillomas and mesenehymal sarcomas of t h e cervix uteri and the vagina in Syrian golden hamster progeny exposed to DES during gestation. The purpose of this study is to show t h e transplacental effect of DES in fem;~Llerats. MATERIALS .~..NDMETHODS Random bred albino rats were provided b y the Rappolovo breeding farm AMS USSR. On the 19th day after conception 11 pregnant rats, 3 - 4 months old, were given a single subcutaneo~s injection of oil solution of DES, (offic. preparation of Rostov Chemical Factory, USSR; 1 mg/kg body wt.). The female offspring at 3 weeks of age were taken from the mothers, randomized, kep~ in stee]: cages in a 14 h light/I0 h dark environment and fed standard laboratory diet and water ad libitum. The offspring were kept for their lifetime under the same conditions as other intact rats o f the colony. The control group was the female offspring of 17 untreated rats. Vaginal smears were examined daffy after the opening of the vagina. To study the functional status of hypothalamic-pituitary-ovarian system a group o f rats aged 3 -.4 months was hemicastrated. Starting on the day of operation they received daily subcutaneous injections of 0.1 m! vegetable oil or 0.57 #g DES dissolved in the same volume of oil for 7 days [2]. On the eighth day after hemicastration the animals were killed and the degree of COH was determined [2,10]. Tolerance. to glucose was studied in 29 experimental and 58 control rats by per oral glucose loading. Blood sugar Ievel was determined by the O-toluidine method [241 in :tats after 18 h fasting and 30, 60 and 120 rain after glucose loadling (3.0 g/kg). The animals not used in the above experiments were allowed to die spontaneously or were kiIled when moribund. Autopsies were performed on all animals. Tumors were fixed in 10% neutral formalin, and 7 ~m paraffin. celloidin sections were stained with hematoxylin and eosin. RESULTS
The exan~Lination of vaginal smears taken daily after the vagina opened showed the reguiar 4 - 5 day estrus cycles in control rats and persistent estrus in the rats prenatally exposed to DES. The age-associated increased incidence of irregular cycles and persistent estrus was found m the control group. DES induced suppression of COH b y 68.5% i~l control hemiovariectomized offspring and did not produce any effect in trm;splacentally estrogen-treated
109 6O
v
5O
40
z
30
20
8
Fig~ 1. The effect of estrogen on the COH in control and prenatally DES-exposed rats: (1) control offspring; (2) offspring prenatally exposed to DES. Unshaded columns, oil injections; Shaded columns, DES injections; Number in columns show the number of animals in groups. Values given are M ± S.E.M.
2.20
1.80
X
1.40 L~ t,O L~
i . O0
/ ---~ 0
~=~ L~
,J
"\
..
\
~ ~ ~0
, ~0
"---. -: ~-~0~o
.60
Fig. 2~ Blood sugar level after per oral glucose loading in the offspring of DES-treated rats. Olycemic index is the ratio of blood sugar level after glucose loading to the level before the loading. Continuous line, control rats; Interrupted line, offspring of DES-treated rats; *, Difference; P < 0.05; control vs, DES-treated offspring~
110
o u9 ¢..
g r
0
O'3
o
<
0'3
k~
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~r3
z
.i
gq
0 Z 0
i. t~
~v ~8
~8
"6
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•- . , 0
0
~
111
~
,
g Fig. 3. Thecafficuioma in the offspring transplacentally treated with DES. H & E. X125.
