Vaginal Adenosis and Adenocarcinoma in Mice Exposed Prenatally Or Neonatally to Diethylstilbestrol

[CANCER RESEARCH 42, 2003-2011, May 1982]

Vaginal Adenosis and Adenocarcinoma in Mice Exposed Prenatally or Neonatally to Diethylstilbestrol

Retha R. Newbold and John A. McLachlan1

Transplacental Toxicology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences. NIH. Research Triangle Park, North Carolina 27709

ABSTRACT carcinoma is low in these DES-exposed females, other genital abnormalities are common. These include the presence of The association of intrauterine exposure to diethylstilbestrol glandular epithelium or its mucinous products in the vagina (DES) and the subsequent development of reproductive tract (vaginal adenosis) or in the portio vaginalis of the cervix (cer abnormalities in young women has been well documented. vical ectropion). Vaginal adenosis and cervical ectropion are Although the incidence of vaginal adenocarcinoma was low in abnormalities observed in unexposed females, including fe the exposed population, vaginal adenosis, a nonmalignant ab tuses and neonates (21); however, they are encountered more normality, was quite common. In order to study the pathogen- often and to much greater extent in prenatally DES-exposed esis of adenocarcinoma and to determine the frequency of women (34). adenosis following prenatal exposure to DES, timed pregnant The frequency of adenosis in women according to several CD-1 mice were treated s.c. with DES (dose range, 5 to 100 studies (2, 18, 36) ranged from 35 to almost 90% of the jug/kg/day) on Days 9 through 16 of gestation. This period exposed population. In addition to this reported high incidence, corresponds to major organogÃ©nesis of the reproductive tract interest in this lesion lies in the hypothesis several investigators in the mouse. Female offspring were sacrificed between 1 and have developed that vaginal adenosis is a premalignant stage 18 months of age. In addition to nonmalignant abnormalities, of adenocarcinoma (3, 14, 20, 34). In a recent report, Antonioli some of which have been described in women exposed pre- ef al. (1) suggested a possible relationship between the 2 natally to DES, two cases of vaginal adenocarcinoma (2%) lesions by showing transitional states between adenosis and were observed in 91 prenatally DES-treated animals. No com adenocarcinoma in the upper portion of the vagina of prenatally parable epithelial lesions were seen in 158 control female mice. DES-exposed human females. One other case of adenocarcinoma of the vagina was reported Reports from this laboratory have described an animal model previously by this laboratory using the prenatally exposed using the CD-1 mouse to evaluate the transplacental effects of animal model. DES (23-26) including the long-term effects on the female In another series of mice treated prenatally with DES, 100 mouse genital tract such as vaginal adenocarcinoma; only one jug/kg/day, 3 of 20 (15%) 1-month-old animals and one of 10 case of vaginal adenosis was demonstrated in these prenatally (10%) 18-month-old treated offspring had glandular epithelium DES-exposed mice (26). Until Plapinger and Bern (30) reported abnormally located in the vaginal fornices (adenosis). Other adenosis-like lesions in the cervicovaginal region of several cervicovaginal abnormalities observed after prenatal DES ex strains of mice, DES- or estradiol-associated vaginal adenosis posure included structural alterations, cervical enlargement, was thought to be confined to the NMRI mouse (11). squamous metaplasia in the endocervical canal, excess kera- The present study was undertaken to provide information on tinization of the ectocervix and vagina, transverse folds and the incidence of adenosis and the relationship of this lesion to basal cell hyperplasia in the upper vagina, and prominent adenocarcinoma in the transplacentally DES-exposed mouse Wolffian duct remnants. Thus, vaginal adenosis in the mouse as well as to compare the incidence of neonatally induced does not appear to be a common abnormality following treat adenosis in the CD-1 mouse strain used in this study with that ment with DES in utero. Neonatal exposure to DES on Days 1 already published (26). to 5, on the other hand, resulted in six of eight (75%) animals with adenosis at 35 days of age. Since perinatal mouse studies have reported high incidences of vaginal adenosis but, to our MATERIALS AND METHODS knowledge, no cases of vaginal adenocarcinoma, the results presented in this report suggest that the stage of cellular Outbred CD-1 mice were obtained from Charles River Breeding differentiation at the time of DES exposure may be critical in Laboratories, Inc., Wilmington, Mass., and were bred to male mice of the final expression of these abnormalities. the same strain in the animal facility at the National Institute of Environ mental Health Sciences. Vaginal plug detection was considered Day 0 of pregnancy. Pregnant mice were housed separately in a room with INTRODUCTION controlled temperature (21-22Â°) and lighting (14-hr light and 10-hr dark periods) and were provided with synthetic bedding, fresh water, The long-term consequences of transplacental exposure to and NIH 31 laboratory mouse chow ad libitum. DES2 on the developing female fetus were originally described Prenatally DES-treated Animal Experiments. Pregnant female mice by Herbst et al. in their report of clear cell adenocarcinoma in were given s.c. injections of DES (Sigma Chemical Co., St. Louis, Mo.) adolescent women (17, 19). Although the incidence of adeno- dissolved in corn oil on Days 9 through 16 of gestation. The daily dose of DES was 100 Â¿ig/kg body weight; the offspring of these animals are ' To whom requests for reprints should be addressed. referred to in this report as DES-100. The total volume of corn oil 2 The abbreviation used is: DES. diethylstilbestrol. administered was 0.01 ml/g maternal body weight. Purity of the DES Received March 25, 1981 ; accepted February 8, 1982. was checked by thin-layer chromatography, high-pressure liquid chro-

