[CANCER RESEARCH 51. 3311-3315. June 15, 1991]

Carcinogenicity of Diethylstilbestrol in the Wistar Rat: Effect of Postnatal OralContraceptive Steroids1

Raymond B. Baggs,2 Richard K. Miller, and Charles L. Odoroff3

Division of Laboratory Animal Medicine and of Pathology and Laboratory Medicine [R. B. B.], Department of Obstetrics and Gynecology [R. K. M.J, Division ofBiostatistics [C. L. O.J, and Environmental Health Sciences Center [R. B. B., R. K. MJ, School of Medicine and Dentistry, University of Rochester, Rochester, New York14642

ABSTRACT

Diethylstilbestrol (DES) has been associated with vaginal neoplasiaand malformations in humans. We have studied a test population of 504female Wistar rats given diethylstilbestrol at from 0.0 to 0.5 mg/kgmaternal body weight on days 18, 19, and 20 of gestation. Animals wereeuthanized in extremis, or at 2 years of age. The incidence of vaginalepithelial tumors was dose related. The types of epithelial tumors of thevagina were adenocarcinoma, squamous cell carcinoma, and mixed carcinoma, containing discrete adenomatous and squamous components. Theincidence of vaginal epithelial tumors was determined to be dose related:rats exposed to 0 mg DES/kg maternal weight had an incidence of 0.6%(1 of 167 rats); 0.1 mg/kg, 4.1%; and 0.5 mg/kg, 4.3% (6 of 140); 25 mg/kg, 1.6% (1 of 63); and 50 mg/kg, 11.5% (3 of 26). Tumors of otherreproductive tissues (mammary gland, ovary, oviduct, cervix, or uterus)demonstrated no discernible DES dose-response relationship. There wasno oncogenic effect of postnatal administration of oral contraceptives (0oral contraceptives, 31.25 Mg/kg diet ethynylestradiol, and 31.25 Mg/kgdiet norethindrone or 104 Mg/kg diet ethynylestradiol and 31.25 Mg/kgdiet norethindrone). Thus, vaginal tumors can be induced in a dose-related manner in the rat following in utero DES exposure. Oral contraceptive treatment did not increase the risk of neoplasia.

INTRODUCTION

Transplacental carcinogenesis, one of the most ominous ofall tumor-inducing processes, predisposes an unborn child tothe latent development of life-threatening neoplasia. DES4 is

one agent that is associated with both vaginal adenocarcinomaand malformations (e.g., adenosis, T-shaped uterus, cervicalhoods, and ridges) in the human female exposed in utero (l).In mice, the long-term effects of neonatal exposure to estrogeninclude hyperplastic vaginal lesions and cervicovaginal cancer.In experiments with appropriate controls, Dunn and Green (2)reported a vaginal tumor incidence of approximately 8% (ourcalculations) for female mice give s.c. injections at birth of 2mg/kg of DES in saline. These tumors were epithelial in originand were observed between 20 and 23 months, but the precisetumor type was not identified. There were no such tumorsreported in the four controls. McLachlan et al. (3, 4) reportedthe occurrence of 3 vaginal adenocarcinomas in mice followingin utero exposure to DES (1 of 29 at 0.005 mg/kg/day; 1 of 57at 0.01 mg/kg/day; 1 of 46 at 0.1 mg/kg/day). Their subsequentstudies (5) using postnatal DES exposure in mice have demonstrated a very high (90%) incidence of uterine adenocarcinoma, but no additional vaginal adenocarcinomas. Walker (6)examined mice exposed in utero to DES. Although none of the

Received 12/1/89; accepted 4/8/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by National Cancer Institute Grant CA22335.! To whom requests for reprints should be addressed, at Division of Laboratory

Animal Medicine, Box 674, School of Medicine and Dentistry, University ofRochester, Rochester, NY 14642.

3The late Professor Odoroff was responsible for the statistical component of

this research.4The abbreviations used are: DES, diethylstilbestrol; OC, oral contraceptives;

EE, ethynylestradiol; NE, norethindrone.

15 animals exposed in utero was reported to have had macro-scopically detectable tumors, of 40 offspring, 10 had uterineadenocarcinoma and 5 had ovarian cystadenocarcinoma.