rats with persistent estrus (Fig. I). Tolerance to glucose was decreased in the offspring of DES-treated rats ~Fig. 2). Data on tumor incidence and site are summarized in Table 1, Twenty-seven tumors were found in 14 of 18 rats prenatally treated with DES (1.93 tumors/ tumor-bearing rat). In the control group 12 tumors were found in 9 of 34 rats (I.33 tumors/tumor-bearing rat}. Neoplasms of the cervix uteri or vagina were n o t observed in either group.. Two thecafolliculomas (Fig. 3), 3 endometrial polyps, and 1 fibroma of the uterus were found in rats treated prenatally with DES; ovarian or endometrial tumors were not observed in the control group. DISCUSSION It is well known that the C&H in hemiovariectomized rats is due to increased activity of the hypothalamic-gonadotropic system in response to a fall in the estrogen level [10]. The dose of DES that sharply inhibited COH in control rats had no effect in rats prenatally exposed to estrogen, These observations suggest that transplacental DES administration increases the threshold of hypothalamic-pituitary sensitivity to a negative feedback of estrogens. Persistent estrus and ol~her changes in homeostatic interrelationships were shown in rodents neonatally treated with se~ hormones [9,14]. Administration of testosterone or estrogen (particularly DES) to pregnant rodents induced persistent estrus and sterility in the offspring [20,22,25,28]. It should be emphasized that ovarian cysts and the absence of corpora lutea were observed
112
in the ovaries transplanted from intact cyclic rats to ovariectomized, persistent estrus off-springof DES-treated mothers [28]. Similar findings were observed in experiments on reciprocal orthotopic ovarian transplantation between young rats with regular cycles and persistentestrus senilerats [8]. Elevation of the threshold of the hypothalamic-pituitary sensitivity to feedback action of estrogens, the decrease of [3H]estradiol uptake in target tissues (includingthe hypothalamus), as well az,changes in the hypothalamic turnover of biogenic amines and increased body weight, were observed in rats with persistentestrus induced by neonatally administered sex hormones [9,14,16 ]. Similar changes, including increased incidence of persistentestrus and decreased tolerance to glucose loading, develop in rats in the course of spontaneous aging [5,6,8,13,21,26]. According to Dilman [11] a gradual elevation of the threshold of the sensitivityof the hypothalamus to feedback control is the key process in the neuroendocrine program of development, aging, and diseasesof compensation, including cancer. It was shown that enhanced tumor incidence in rodents in which persistent estrus was induced by various methods [1,4,29] was followed by signs of intensified aging [1,6]. Persistentestrus induced by various methods m a y be observed in animals with qualitativeand quant!~tativedifferences in levelsof estrogens [3]. This probably determines the spectrum of tumors developed [4]. For example, V~ha-Perttula and Hopsu [29] observed granulosa cell ovarian tumors in 2 3 % of rats neonatally treated with androgens; Vorherr et al. [30] observed 2 squamous cell vaginal carcinomas, endometrial and ovarian adenocarcinomas in rats with persistentestrus induced by prenatal exposure to DES. The data reported here demonstrate that tumor incidence in the offspring of rats treated with D E S was 3 times higher than that in the control offspring. Tumors of the ovary of the corpus uteri were found only in the experimental group; none were observed in control rats. In the large group o~ intact female rats of our colony the incidence of ovarian tumors did not exceed 0.4% [7]. Other tumors were found more frequently in the experimental group as com.. pared with the control group; however, there was no significant.;difference in histologicaltype, perhaps because of the small number of animals used in these preliminary observations. It should be emphasized that Nomura and Kanzaki [25] observed an increased incidence of spont$,neous tumors in mice prenatally exposed to DES. To elucidate 1~hesediscrepanc~:esit would be worthwhile to examine hormonal patterns in .an experimental study of dyshormona~ carcinogenesis according ~o our previous data [4]. It m a y be supposed that the increase in tumor incidence that occurs in female offspring of DES-treL~ted rodents might depend on the hormonal-metabolic changes induced by damage of the hypothalamie sex differentiationduring the perinatal period. ACKNOWLEDGEMENTS A u t h o r s are in d e b t t o Dr. N.M. G o r o d i l o v a for e x c e l l e n t e s t i m a t i o n of b l o o d sugar..
