Long-Term Alterations in Histology and Steroid

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[CANCER RESEARCH 47, 4165-4172, August 1, 1987] Long-Term Alterations in Histology and Steroid Receptor Levels of the Genital Tract and Mammary Gland following Neonatal Exposure of Female BALB/cCrgl Mice to Various Doses of Diethylstilbestrol1

Howard A. Bern,2 Marc Edery,3 Karen T. Mills, Arthur F. Kohrman, Takao Mori, and Lisa Larson

Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720 [H. A. B., M. E., K. T. M., L. L.J; Department of Pediatrics, The University of Chicago and LaRabida Children's Hospital and Research Center, Chicago, Illinois 60649 Â¡A.F. K.J; and Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan [T. M.]

ABSTRACT Both ovary-dependent and ovary-independent alterations have been established with natural sex hormones. DES, a synthetic The relation of the dosage of diethylstilbestrol (DES) administered estrogen, given to the pregnant mother or directly to the new neonatally to the incidence and severity of genital tract and mammary born, is also capable of producing reproductive and mammary gland lesions and to the levels of sex hormone receptors was examined using a mouse model for human intrauterine DES exposure. Female abnormalities in laboratory mammals (as examples of the range BALB/cCrgl mice received various doses of DES (ranging from 5 x of observations that have been made, see selected Refs. 3 and 10 'III 5/ig daily for the first 5 days of life) or the sesame oil vehicle 8-25). To date, the few dose-related experiments with DES alone. In the vagina, at all ages examined (1, 2, 6, and 12 months) have been concerned with cervicovaginal lesions in outbred cytosolic estrogen receptors are consistently decreased after high doses strains of mice, NMRI (26), and CD-I (27). of neonatal DES (IK"1 and 1 Â«Â¿g).Incontrast, at the same ages, vaginal The present experiments were designed to establish the min cytosolic progestin receptors increase after identical doses. In the uterus, imal amounts of neonatally administered DES required to the 1-Mgdose of neonatal DES also consistently decreases cytosolic produce ovary-dependent and ovary-independent reproductive estrogen receptors while increasing cytosolic progestin receptors at 1, 2, and 6 months of age. Histologically, neonatal doses of 5 x 10 - /tg DES abnormalities and to alter cytosolic ER and PR levels in inbred mice of the BALB/cCrgl strain. result in vaginal lesions at 2 months. With age, this threshold level decreases, implying interaction with an altered hormonal milieu. The uterus shows a sensitivity similar to that of the vagina in regard to the MATERIALS AND METHODS histopathological effects of neonatal DES. The ovary and mammary glands are 10- to 100-fold more sensitive to neonatal DES exposure. Histopathological Studies