The rat has proven to be highly susceptible to the abortifa-cient and teratogenic effects of DES (7-9). These reproductiveeffects are dependent upon when the exposure period occursduring development. Before day 18 of gestation, a high incidence of pregnancy loss is observed. Between 18 and 20 days,female hypospadias, uncoiled oviducts, and vaginal uroliths canbe produced in a dose-related manner (10). Postnatal exposureto DES did not produce these effects, but rather an increasedincidence of adenosis. Branham et al. (Il, 12), studying theeffect of early postnatal DES on uterine cell populations in rats,found that decreased uterine growth was a consequence ofcombined hypertrophy and hypoplasia in all cell types exceptlongitudinal muscle.

Besides the issue of in utero exposure to diethylstilbestroland induction of vaginal tumors and malformations, there hasbeen increasing interest in postnatal confounding factors.Among these confounding postnatal factors is the use of oralcontraceptives by women who were exposed in utero to diethylstilbestrol. Therefore, this rat model was evaluated to determine the incidence of prenatal DES exposure when the miiller-ian duct is developing (18-20 days). We then used this in uteroexposed DES population to determine risk for carcinogenicitywith postnatal exposure to oral contraceptives.

MATERIALS AND METHODS

Virgin female Wistar [Crl:(WI)BR] rats (Charles River Laboratories,Wilmington, MA) were treated with DES (Sigma Chemical Co., St.Louis, MO) in corn oil s.c. on days 18-20 of gestation (presence ofvaginal plugs in the animal or in the cage debris, or sperm in the vaginalsmear used as the index of a positive pregnancy). The determination ofpregnancy was considered day 0 of pregnancy. The doses of DES usedin this experiment are quite analogous to those used in the humanexperience (13). The daily dose of DES received by women ranged from5 mg/day (7-8 weeks pregnant) to 150 mg/day (34-35 weeks pregnant);thus the dose for a 50-kg woman ranged from 0.1 to 3 mg/kg. The ratswere given DES at 0.0 to 0.5 mg/kg maternal body weight on days 18,19, and 20 of gestation and at 25 and 50 mg/kg on days 19 and 20 ofgestation (Table 1).

Beginning at 10 weeks of age, two groups of animals were investigated, DES and non-DES exposed. Using a computer-generated random assignment of animals, female offspring were fed OC in diet for 5days/week at doses of control (no OC), low (31.25-Mg/kg feed of EE,31.25-Mg/kg feed of NE) or high (104-Mg/kg feed of EE, 31.25-Mg/kgfeed of NE). The rats weighed 0.25-0.45 kg and ate between 10 and 15g of diet/day; the p.o. oral dose was thus 1-1.2 Mg/kg/day EE, 1-1.2Mg/kg/day NE for the low dose OC group and 3-4 Mg/kg/day EE, 1-1.2 Mg/kg/day NE for the high dose OC group. The EE doses arecomparable to those used in women for contraception (0.03 mg EE/day for a 50-kg individual, or 0.6 Mg/kg/day). The NE doses are low bycomparison with modern "low dose" formulation, in which women

would receive 10 Mg/kg/day (14). The oral contraceptives were dissolvedin corn oil, diluted in 95% ethanol, and mixed with the powdered diet(Purina 5001). The ethanol was allowed to evaporate thoroughly at

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ORAL CONTRACEPTIVES AND DES CARC1NOGENICITY

Table 1 Incidence of tumors infernale rats exposed to diethylstilhestrol in utero

Diethylstilbestrol(mg/kg maternalwl)Litters'Total

born (male andfemale)Aliveat weaning (male andfemale)Female

progeny ontrial%of female surviving (no.)for>

120daysTotaldays atriskDaysat risk after 120 daysfrombirthTumorsAll

vaginaltumors''CasesRate,

0'Rate,

Utf%oflesions*Vaginal

adenocarcinomaCasesRate,

0Rate,120%

oflesions0.0°37403364147100(147)88.30670.73722.32.81.400000.1°141271104996(47)29,82323,97426.78.34.226.78.34.2Vaginal

squamous cellcarcinomasCasesRate.