i J3 REFERENCES 1 Anisimov, V.N. (1971) Bla.,;tomogenesis in persistent estrous rats. Vopr. Onkol., (In Russ.), 8, 67--75. 2 AnJsimov, V.N. (1978) Pharmacological study of cholinergic mechanisms of compensatory ovarian hypertrophy in rats. Endokrinologie, 71, 149--153. 3 Anisimov, V.N. and Pavlova, M.V. (1970) Some peculiarities of steroid hormones excretion in rats with persistent estrus. Vopr. Onkol., (In Russ.), 12, 26--81. 4 Anislmov, V.N. and Pavlova, M.V. (1972) Hormonal shifts and tumor lOCalization in rats with persistent estrus. Vopr. Onkol., (In Russ.), 5, 68--71. 5 Anisimov, V.N., Ostroumova, M.N. and Dilman, V.M. {1974) Age increase in the hypothalamic-hypophyseai threshold to the inhibitory action of estr,)gens and the effect of an epiphyseai extract on this process. Bull. Exp. Biol. Med., (In Russ.), 77, 100--102. 6 Anisimov, V.N. and Lvovich, E.G. {1976) Disorders in carbohydrate and }'at metabolism in rats in ageing, persistent estrus and DMBA injection. Vopr.Onkol., (In Russ.), 2, 55--58. 7 Anisimov, V.N., Alexandrov, V.A., Klimashevsky, V,F., Kolodie, V.I., Likhachev, A. Ya., Okulov, V.B., Pozharisst::i, K.M., and Savelieva, O.P. (1978) Spontaneous tumors in rats bred at the "Rappolovo" breeding farm of the USSR Academy of Medical Sciences. Vopr.Onkol., (In Russ.), 1, 64--70. 8 Aschheim, P. (1976) Aging in the hypothaIamic-hypopbyseal-ovarian axis in the rat. In" Hypothalamus, Pituitary and Aging, pp. 378--418. Editors: A.V. Everitt and J.A. Burgess. C.C. Thomas, Springfield, Illinois. 9 Barraclough, C.A. (1966) Modification in the CNS regulatic.n of reproduction after exposure' of prepuberta[ rats to steroid hormones. Recent. Pragr. Horm. Res., 2 2 , 5 0 3 - 539. 10 B~teher, R.L. (I977) Changes in gonadotropins and steroids associated with unilateral ovariectomy of the rat. Endocrinology, 1 0 1 , 8 3 0 ~ 8 4 0 . 11 Dilman, V.M. (1971) Age.associated elevation of hypothalamic threshold to feedback control and its role in development, a~eing and disease. Lancet, 1, 1211~1219. 12 Dunn, T.B. and Green, A.W. (1963) Cysts o f the epid[dimya, cancer of the cervix, granular ceil myoblastoma and other lesions after estrogen in~ection in newborn mice. J. Natl. Cancer Inst., 31, 425--455. 13 Finch, C.E. ( 1 9 7 6 ) T h e regulation of physiological changes during mammalian aging. Q. Rev. BioL, 51, 79--83. 14 Flerko, B. (1975) Perinata[ androgen action and the differenti:ationof the hypothalamus. In: Growth and Development of the Brain. Editor: M.A.B. Brazier. Raven Press, New York. 15 Forsberg, J.G. (1975) Late effects in the vaginal and cervical epithelia after injections of diethylstilbestrol into ~eonatal mice. Am. J. Obstet. Gynecol ~ 121, 1(~1--104. 16 Harris, G.W. and Levine, J. (1965) Sexual differentiation of t:~e brain and its experimental control. J. Physiol., 1 8 1 , 3 7 9 ~ 4 0 0 , 17 Herbst, A.L. (1976) Summary on the changes in the human female genital tract as a consequence of materna| diethylstilbestrol therapy. J. Toxicol. Environ. Health, Suppl. 1, 13--20. 18 IARC (1974) Se:~ hormones. In: IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol. 6. IARC, Lyon, 19 Kimura, T. and Nandi, S. (1957) Nature of induced persistent vaginal cornifieation in mice. IV. Changes in the vagina! epithelium of old mice truated neonatally with estradiol or testosterone. J. Natl. Cancer Inst., 39, 75--93. 20 Laszlo, J. and Gyorgy, G. (1963) Tierexperimentellc Versuehe zur Klarung der Pathogenese des Stein-Leventhal-Syndroms. Arch. Gynaekoi., 198, 504--506. 21 Lu, K,H., Huang, H.H., Chen, H.T, Karcz, M., Mioduszewski, R. and Meites, J. (1977) Positive feedback by estrogen and progesterone on LH release in old and y o u n g rats. Proc. Soc. Exp. Biol. Med., 154, 82--85.