Newborn BALB/cCrgl female mice were given daily s.c. injections INTRODUCTION of DES (Sigma, St. Louis, MO) in 0.02 ml sesame oil (cold pressed, Main's; Los Angeles, CA) or the vehicle alone for the first 5 days of Fifteen years ago, Herbst et al. (1) reported on a cluster of life. A range of doses was used: 5 x 10"'; 2 x 10~'; 10~'; 5 x 10~2; cases involving clear cell adenocarcinoma of the vagina in young 10~2;5 x IO'3; IO'3; 5 x 10~*; KT4; 5 x 10~5;and 10"' MgDBS/day. women, which were linked to intrauterine DES4 exposure. The The initial injection was given within the first 24 h after birth. Some daily dosages of DES administered to pregnant women ranged mice in groups receiving the higher DES dosages (>5 x 10~4/ig) were from 1.5-150 mg (2). The minimum dose of DES resulting in ovariectomized at 40 days of age. All experimental animals were fed developmental abnormalities in humans is unknown. The neo water and a standard laboratory chow (Berkeley Diet; Feed Stuffs natal mouse shows genital tract development similar to that of Processing, San Francisco, CA) ad libitum and housed in a temperature- a human fetus at the end of the first trimester (the critical controlled room with an artificially illuminated 12-h day. Mice were killed at either 2 or 15 months of age. Reproductive tract, period for later adenocarcinoma occurrence in humans after ovaries, and adrenals were fixed in Bouin's fluid; 1-nm parasagittal DES exposure) and thus provides a model for exploration of the dose-related effects of early DES exposure. paraplast (Sherwood Medical, St. Louis, MO) sections of the cervico vaginal regions, along with transverse sections of uteri, ovaries, and The neonatal mouse model has been utilized to demonstrate adrenals were prepared and stained with hematoxylin and eosin. Mam the long-term effects of early sex hormone exposure on the mary glands attached to the skin from 15-month-old mice were fixed female reproductive tract (2, 3). Although the embryonic origin in 10% neutral formalin. After removal from the skin and staining with of the cervicovaginal epithelial abnormalities (mÃ¼llerianduct iron hematoxylin, whole mounts of the mammary glands were coded versus urogenital sinus) in the mouse (4-7) remains partly and examined under a dissecting microscope. A second group of newborn BALB/cCrgl female mice received 5 controversial, the incidence and extent of vaginal anomalies are daily s.c. injections of 10~2MgDES or the control sesame oil vehicle dependent upon the amount of sex hormones administered. within the first 24 h after birth. These animals were housed and fed as Received 10/15/86; revised 5/5/87; accepted 5/8/87. in Series 1 and were ovariectomized at 40 days of age and killed at 15 The costs of publication of this article were defrayed in part by the payment months of age. The data from this group were combined with Series 1. of page charges. This article must therefore be hereby marked advertisement in Histological material was processed as above. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by Grant CA05388, awarded by the National Cancer Institute, Mice were examined every 2-3 weeks and at autopsy for the presence Department of Health and Human Services. Dedicated to the memory of Dr. of vaginal concretions. When concretions were found, they were re Georges Rudali, eminent tumor biologist and great friend. moved with blunt forceps. 2To whom requests for reprints should be addressed. Data were analyzed utilizing the x2 test. 3 Recipient of a fellowship from the Ligue Nationale FranÃ§aisecontre le Cancer. 4The abbreviations used are: DES, diethylstilbestrol; ER, (cytosolic) estrogen Receptor Studies receptor; PR, (cytosolic) progestin receptor; [3H]R5020, 117a-mÂ«A^/-3H]promegestone; R5020, promegestone (17,21-dimethyl- 19-nor-4,9-pregnadiene-3,20- Animals. Newborn BALB/cCrgl female mice were used. Litter size dione); [3H]R2858, [\l0-methoxy-3H] moxestrol; R2858, moxestrol [110-meth- was adjusted to between 4 and 6 pups/mother with the addition of male oxy-17a-ethinyl-1,3,5 (10)-estratriene-3,17-diol]; Buffer A, 25 mM Tris-HCl, 1.5 pups born on the same day. Animals were given s.c. injections of 1, mM EDTA, 10 mM thioglycerol, and 10 mm sodium molybdate, pH 7.4; Buffer IO"1, IO"2, IO"3, 10-", and 10"' /ig DES in 0.02 ml sesame oil or the B, 10 mM Tris-HCl, 1.5 mM EDTA, 10 mM thioglycerol, and 10 mM sodium molybdate, pH 7.4. sesame oil vehicle for 5 consecutive days, starting within 18 h after 4165 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1987 American Association for Cancer Research. RECEPTOR/GENITAL TRACT CHANGES AFTER NEONATAL DES birth. These mice were ovariectomized 9-10 days prior to sacrifice and equilibrium or steady-state conditions and allow complete exchange of were killed at 1,2, 6, and 12 months of age. Another series of mice bound hormones in mouse tissues (33-36). A 25-fold excess of dexa- received 1(T2, 10"', or l MgDES daily along with sesame oil controls; methasone was also added to the [3H]R5020 solution to saturate glu- these mice were ovariectomized at 30 days and killed at 2 or 6 months cocorticoid receptors. Under the assay conditions binding of dexameth- of age. Tissues pooled from 4-8 mice/experiment were collected asep- asone to PR is negligible. tically and kept on ice until homogenization. The uterine cervix was For estrogen receptor determinations, 0.3 ml protamine sulfate so dissected from each reproductive tract and discarded, thus leaving uteri lution (2 mg/ml in Buffer B) was added for 10 min at 4Â°Cfollowing and vagina for separate study. Reagents [3H]R5020 (87 Ci/mmol), the initial incubation. The resultant precipitate was filtered onto Gel- unlabeled R5020, [3H]R2858, (87 Ci/mmol), and unlabeled R2858 man type AE glass filters which were then washed three times with 5 ml Buffer B. For the [3H]R5020 binding assay, unbound steroids were moxestrol were purchased from New England Nuclear, Boston, MA. removed by incubation for 10 min at 0-4Â°Cwith 0.3 ml 0.5% Norit A- All other chemicals were from Sigma. Homogenization and Cytosol Preparation. All procedures were per 0.05% Dextran T-70 (dextran-coated charcoal) in Buffer B and centri formed at 4Â°C.Tissues were homogenized with two 10-s bursts of a fuged at 2000 x g for 10 min. Aliquots of the supernatant (0.3 ml) were polytron PT-10-ST (Brinkman Instruments, Westbury, NY) in 8 ml of removed and after addition of 8 ml Aquasol, the radioactivity was Buffer A/g tissue. The homogenate was centrifugea at 13,000 x g for measured in a Beckman liquid scintillation spectrometer with a count 15 min, and the resulting supernatant was centrifuged at 105,000 x g ing efficiency of 42%. The glass filters were also placed in 8 ml Aquasol, for 90 min to obtain the cytosol (supernatant). Protein content was and the residual radioactivity was determined. Specific binding was obtained by subtracting nonspecific from total bound radioactivity and determined by the method of Lowry et al. (28). expressed as ("molesper mg protein. The labeled ligands for determining ER and PR concentrations were [3H]R2858 and ['HJR5020, respectively. In a standard assay, 0.2-ml Statistical Analysis. The significance of differences among the results was determined using the Duncan multiple comparison test. aliquots of cytosol were incubated with 0.1 ml labeled R2858 or R5020 in Buffer B. Cytosols were incubated with saturating concentrations of ligands (10 nin [3H]R2858 or 20 nM [3H]R5020) in the presence or RESULTS absence of a 200-fold M excess of the appropriate unlabeled synthetic steroid (cf. Ref. 29). Incubations were conducted for 5 h at 23Â°C(ER) Cervicovaginal Tract or 4 h at 0-4Â°C(PR), which represent optimal exchange conditions Histopathology. Cervicovaginal histopathology was analyzed (30-32). Time-course studies have shown that these times represent at 2 and 15 months in regard to occurrence of the following