0Rate,120%

oflesionsMammary

adenomasandfibroadenomas'CasesRate,

0Rate.120%

oflesionsPituitary

chromophobeadenomas'CasesRate,

0Rate,120%

of lesions00001719.324.011

J33.44.22.00000826.833.417.026.78.34.30.5°211871728052(42)25,07117.058416.023.49.54*16.023.49.51*4.05.92.4620.135.214.3323.517.67.125"211631396341(26)12,7458,17917.812.23.8000017.812.23.8539.261.119.2000050*10682611100(11)5,4264,106355.373.127.30000355.373.127.3118.424.49.10000

" Dams were treated on days 18, 19. and 20 of gestation.* Dams were treated on days 19 and 20 of gestation.f All litters but 0.1 mg/kg (13 of 14) and 50 mg/kg (5 of 10) had live born.* No animals with vaginal tumors were littermates.' N umber of lesions per 100.000 days at risk./ Number of lesions per 100,000 days at risk assuming animals were placed on

trial at 120 days.* Incidence of lesions (as %) in animals which survived >120 days.* One animal has a mixed tumor and is counted in both categories.1No animals have vaginal tumors.

room temperature before feeding. Fresh diet was prepared biweekly,with storage at 5°C.Purina 5001 in pellet form, devoid of exogenous

steroid, was fed 2 days/week to maintain the cyclic nature of exposureand to prevent incisor overgrowth. Additional animals were subsequently generated for the oral contraceptive study. Thus, Table 2, 0.0DES, no OC, contains the 147 animals in Table 1, plus an additional18 animals, and the 0.5 DES in utero, no OC is composed of the 80reported in Table 1. plus an additional 60 generated concurrently.

In the studies involving the postnatal administration of oral contraceptives, the litters were separated to randomly distribute the membersof the litter across treatments via computer-generated random numberassignment. These animals were treated and monitored for 2 years.Moribund animals were euthanized; all others were euthanized at 2years. A complete necropsy examination included gross dissection andhistológica! examination of the following tissues: vagina (posterior,mid-, and anterior, including cervical canal); uterus (posterior, includingcervix, body, and anterior); oviduct; ovary; urinary bladder; urethra;ureters; kidney; mammary gland; brain (including pituitary); heart,lung; liver; spleen; digestive tract (stomach, duodenum, jejunum, cecum,colon); pancreas; skeletal muscle; skin; thyroid; adrenal gland. Thevagina was fixed in 0.2-cm-thick transverse blocks, sectioned at 5 ^m,

and routinely stained with hematoxylin and eosin. The majority of thetumors were detectable as small masses between 1and 2 cm in diameter.Histopathological observations were reviewed by outside experts. Radiographie evaluation to document hypospadias (10) was performed onall animals.

RESULTS

Table 1 reports the observations in this study of 948 offspringborn, of which 350 female progeny are studied. The highestDES dose group (50 mg/kg) had the highest incidence ofpreweaning mortality (62% as compared to 10 to 15% for theother treatments). Mortality prior to 120 days of age wasassociated with reproductive tract malformations, such as hypospadias, cleft phallus, vaginal calculi, vaginal hyperkeratosis,and adenosis (15). The female progeny were entered onto thestudy at weaning.

Because of the early mortality in only the DES-exposedpopulation, the tumor data are expressed in two forms. "Rate"

reflects the number of tumors per 100,000 days at risk. Thisvalue includes all animals placed on trial. A second calculationwas needed to better reflect the latency period for tumor development. Since the earliest appearance of a mammary or vaginaltumor was 6.5 months, "percentage surviving" is the percentage

of female animals that survived for more than 120 days.Prenatal administration of DES on days 18, 19, and 20 of

gestation to rat dams resulted in the production of reproductivetract tumors in the female progeny (Table 1). The majority ofthe tumors were detectable as small masses between 1 and 2cm in diameter. At 0.1 and 0.5 mg/kg DES, five adenocarci-nomas (Fig. 1) and one mixed carcinoma, containing discreteadenomatous and squamous components (Fig. 2), were observed. At high doses only squamous cell carcinomas (Fig. 3)were present. Using the corn oil control groups as a standard,the relative odds of observing a tumor at each of the doses were:0.1 mg/kg, 3.2; 0.5 mg/kg, 5.9; 25 mg/kg, 20; and 50 mg/kg,95. With the exception of the 0.1-mg/kg dose, these relativeodds are all significant (P < 0.05; nonsimultaneous comparisons). It should be noted, however, that the two tumors in thecorn oil controls were nonepithelial in origin (leiomyosarcomaand leiomyoma) and may not be directly comparable to vaginalepithelial tumors. Rat days at risk for all vaginal tumors weredose related, increasing from 6.7 to 55.3 tumors/100,000 daysat risk.