114
22 McLachlan, J.A., Shah, H.C., Newbold, R.R. and Bullock, B.C, (1975) Effect of prenatal exposure of mice to diethylstilbestrolon reproductive tract function in the offspring. Toxic0 I.Appl. Pharrn~col., 33, 190. 23 Mori, T. (1968) Changes in reproductive organs and some other glands in old C 3 H / M S mice treated ne0natally with low doses of estrogen. Ann. ZooL Jpn., 41, 43--52. 24 M{iller,G. (1965) Zur Blutzuckerbestimmung mit o-toluidln. Dtseh. Z. Verdau Stoffwechselkr., 25, 76--80. 25 Nomura, T. and Kanzaki, T. (1977) Induction of urogenital anomalies and some tumors in the progeny of mice receiving diethylstilbestrolduring pregnancy. Cancer Res., 37 1099--1104.
26 Pen~g, l~I.T,and Peng, Y.M. (1973) Changes in the uptake of tritlatedestradiol in the hypothalamus and ~denohypophysis of old female rats. Fertil. Steril.,24, 534--539. 27 Rustia, M. and $hubik, P. (1976) Transplacental effects of diethylstilbestrol on the genital tract of hamster offspring. Cancer Letters, I, 139--146. 28 Smith, W.N.A. (1969) Transplacental influence of estrogen upon ovulatory mechanisms in the rat. J. Endocrinol., 45,143--144. 29 VavhwPerttula, T. and Hopsu, V.K. (1965) High incidence of malignancies in persistent estrous rats.Acts Pathol. Microbiol. Seand., 64,286--288. 30 Vorherr, H,, Messer, R.H., Vorherr, U.F., Jordan, S.W. and Kornfeld, M, (1976) Diethyistilbestrol-indueed teratogenesisand carcinogenesis in rats. Fed. Proc., 35, 567.
107
© Elsevier/North-Holland ScientificPublishers Ltd.
TRANSPLACENTAL EFFECT' O F DIETHYLSTILBESTROL IN FEMALE RATS
N.P. N A P A L K O V
and V.N. A N I S I M O V
Petrov Research Institute o f Oncology, 68 Leningradskaya St., Pesoehny.2, Leningrad 188~46 (U.S.S.R.)
(Received 27 April 1978} (Revised version received 27 September 1978) (Accepted 28 Septersber 1978}
SUMMARY
The effect of diethylstilbestrol propionate (DES; 1 mg/kg body wt. was studied in the female offspring of rats exposed subcutaneously on the 19th day o f gestation. Examination of vaginal smears showed persistent estrus in adult rats treated prenatally with DES. Compensatory ovarian hypertrophy (COH) induced by hemicastration of these rats was not suppressed by estrogens. A per oral test for glucose-tolerance revealed decreased utilization of glucose in the offspring o f DES-treated r a t s Preliminary observations demonstrated that tumors developed in 14 of 18 (77.8%) progeny transpiacentally exposed to DES and in 9 o f 34 (26.5%) intact control rats. Tumors of the ovary and the endometrium were found only in the rats treated prenatally with DES; no tumors of the uterine cervix or vagina were observed in either experimental or control groups. It is suggested that a transplacental effect of DES in the female is impairment of the sex differentiation o f the hypothalamus. The resulting hormonal and metabolic shift:~ might promote tumorigenesis.
INTRODUCTION During the last decade, reports on she development of cervical and vaginal adenocarcinomas in human females exposed in utero to DES have been pub?,ished [17,18]. Hyperplastic ~esions in the uterine cervix and vagina were induced in mice treated with estrogens during: the first week of ilfe [12,15,19,23]. A single administration of DES to mice on the :~.5--19th day of pregnancy led ~o hypertrophy of the vaginal portio of the cervix and to persistent estrus in the A bbreviations: COH, compensatory ovarian hypertrophy; DES, diethyIstilbestrol propionate.