Fig. 1. A, cervix (left) and upper vagina from a control intact 15-month-old female BALB/c mouse. H&E. x 40. Bar, 200 urn. B, cervix from an intact 15-month- old female BALB/c mouse given Sx 10~' ^g DES daily for the first 5 days after birth. Note the adenosis-like lesion (right). H&E. x 85. Bar, 100 >im. C. cervix and upper vagina from an intact 15-month-old female BALB/c mouse given IO'3 ^g DES daily for the first 5 days after birth. Note hyperplastic stratified squamous epithelium and downgrowths, especially in the cervical region. H&E, x 17. Bar, 400 Â¡Â¡m.D,vagina from an intact 15-month-old female BALB/c mouse given 5 x 10"'"' fig DES daily for the first 5 days after birth. Note invasive lesion (Â¿).Masson's trichrome, x 130. Bar, 100 Â¡an.

4166

Table 1 Incidence of cervicovaginal abnormalities in 2-month-old female Table 3 Overall incidence of vaginal concretions infernale BALB/cCrgl mice BALB/cCrgl mice neonatally treated with DES for 5 days neonatally treated with DES for 5 days No. of mice with Dose of No. of mice with (xgDES)5 females(%)Intact343218322022472426262676Ovariectomized421317362526214410(29)4(13)2(11)1 vaginal concretions Cervicovaginal Dose No. of Hyperstratification" downgrowths and (xg DES) females survival lesions (%) x10-'2 x10-'io-15 A. Intact 5 xIO'12 xIO'2io-25 xIO"'10-'5 15(100) 7(47) 1(34)4(20)4(18)1(2)0(0)0(0)0(0)0(0)0(0)6(14)2(15)4(24)8(22)2(8)3(12)0(0)0(0) 4(80) 2(40) xIO'3io-35 xIO'2io-25 4(67) 4(67) 13(87) 2(13) xIO-"io-45 xIO'3io-35 10(100) 0(0) 5(100) 0(0) x10-'io-'Control5 xIO"1io-15 11 (50) 1(5) 0(0) 0(0) x10-'io-5Control15561510522121212122883100100100100100100100100100100%0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(4) 0(0) xIO"12 B. Ovariectomized x10-'io-'5 5 x10-'2 xIO'2io-25 x10-'10-'5 xIO-3io-3ControlNo. xIO-2io-25 (77)9(90)6(40)0(0)0(0)10(67)2(33)6(60)10(56)0(0)0(0)0(0)0(0) xIO"3io-3ControlIS61018101510166010010095831009110014(93)6(100)8(80)14