Of 165 animals given only corn oil in utero, there was oneanimal with a vaginal epithelial neoplasm. Histologically (Fig.4), this was an adenocarcinoma. The animal was euthanized inextremis at 419 days of age. Grossly, the uterus and vagina wereincarcerated in an irregular 2.5- x 2.0- x 1.9-cm firm tan mass.

Table 2 Vaginal tumor type and incidence by treatment0.0DES"in

uteroNo.

of females intrialVag.SCCVag.AdenoCAVag.

sarcomaNoOC165012HiOC220010.1

DESinuteroNoOC49020HiOC151000.5

DES inuteroNoOC140423LoOC8000HiOC80312

"0.0 DES. 0.0 mg/kg maternal body weight days 18, 19, and 20 of gestation;

0.1 DES, 0.1 mg/kg maternal body weight days 18, 19, and 20 of gestation; 0.5DES, 0.5 mg/kg maternal body weight days 18, 19. and 20 of gestation; No OC,no oral contraceptive; Lo OC. 31.25 wg/kg diet ethynylestradiol and 31.25 Mg/kgdiet norethindrone; Hi OC. 104 Mg/kg diet ethynylestradiol and 31.25 Mg/kg dietnorethindrone; Vag., vaginal: SCC, squamous cell carcinoma; AdenoCA, adenocarcinoma.

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ORAL CONTRACEPTIVES AND DES CARCINOGENICITY

Fig. 1. Adenocarcinoma in vaginal wallfrom rat treated with DKS in utero. I ', vaginal

lumen; A, ncoplaslic epithelial cells with adenomatous differentiation. Bar, 50 ^m.

Fig. 2. Mixed carcinoma in vaginal wallfrom rat treated with DES in ulero. I, vaginallumen; A, neoplastic epithelial cells with ade-nomatous differentiation; S, neoplastic epithelial cells with squamous differentiation. Bar,50 urn.

The severely invasive characteristics of the tumor obscured theprecise tissue of origin, with local extension to uterine serosaand peritoneum and metastasis to lung. Including this animal,the incidence of vaginal epithelial tumors was determined to bedose related: rats exposed to 0 mg DES/kg maternal weighthad an incidence of 0.6% (1 of 167 rats); 0.1 mg/kg, 4.1 %: and0.5 mg/kg, 4.3% (6 of 140), 25 mg/kg, 1.6% (1 of 63), and 50mg/kg, 11.5% (3 of 26).

When the data for all vaginal tumors were analyzed by themethod of Laird and Oliver (16), a significant dose responsewas observed (x2\ = 19.31, P = 0.001). The function increased

from 0 to 0.5 mg/kg. dropped at 25 mg/kg, and then rosesharply. In view of the different tumor types observed, this may

suggest different induction processes at each dose level.The sarcomas, unlike vaginal epithelial tumors, did not show

an obvious relationship to treatment. There was no litter effectin the tumor incidence. Tumors of other reproductive tissues(mammary gland, ovary, oviduct, or uterus) demonstrated nodiscernible dose-response relationship for DES (Table 1). Thefemale offspring treated in utero with 0, 0.1, or 0.5 mg DES/kg were fed oral contraceptives at doses up to 104 Mg/kg feedof ethynylestradiol: 31.25 Mg/kg of norethindrone for 5 daysper week and demonstrated no significant effects of the oralcontraceptive steroids on the incidence of vaginal tumors (Table2), or in any other reproductive tissue following 2 years ofexposure.

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ORAI. CONTRACEPTIVES AND DES CARCINOGENICITY

". v5 '

-fc «

Fig. 3. Squamouscell carcinoma in vaginalwall from ral treated with DES in utero. I',

vaginal lumen; S. ncoplastic epithelial cellswith squamous differentiation. Bar. 50 ¡an.

<s

' I

V

Fig. 4. Adenocarcinoma in rat treated withcorn oil vehicle only. V, vaginal lumen; A,ncoplastic epithelial cells with adenomatousdifferentiation. Bar, 50 um.

•

DISCUSSION

Many similarities among species are reported concerningperinatal exposure to diethylstilbestrol (17, 18). Takasugi andBern (19) demonstrate that persistent estrus in rodents is analteration in the hypothalamo-hypophysial system. Althoughthere are dramatic alterations in the morphology of the vaginaland uterine epithelium, extrapolations are not successful because of the low incidence of vaginal tumors. Both the terato-

genic and carcinogenic aspects of DES indicate the importanceof the epithelial/stromal components of the reproductive tract(20). Different cell types assumed to be exposed in these studiesundergo growth and differentiation at different gestational ages.Thus it is important to correlate the development of the repro

ductive tract, in particular the vagina, with the particular experimental model used.