208
progeny; however, development of cervical and vaginal adenocarcinomas was n o t observed [25]. Vorherr et al, [30] obsevped genital abnormalities, squamous cell vaginal carcinomas, endometriaI and ovarian adenocarcinomas in rats transplacentally exposed to DES: g u s t i a and Shubik [27] showed the development of hyperkeratcsis and parakeratosis and o f squamous cell papillomas and mesenehymal sarcomas of t h e cervix uteri and the vagina in Syrian golden hamster progeny exposed to DES during gestation. The purpose of this study is to show t h e transplacental effect of DES in fem;~Llerats. MATERIALS .~..NDMETHODS Random bred albino rats were provided b y the Rappolovo breeding farm AMS USSR. On the 19th day after conception 11 pregnant rats, 3 - 4 months old, were given a single subcutaneo~s injection of oil solution of DES, (offic. preparation of Rostov Chemical Factory, USSR; 1 mg/kg body wt.). The female offspring at 3 weeks of age were taken from the mothers, randomized, kep~ in stee]: cages in a 14 h light/I0 h dark environment and fed standard laboratory diet and water ad libitum. The offspring were kept for their lifetime under the same conditions as other intact rats o f the colony. The control group was the female offspring of 17 untreated rats. Vaginal smears were examined daffy after the opening of the vagina. To study the functional status of hypothalamic-pituitary-ovarian system a group o f rats aged 3 -.4 months was hemicastrated. Starting on the day of operation they received daily subcutaneous injections of 0.1 m! vegetable oil or 0.57 #g DES dissolved in the same volume of oil for 7 days [2]. On the eighth day after hemicastration the animals were killed and the degree of COH was determined [2,10]. Tolerance. to glucose was studied in 29 experimental and 58 control rats by per oral glucose loading. Blood sugar Ievel was determined by the O-toluidine method [241 in :tats after 18 h fasting and 30, 60 and 120 rain after glucose loadling (3.0 g/kg). The animals not used in the above experiments were allowed to die spontaneously or were kiIled when moribund. Autopsies were performed on all animals. Tumors were fixed in 10% neutral formalin, and 7 ~m paraffin. celloidin sections were stained with hematoxylin and eosin. RESULTS
The exan~Lination of vaginal smears taken daily after the vagina opened showed the reguiar 4 - 5 day estrus cycles in control rats and persistent estrus in the rats prenatally exposed to DES. The age-associated increased incidence of irregular cycles and persistent estrus was found m the control group. DES induced suppression of COH b y 68.5% i~l control hemiovariectomized offspring and did not produce any effect in trm;splacentally estrogen-treated
109 6O
v
5O
40
z
30
20
8
Fig~ 1. The effect of estrogen on the COH in control and prenatally DES-exposed rats: (1) control offspring; (2) offspring prenatally exposed to DES. Unshaded columns, oil injections; Shaded columns, DES injections; Number in columns show the number of animals in groups. Values given are M ± S.E.M.
2.20
1.80
X
1.40 L~ t,O L~
i . O0
/ ---~ 0
~=~ L~
,J
"\
..
\
~ ~ ~0
, ~0
"---. -: ~-~0~o
.60
Fig. 2~ Blood sugar level after per oral glucose loading in the offspring of DES-treated rats. Olycemic index is the ratio of blood sugar level after glucose loading to the level before the loading. Continuous line, control rats; Interrupted line, offspring of DES-treated rats; *, Difference; P < 0.05; control vs, DES-treated offspring~
110
o u9 ¢..
g r
0
O'3
o
<
0'3
k~
Cq
~r3
z
.i
gq
0 Z 0
i. t~
~v ~8
~8
"6
:2
•- . , 0
0
~
111
~
,
g Fig. 3. Thecafficuioma in the offspring transplacentally treated with DES. H & E. X125.