controls. Doses higher than 10 2 /Â¿gDES resulted in an en " More layers of epithelial cells than are usually seen in the controls. hanced incidence of epithelial abnormalities (Table 1A). At 2 Table 2 Incidence of cervicovaginal abnormalities in 15-month-old female months of age, the DES-treated Ovariectomized females had a BALB/cCrgl mice neonatally treated with DES for 5 days significantly greater incidence of epithelial hyperstratification at all doses above 10~3MgDES (Table IB). As in intact females, A. IntactDose(Mg doses greater than IO"2Â¿tgDEStended to increase the incidence of mice with cervicovaginalsurvival63156747409492837979SO94Downgrowthsof downgrowths and lesions. offemales1048841623101111744No. Fifteen-month-old intact females, regardless of the amount DES)5 (%)8(80)3(75)2(25)6(75)3(75)14(%) Lesions x10-'2 of DES injected, had a high incidence of vaginal downgrowths x10-'io-'5 (Table 2A). Neonatal doses higher than 5 x 10~s Mg DES

xIO'2io-25 resulted in a significantly increased incidence of cervicovaginal lesions. xIO'3io-35 (88)23(100)7(70)8(73)9(82)7(100)28 DES-treated Ovariectomized female mice, 15 months of age, showed a significantly greater incidence of hyperstratification xIO-410-5 at all doses greater than 5 x 10~3 Â¿igDES (Table 2B). The x10-*io-'ControlNo. incidences of downgrowths and of lesions tended to increase at neonatal doses greater than 5 x 10"' and IO"2 ^g DES, respec (64)8(80)3(75)4(50)6(75)2(50)5(31)12(52)2(10)3(27)1(9)1(14)1(2)B. tively. OvariectomizedNo. Vaginal concretions were present in all DES treatment cervicovaginalDosekg of mice with groups, regardless of ovariectomy, which received neonatal doses higher than 10~3 ng DES daily (Table 3). Concretions offemales87791391017 fication(%)7(88)6(86)7(100)8(89)5(38)2(22)1(10)1(4)Down-growths(%)1(12)3(43)2(28)4(44)2(15)1(11)1(10)0(0)Lesions(%)0(0)1(14)1(14)3(33)0(0)0(0)0(0)0(0) DES)5 never occurred in control mice of any age. xIO'12 Receptors. The effects of increasing doses of neonatal DES xIO-'io-'5 treatment on cytosolic receptor levels at different ages in the vagina are shown in Fig. 2. ER levels were decreased at doses xIO-2io-25 greater than 10~2(1 and 2 months) or 10~3 (6 and 12 months) xIO-3io-3ControlNo. Mg DES. In contrast, the higher doses of DES (>10~2 ^g) increased PR at all ages. In control animals, the levels of + 10Â°%survival4710010053558210094+ 82Hyperstrati Â°Mean Â±SE. cytosolic ER and PR in the vagina were similar at all ages. In the Ovariectomized mouse series (Fig. 3), the ER levels were not changed, but the PR levels were again significantly higher features: vaginal hyperstratification/cornification, wherein the with the IO"2- and 1-Mgdoses of DES. vaginal epithelium preponderantly included an additional 4-10 cell layers (often cornified) over those usually seen in the Uterus appropriate controls; epithelial downgrowths typified by an irregular stratum basale with interruptions of the basal lamina; Histopathology. The major uterine epithelial change observed cervicovaginal lesions, large hyperplastic and metaplastic epi was the appearance of epithelial stratification ranging from a thelial foci, predominantly squamous cell carcinoma-like in pseudostratified columnar luminal epithelium to severe squa- appearance, with occasional glandular elements (Fig. 1). mous metaplasia of this epithelium (four or more layers) also DES-exposed intact females, 2 months of age, generally involving one or more glands (Fig. 4Â£>).At 2 months of age, exhibited significant stratification/cornification compared with intact mice showed a significant amount of epithelial stratifi- 4167 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1987 American Association for Cancer Research. RECEPTOR/GENITAL TRACT CHANGES AFTER NEONATAL DES

700|- Vagina DER 500- fcntorJL 400 Jl:1 300 TÃ•*_LJ*._!_ 200 100 (4THÃ±DFnâ€¢R* 4-St