A variety of regimens have been used to induce vaginalepithelial changes in mice. The highest incidence of adenosisoccurred following postnatal exposure, whereas the only descriptions of vaginal adenocarcinomas associated with DEShave occurred following prenatal exposure. Following prenatalexposure to DES on days 9-16, three vaginal tumors had beeninduced in mice; however, no dose-response relationship had as

yet been demonstrated (4). Besides these vaginal tumors, a highincidence of ovarian lesions was noted. No such ovarian lesionswere noted in the present rat study. These differences may notbe species dependent but rather may be due to the timing of theexposure. The current rat study specifically emphasized the

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ORAL CONTRACEPTIVES AND DES CARCINOGENICITY

period of vaginal and uterine tract development, while in themouse studies, earlier treatment was utilized.

The immune system may be affected by DES. Kailand andHolmdahl (21) reviewed the complex literature of murinemodels and concluded that neonatally DES-exposed mice dohave impaired natural killer lymphocyte function. In that study,transplantation of target tissue from DES-treated to untreatedthymectomized mice did not increase the frequency of neoplasia(22); T-cell function is apparently irrelevant.

It is important to note that the development of the rodent(rat and mouse) reproductive tract is truly perinatal. Thus, todocument teratogenesis/carcinogenesis of the reproductivetract, exposure only until day 14 of gestation in the rodent isnot appropriate. Vorherr et al. (23) reported, in ten Sprague-Dawley rats exposed to five different pre- and postnatal regimens of DES, four reproductive tract tumors and associatedmalformations, but no vaginal adenocarcinomas. Unfortunately, no controls were reported. Boylan (24) observed abnormal nipple development, ovarian cysts, and reproductive tractmalformations in the rat, following prenatal exposure to DES.No tumors were reported, however. It should be noted that thecurrent study indicates that the lack of tumors in the Boylanstudy may have been due to the short postnatal observationperiod. We observed similar precocious development of thenipples in the current rat study; however, this precocious neonatal mammary response did not result in an increase in mammary tumors (Table 1). The Wistar rat studies identified adefinite "window" during gestation when DES induced vaginal

epithelial changes, including adenocarcinomas. Administrationprior to day 15 produced abortion; administration after day 20induced lactation suppression in the dam.

The presence of epithelial tumors of the vagina in the Wistarrat and their appearance in a dose-related manner add furthersupport to the general association in the human between DESand vaginal tumors ( 1) as well as with the dose-related incidenceof reproductive tract malformations in the rat.

In conclusion, vaginal tumors of epithelial origin (adenocarcinomas and squamous cell carcinomas) can be induced inWistar rat offspring in a dose-related manner by DES administration between days 18 and 20 of gestation. Later administration of oral contraceptive steroids to those offspring did notenhance the risk of neoplasia due to DES in this model.

ACKNOWLEDGMENTS

We wish to thank Dr. Norval King, New England Regional PrimateResearch Center, Harvard Medical School, and Dr. Jerry Rice, NationalCancer Institute, NIH, for their review of the histological diagnoses,and we gratefully acknowledge the technical assistance of Linda John-stone, Roger McKenzie, Sue Higgins. and Robert Baitchman and thedata management assistance of Sandra Plumb.

REFERENCES

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(eds.), Developmental Effects of Diethylstilbestrol (DES) in pregnancy, pp.26-45. New York: Thieme-Stratton. Inc.. 1981.

2. Dunn. T. B.. and Green. A. W. Cysts of the epididymis. cancer of the cervix,granular cell myoblastoma and other lesions after estrogen injection innewborn mice. J. Nati. Cancer Inst.. 31: 425-455. 1963.

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4. Newbold, R. R., and McLachlan. J. A. Vaginal adenosis and adenocarcinomain mice exposed prenatally or neonatally to diethylstilbestrol. Cancer Res..«.•2003-2011,1982.

5. Newbold. R. R., Bullock. B. C.. and McLachlan. J. A. Uterine adenocarcinoma in mice following developmental treatment with estrogens: a modelfor hormonal carcinogenesis. Cancer Res.. 50: 7677-7681. 1990.