rats with persistent estrus (Fig. I). Tolerance to glucose was decreased in the offspring of DES-treated rats ~Fig. 2). Data on tumor incidence and site are summarized in Table 1, Twenty-seven tumors were found in 14 of 18 rats prenatally treated with DES (1.93 tumors/ tumor-bearing rat). In the control group 12 tumors were found in 9 of 34 rats (I.33 tumors/tumor-bearing rat}. Neoplasms of the cervix uteri or vagina were n o t observed in either group.. Two thecafolliculomas (Fig. 3), 3 endometrial polyps, and 1 fibroma of the uterus were found in rats treated prenatally with DES; ovarian or endometrial tumors were not observed in the control group. DISCUSSION It is well known that the C&H in hemiovariectomized rats is due to increased activity of the hypothalamic-gonadotropic system in response to a fall in the estrogen level [10]. The dose of DES that sharply inhibited COH in control rats had no effect in rats prenatally exposed to estrogen, These observations suggest that transplacental DES administration increases the threshold of hypothalamic-pituitary sensitivity to a negative feedback of estrogens. Persistent estrus and ol~her changes in homeostatic interrelationships were shown in rodents neonatally treated with se~ hormones [9,14]. Administration of testosterone or estrogen (particularly DES) to pregnant rodents induced persistent estrus and sterility in the offspring [20,22,25,28]. It should be emphasized that ovarian cysts and the absence of corpora lutea were observed
112
in the ovaries transplanted from intact cyclic rats to ovariectomized, persistent estrus off-springof DES-treated mothers [28]. Similar findings were observed in experiments on reciprocal orthotopic ovarian transplantation between young rats with regular cycles and persistentestrus senilerats [8]. Elevation of the threshold of the hypothalamic-pituitary sensitivity to feedback action of estrogens, the decrease of [3H]estradiol uptake in target tissues (includingthe hypothalamus), as well az,changes in the hypothalamic turnover of biogenic amines and increased body weight, were observed in rats with persistentestrus induced by neonatally administered sex hormones [9,14,16 ]. Similar changes, including increased incidence of persistentestrus and decreased tolerance to glucose loading, develop in rats in the course of spontaneous aging [5,6,8,13,21,26]. According to Dilman [11] a gradual elevation of the threshold of the sensitivityof the hypothalamus to feedback control is the key process in the neuroendocrine program of development, aging, and diseasesof compensation, including cancer. It was shown that enhanced tumor incidence in rodents in which persistent estrus was induced by various methods [1,4,29] was followed by signs of intensified aging [1,6]. Persistentestrus induced by various methods m a y be observed in animals with qualitativeand quant!~tativedifferences in levelsof estrogens [3]. This probably determines the spectrum of tumors developed [4]. For example, V~ha-Perttula and Hopsu [29] observed granulosa cell ovarian tumors in 2 3 % of rats neonatally treated with androgens; Vorherr et al. [30] observed 2 squamous cell vaginal carcinomas, endometrial and ovarian adenocarcinomas in rats with persistentestrus induced by prenatal exposure to DES. The data reported here demonstrate that tumor incidence in the offspring of rats treated with D E S was 3 times higher than that in the control offspring. Tumors of the ovary of the corpus uteri were found only in the experimental group; none were observed in control rats. In the large group o~ intact female rats of our colony the incidence of ovarian tumors did not exceed 0.4% [7]. Other tumors were found more frequently in the experimental group as com.. pared with the control group; however, there was no significant.;difference in histologicaltype, perhaps because of the small number of animals used in these preliminary observations. It should be emphasized that Nomura and Kanzaki [25] observed an increased incidence of spont$,neous tumors in mice prenatally exposed to DES. To elucidate 1~hesediscrepanc~:esit would be worthwhile to examine hormonal patterns in .an experimental study of dyshormona~ carcinogenesis according ~o our previous data [4]. It m a y be supposed that the increase in tumor incidence that occurs in female offspring of DES-treL~ted rodents might depend on the hormonal-metabolic changes induced by damage of the hypothalamie sex differentiationduring the perinatal period. ACKNOWLEDGEMENTS A u t h o r s are in d e b t t o Dr. N.M. G o r o d i l o v a for e x c e l l e n t e s t i m a t i o n of b l o o d sugar..
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