2 MONTHS Uterus w 400 JL JL J, * JL T 900 300 c 200 E 700

| '00 500 a. bLo 300

o 6 MONTHS * 100 E 400 X JL Â« 300 C IO"1-2 IO'1 1 C IO" 10" 1 200 Neonatal Treatment with DES (days 1-5) sig Fig. 3. Effects of neonatal DES treatment on the levels of ER and PR in the 100 uterus and vagina of 2- (left) and 6- (right) month-old BALB/c mice ovariectomized at 30 days. Numbers at bottom of columns [mean Â±SE (bars)], number of U experiments (mice per experiment). â€¢,significant difference from control (P < 0.01). MONTHSJ_4h~~ 12 observed with age [approximately, 1000, 750, 650, and 650 fmol/mg protein at 1, 2, 6, and 12 months of age, respectively, 400 in the first series (Fig. 5); 550 and 300 fmol/mg protein at 2 300 *LX_i4Tug of age: hyperplastic alveolar-Iike dysplasias (Fig. 4/4) and the presence of an aberrant secretory state (Fig. 45). This latter Fig. 2. Effects of neonatal DES treatment on the levels of ER and PR in the condition was marked by abnormally dilated and "beaded" vagina of 1-, 2-, 6-, and 12-month-old BALB/c mice. Numbers at bottom of columns, [mean Â±SE (bars)], number of experiments (4-8 mice/experiment). â€¢, ducts and enlarged cystic lobuloalveolar structures (cystic alveo significant difference from control (P < 0.02). lar adenosis). cation at most dose levels higher than 5 x 10~3fig DES (Table All ovariectomized animals regardless of neonatal treatment 4). All mice receiving doses greater than 5 x 10~3MgDES had consistently exhibited mammary gland development character istic of the castrate animal: minimal development of a fine duct significantly stratified uterine epithelium as compared with system and no terminal buds or alveoli. Consequently, the data ovariectomized controls (Table 5). The threshold dose for the from these groups have been omitted from Table 6. The inci appearance of significant uterine stratification was 10~3^g DES dence of mammary dysplasias is highest in the 10~3-^ig DES in intact mice at this age. All neonatal doses of DES greater group in Table 6 and is significantly different from controls than 10~3 Mg DES resulted in a highly significant increase in and from groups receiving 2 x 10~', 10~', 5 x 10~3, and 10~3 stratification in intact animals (Table 5). The incidence of Mg DES. Two nodule "ghosts," the apparent remnants of re stratification was increased markedly at 15 months in ovariec gressed nodules, were identified in control animals. The inci tomized mice. dence of the abnormal secretory state is significantly greater Receptors. Fig. 5 illustrates the effects of increasing doses of than in the controls at all DES doses higher than 10~4ng (Table neonatal DES treatment on the cytosolic receptor levels in the 6). One control animal also exhibited an abnormal secretory uterus. The highest neonatal dose (l MgDES) consistently and state. significantly decreased ER and PR levels until 12 months of age when there was no longer a difference when compared with controls. ER was also decreased by lower doses of DES (10~2 Adrenals and 10"' /jg) at 1 month of age, and PR was reduced by these Adrenals in older intact and ovariectomized mice exhibit doses at 6 months of age. In ovariectomized mice, only the 1- dense circummedullary ceroid deposits, B-cell nodular hyper- ng dose at 1 month of age significantly lowered ER levels, and plasia, and spindle-cell proliferation in the zona glomerulosa, no significant effect was seen on PR levels at any dose. regardless of DES exposure. Adrenocortical carcinoma was In control animals, PR levels in the uterus were similar at 1, evident in about 30% of the control mice and in about 15% (8- 2, and 6 months of age. However, a decline in uterine ER was 57%) of the DES-treated mice. 4168

Fig. 4. A, mammary gland whole mount from an intact 15-month-old female BALB/c mouse given 5 X 10 ' jig DES for the first 5 days of life. Note the hypcrplastic alveolar nodule (HAN). Iron-hematoxylin, x 22. Bar, 400 ^m. B, mammary gland whole mount from an intact 15-month-old female BALB/c mouse given 10'* fi%DES for the first 5 days of life. Note the lobuloalveolar development and ductal dilation. Iron-hematoxylin, x 22. Bar, 400 Â¿im.C,transverse section of a uterus from a control 15-month-old female BALB/c mouse. Note the normal columnar epithelium of the uterine glands. H&E, x 85. Bar, 100 Â»im.D,transverse section of a uterus from a 15-month-old female BALB/c mouse given 5 x 10"' ng DES for the first 5 days of life. Note squamous metaplasia of uterine glands. H&E, x 130. Bar, lOO^m.