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8. Henry. E. C., Miller, R. K., and Baggs. R. B. Direct fetal injections ofdiethylstilbestrol and 17fi-cstradiol: a method for investigating their terato-genicity. Teratology. 29: 297-304. 1984.

9. Rothschild. T. C., Calhoon, R. E., and Boylan. E. S. Genital tract abnormalities in female rats exposed to diethylstilbestrol in utero. Reprod. Toxicol.,/.- 193-202. 1988.

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12. Branham. W. S., Zehr, D. R.. Chen. J. J.. and Sheehan, D. M. Uterineabnormalities in rats exposed neonatally to diethylstilbestrol. ethynylestra-diol, or clomiphene citrate. Toxicology. 51: 201-212. 1988.

13. Hubby. M. M.. Haenszel, W. M., and Herbst. A. L. Effects on the motherfollowing exposure to diethylstilbestrol in pregnancy. In: A. L. Herbst andH. A. Bern (eds.). Developmental Effects of Diethylstilbestrol (DES) inPregnancy, pp. 120-128. New York: Thieme-Stratton, Inc.. 1981.

14. Stubblefield. P. G. Selection of steroid combinations for oral contraceptivesof maximum benefit. J. Reprod. Med.. 31 (Suppl.): 922-928. 1986.

15. Baggs, R. B., Miller, R. K., Garman. R., and McKenzie, R. D. Teratogenicand neoplastic lesions of the genitourinary system in Wistar rats exposed inutero to DES. Teratology. 21: 26A, 1980.

16. Laird, N., and Oliver, D. Covariance analysis of censored survival data usinglog-linear analysis techniques. J. Am. Slat. Assoc., 76: 231-240, 1981.

17. Bern. H. A., and Talamanties, F. J. Neonatal mouse models and their relationto disease in the human female. In: A. L. Herbst and H. A. Bern (eds.).Developmental Effects of Diethylstilbestrol (DES) in Pregnancy, pp. 129-147, New York: Thieme-Stratton, Inc., 1981.

18. Forsberg. J. G.. and Kaliami. T. Embryology of the genital tract in humansand rodents. In: A. L. Herbst and H. A. Bern (eds.). Developmental Effectsof Dicthylstilbestrol (DES) in Pregnancy, pp. 4-25. New York: Thieme-Stratton. Inc., 1981.

19. Takasugi. N.. and Bern. H. A. Introduction: abnormal genital tract development in mammals following early exposure to sex hormones. In: T. Moriand H. Nagasawa (eds.). Toxicity of Hormones in Prenatal Life, pp. 1-7.Boca Raton. FL: CRC Press. Inc.. 1988.

20. Cunha, G. R.. and Fujii, H. Stromal-parenchymal interactions in normal andabnormal development of the genital tract. In: A. L. Herbst and H. A. Bern(eds.). Developmental Effects of Diethylstilbestrol (DES) in Pregnancy, pp.179-193. New York: Thieme-Stratton, Inc., 1981.

21. Kalland, T.. and Holmdahl. R. Estrogens and immunity: long-term consequences of neonatal imprinting of the immune system by diethylstilbestrol.In: T. Mori and H. Nagasawa (eds.), Toxicity of Hormones in Prenatal Life,pp. 112-126. Boca Raton. FL: CRC Press, Inc.. 1988.

22. Ways. S. C.. Bern, H. A., and Blair. P. B. Effect of immunosuppression onneonatally diethylstilbestrol-induced genital tract lesions and tumor development in female mice. J. Nail. Cancer Inst., 73: 863-870. 1984.

23. Vorherr, H.. Messer. R. H., Vorherr, U. F., Jordan, S. W., and Kornfeld, M.Teratogenesis and carcinogenesis in rat offspring after transplacental andHansm.iiMin.ii> exposure to diethylstilbestrol. Biochem. Pharmacol.. 28:1865-1877, 1979.

24. Boylan. E. S. Morphological and functional consequences of prenatal exposure to diethylstilbestrol in the rat. Biol. Reprod.. 19: 854-863, 1978.

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1991;51:3311-3315. Cancer Res   Raymond B. Baggs, Richard K. Miller and Charles L. Odoroff  Postnatal Oral Contraceptive SteroidsCarcinogenicity of Diethylstilbestrol in the Wistar Rat: Effect of

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