Table 4 Incidence of uterine epithelial stratification in 2-month-old female Table 5 Incidence of uterine epithelial stratification in 15-month-old female BALB/cCrgl mice neonatally treated with DES for 5 days BALB/cCrgl mice neonatally treated with DES for 5 days No. of of mice of of mice No. of of mice of of mice Dose ovariectomized with uterine intact with uterine Dose ovariectomized with uterine intact with uterine (MgDES)females5 stratification(%)0(0)1 females145615105221212121227No.stratification(%)4 (fig DES)females5 stratification(%)4 females1048841623101111744No.stratification(%)9 x10-'2 (29)Â°3 x10-'2 (57)"2 (90)"4(100)Â°7 xIO"110-'5 (25)*1(10)0(0)0(0)0(0)0(0)0(0)No.(60)Â°0(0)4 xIO'110-'5 (27)Â°5(71)"2 (88)Â°6 xIO'2io-25 (27)Â°6 x10~2io-25 (22)*3 (75)Â°4(100)Â°12(75)Â°7 (60)Â°0(0)0(0)0(0)0(0)0(0)0(0)0(0) (23)f0(0)0(0)0(0)No. xIO-3io-35 xIO"3io-35 (30)Â°0(0)0(0)0(0)0(0)0(0) xIO-1io-15 xIO"1io-45

xIO-5io-'Control124101710151016No. xIO'5io-'Control77791391027No.

* Significantly greater than controls, at P < 0.005 (x2). Â°Significantly greater than controls, at P < 0.005 (x2). * Significantly greater than controls, at P < 0.05 (x2). * Significantly greater than controls, at P < 0.05 (x2). c Significantly greater than controls, at P < 0.01 (x2).

Ovary DISCUSSION In about 30% of normally cycling female mice, ovaries con tain corpora lutea (Tables 7 and 8). Neonatally DES-treated The present study collates the differential responses of var mice exhibit an anovulatory syndrome characterized by few ious organs of female mice of the BALB/c strain to neonatal follicles, ceroid deposition, and a lack of corpora lutea. The exposure to graded doses of diethylstilbestrol, a synthetic estro threshold dose for the anovulatory syndrome was 5 x 10~3 pg gen. The general histopathological changes recorded are the DES at 2 months of age (Table 7) and 5 x 1(T4 Mgat 15 months same as those reported since 1962 as occurring following neo of age (Table 8). natal exposure to sex steroids (3). In addition, the nature of the 4169 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1987 American Association for Cancer Research. RECEPTOR/GENITAL TRACT CHANGES AFTER NEONATAL DES Table 7 Incidence of corpora lutea in 2-month-old intact female BALB/cCrgl MONTHrjER*-_L_JL4.-JLT~L5T3JLDPRT5*T_L3fh3X mice neonatally treated with DES for 5 days 1000750 Dose of of mice with ovaries (MgDES)5 females15561595211212121226No.bearing corpora lutea(%)0(0)0(0)0(0)0(0)0(0)0(0)3(14)1(8)1(8)2(17)3(25)8(31) xIO'12 XT x10-'10-'5 5002501TpH'T xIO-2io-25

il n ir n~ n xIO'3io-M5

MONTHS750500250---TJL4TL3JL_L4TJ_4Li3T_E_2 xIO-1io-15 xIO'5io-*ControlNo.

*4T Â°Not significantly different from control, IO"3and lower doses. xT5 Table 8 Incidence of corpora lutea in 15-monlh-old intact female BALB/cCrgl mice neonatally treated with DES for 5 days Dose of of mice with ovaries OigDES5x )females104784162310* bearing corpora lutea(%)0(0)"0(0)1 10-2 l x10-10~5x a (14)"0(0)0(0)0(0)*2(9)0(0)4(36)3(27)3(43)16(36)Â° o 10-10-5x 6 MONTHS "-J_Cj_i_L4i}JLaiÂ»n_i._L314x10-10-5 x10-10-5x 1111â€¢j44No. *Â« 10-10-ControlNo. 50O- ., TJ_t-rT4rS*Fi312 -r- "K

250 - " One female with granulosa cell tumor. '' Two females with granulosa cell tumors. c Not significantly different from control, 10~4and lower doses.

change essentially agrees with that observed after antenatal exposure to DES (9, 18). The doses of DES needed to induce MONTHS--JLestradiol, owing to the inability of murine a-fetoprotein to bind the synthetic 500- 1iTÃŒIO'4iiitIO'3aJILÂ»IO'2T4IO'1T4testrogen and thus protect the neonate (37, 38). A series of thresholds can be derived from the appearance of 250- various changes in the vagina, uterus, mammary gland, and ovary. Fig. 6 presents these thresholds in diagrammatic fashion. Ovary-dependent (hypothalamus/hypophysis-mediated) lesions increase significantly (P < 0.005) in the vagina of 15-month- old mice with as little as 10~4Â¿igdailyfor 5 days; 5 x 10~2 jÂ¿g Neonatal Treatment with DES (days 1-5) jug are required to induce ovary-independent (direct) lesions in the vagina at 15 months of age. In the uterus, 10~3tig DES induced Fig. 5. Effects of neonatal DES treatment on the levels of ER and PR in the uterus of I-, 2-, 6-, and 12-month-old BALB/c mice. Numbers at bottom of significant epithelial stratification in 15-month-old intact mice; columns [mean Â±SE (oars)], number of experiments (4-8 mice/experiment). Â«, a 10-fold higher dose was affective in ovariectomized mice significant difference from control (P < 0.02). (direct effect). Based on the occurrence of corpora lutea in the ovary, anovulatory conditions result from as little as 5 x 10~4 MgDES at 15 months of age (5 x 10~3 Â¿tgDES at 2 months). Table 6 Incidence of mammary gland abnormalities in 15-month-old intact Significant hypoplasia in the mammary gland is seen with 10 ' female BALB/cCrgl mice neonatally treated with DES for 5 days pg or larger amounts of DES at 15 months of age; lower doses No. of mice with of mice with (threshold = 5 x 10~4Â¿igDES) stimulate significant abnormal Dose No. of hyperplastic alveolar abnormal (MgDES)(%)5x females nodule-like lesions secretory state(%)5 secretory activity (Table 6). These effects are ovary dependent 10-2x (56)Â°2 (3, 39). 10-10-5 (25)Â»2 (50)Â°5 The present data demonstrate that neonatal treatment of (25)'1(13)0(0)5 (63)Â°4 x10-10-5 (50)Â°3 mice with DES can quantitatively modify ER and PR levels in (75)Â°12(86)Â°18(90)Â°3the female genital tract. While recognizing that the cytosolic x10-10-5 (36)"9 (45)"2(20)2(18)1(19)0(0)2(4)No. and nuclear compartmentalization of receptors is probably x10-10-5x (30)"2(18)0(0)1(14)1(2)largely artificial (40) we discuss our cytosolic receptor data without reference to this situation and consider the cytosolic 10-94884142010111110-Control levels as a reflection of the total tissue levels. Consistently from 1-12 months of age in the vagina, and from 1-6 months of age 451(11)1 " Significantly greater than control, at P < 0.005 (x2). in the uterus, cytosolic ER was decreased, as also may occur in * Significantly greater than control, at P < 0.05 (x2). the rat uterus (41-43; for exception, see Ref. 44), whereas PR 4170

DESyug/doy for 5 days the increased PR level could reflect a real sensitivity to proges 1 IO"1 IO"2IO"3 IO'4 IO"5 tin. l 1 1 1 1 1 If the higher doses of DES result in more severe epithelial VAGINA lesions in the vagina and uterus, the failure of these doses to 2 months hyperstratificotion cause PR decrease in the uterus at 12 months of age may reflect downgrowths the development of uterine squamous metaplastic lesions com lesions parable to the vaginal lesions. Thus, if the uterus had been hyperstrat if Â¡cation assayed at older ages (>15 months) when the metaplastic le downgrowths sions were even more extensive, a significant rise in PR might lesions have been seen, as occurred in the vagina at younger ages. 15months hyperstrat if Â¡cation The difference in response observed between the uterus and downgrowths vagina in our study is comparable to the results of a previous lesions report from our laboratory (32) indicating that animals neona .concretions hyperstrat if Â¡cation tally treated with high doses of estradiol (20 ng) exhibit lower downgrowths vaginal nuclear estrogen-binding capacity compared with un lesions treated animals but that uterine nuclear ER levels in estrogen- concretions treated animals were not different from controls. The fact that MAMMARY GLANDS uterus and vagina respond differently to hormonal stimulation 15 months {hyperplastic alveolar has been demonstrated by Krueger et al. (50) studying DNA nodule replication in the rat vagina and lower fornix following estrogen abnormal secretory and progestin treatment. Forsberg (51 ) also obtained differen state tial results when studying the mitotic rate in the uterus, vagina, OVARY and cervix of neonatally estrogen-treated mice. In contrast, 2 months( absence of corpora however, to Krueger's results in the adult rat, Forsberg (51) 15months{ lutea UTERUS reported that 18 h after administration of 5 Â¿tgestradiol the 2 months mitotic rate was increased in the uterus but decreased in the stratification vagina and cervix, indicating that the cervix responds in a manner similar to the vagina but differently from the uterus. 15 months The persistent alterations of ER and PR levels observed stratification through 6 months of age in the uterus and 12 months of age in the vagina are difficult to explain. Neonatal DES may influence the ontogeny of ER and PR resulting in permanent changes in Fig. 6. Diagrammatic summary of minimal daily neonatal dosage of DES needed to induce change tested. ER and PR levels later in life (a direct effect of DES on the genital tract) (43). Aihara et al. (29) have shown that in 2- was decreased in the uterus and increased in the vagina of mice month-old mice, neonatal estradiol administration reduces the treated neonatally with high DES doses, as compared with levels of uterine cytosolic ER and that the induction of new controls. Thus, neonatal DES exposure disrupts the ER/PR uterine ER under the influence of estrogen may be altered by balance in the vagina, which may lead to an abnormal response neonatal estrogen treatment. Results from our laboratory indi of this organ to hormonal stimulation later in life. It has been cate that neonatal estradiol treatment alters ER and PR levels reported (44, 45) that perinatal DES acts to influence the in the uterus and vagina in a manner similar to that observed with high doses of DES.5 Another possibility is that the neu- ontogeny (before 30 days of age) of cytosolic ER and PR in the vagina and uterus of mice and modifies the nuclear retention roendocrine pathways are permanently altered by neonatal es of ER and PR in both tissues. Our data further demonstrate a trogen administration (39, 52, 53) and that later changes are persistence of the cytosolic receptor modifications throughout mediated through a modified hypothalamo-hypophysio-ovarian the first 12 months of age in the vagina and through at least axis (53-57). Changes may also reflect, in part, alterations in the first 6 months of age in the uterus. In the mammary gland tissue composition of the organ under study (for example, also (46), a high dose of DES administered neonatally results extensive epithelial lesions develop progressively in the vagina in significantly decreased ER (at 1, 2, 6, and 12 months of age). after neonatal estrogen treatment; cf. RÃ©f.3). In contrast, The decrease in cytosolic ER associated with decreased PR may neonatal exposure of hamsters to DES did not result in any change "in the physiochemical, functional properties of the indicate that neonatal DES down regulates ER in the uterus uterine estrogen receptor system" (58). without nuclear accumulation, as Aihara et al. (29) suggest. A somewhat different situation is observed in the vagina, but since nuclear receptors were not measured in this study, we do not ACKNOWLEDGMENTS know whether the decrease in cytosolic ER is followed by a nuclear accumulation, as the concomitant increase in PR levels We are indebted to Jimmy K. L. Choy for technical assistance, to would suggest (47). Aniko Mos for microtechnique, and to Suzie Castillo for manuscript preparation. The increase in PR accompanying the decrease in ER in the vagina should also occur in the uterus, if endogenous estrogen levels are acting to up-regulate PR. The inability to generalize REFERENCES this phenomenon leaves such an explanation unlikely. It should 1. Herbst, A. L., Ulfelder, H., and Poskanzer, D. C. Adenocarcinoma of the be noted that the persistently cornifying vagina is responsive to vagina: association of matemal stilbestrol therapy with tumor appearance in progestin administration in adult life (48); in addition, postnatal young women. N. Engl. J. Med., 284: 878-881, 1971. progesterone administration reduces the severity of the histo- 2. Herbst, A. L., and Bern. H. A. (eds.). Developmental Effects of Diethylstil- pathological changes induced by neonatal estrogen (49). Thus, 1M. Edery, K. T. Mills, L. Larson, and H. A. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1987 American Association for Cancer Research. Long-Term Alterations in Histology and Steroid Receptor Levels of the Genital Tract and Mammary Gland following Neonatal Exposure of Female BALB/cCrgl Mice to Various Doses of Diethylstilbestrol

Howard A. Bern, Marc Edery, Karen T. Mills, et al.

Cancer Res 1987;47:4165-4172.

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