MAY 1982 2003

matography, and gas chromatography-mass spectrometry and ex nants (Fig. 1) observed in serial cross-sections of the cervico ceeded 99% (27). vaginal region. The incidence of these remnants in 1-month- Pregnant mice delivered their young, and all litter sizes were ran old animals was 11 in 20 and in 18-month-old animals was 3 domly reduced to 8. At 25 days of age, the mice were weaned and in 10; the ductal remnant in one of the 18-month-old animals housed in groups of 5/cage. At 1 month (20 control and 20 DES-100) or 18 months of age (10 control and 10 DES-100), female offspring was cystic. In contrast, no prominent Wolffian remnants were were sacrificed by cervical dislocation. Only one to 2 mice per litter observed in this series of control females. were included in each group. Reproductive tract tissues were removed, Another common feature observed in these mice after in fixed, embedded, and serially sectioned according to the method utero exposure to DES was excessive vaginal keratinization. described by Plapinger and Bern (30). Briefly, a transverse cut was The squamous epithelium throughout the vagina of most DES- made through the cranial cervix approximately 0.5 mm caudal to the 100 mice was composed of many layers of immature and junction of the uterine horns. The portion of the reproductive tract mature cells covered by sheets of keratin. Leukocytes were caudal to that cut was fixed in Bouin's solution. The fixed tissue was commonly seen in the superficial layers. In 4 of 10 (40%) 18- embedded in paraffin and 6-fim serial transverse sections were made, month-old animals, the increase in keratinization combined starting at the cranial end and stopping after approximately three- with basal cell hyperplasia resulted in irregular rete pegs of fourths of the tissue was sectioned. This procedure provided serial epithelium which extended into the subjacent stroma (Fig. 2). sections through the cervical canal, vaginal fornices, and upper por tions of the vaginal canal. A light microscope was used to evaluate In another case, the vaginal epithelium formed papillary projec hematoxylin and eosin-stained sections of reproductive tract as well as tions into the lumen (Fig. 3). representative sections of liver, lung, kidney, adrenal, and spleen. Urethral openings, abnormally located anterior to the vulva Cervical measurements were made as described previously (26). (persistent urogenital sinus), were found in 20% of the 1- An additional series of pregnant animals was similarly exposed to month-old DES-100 animals. "Gland-like structures" associ daily s.c. DES doses of either 5, 10, or 100 M9/kg of body weight on ated with this abnormality were of urothelial origin. Days 9 to 16 of gestation. Their offspring are referred to as DES-5, All of the prenatally DES-100 mice showed hypertrophy in DES-10, and DES-100, respectively. Animals sacrificed included 158 the structure of the portio vaginalis (cervix) (Chart 1). The controls (72 six- to 11-month-old mice, 53 twelve to 18 months old, 33 outside diameter of the cervical area in 1-month-old animals older than 18 months), 29 DES-5 (zero to 11 months old, 26 twelve to averaged 3.6 Â±0.5 (S.D.) mm for DES-100 compared to 2.85 18 months old, 3 older than 18 months), 16 DES-10 (zero 6 to 11 months old, three 12 to 18 months old, 13 older than 18 months), and Â±0.7 mm for controls and at 18 months averaged 4.37 Â±0.45 46 DES-100 (19 six to 11 months old, 17 twelve to 18 months old, 10 mm for DES-100 and 2.8 Â±0.4 mm for controls. Histological older than 18 months). evaluation of tissues from this area showed that both the stroma The reproductive tract tissues of these animals were fixed in 10% and epithelium were affected by DES and contributed to the neutral buffered formalin, embedded in paraffin, and serially sectioned enlargement seen macroscopically. Stromal edema in this area at 6 firn as described previously (26). In this study, the vagina and was seen in 75% of the 1-month-old and 80% of the 18-month- cervix were cut in the midsagittal plane. Both halves were embedded old animals. In cross-section, the epithelial mucosa of the DES in the same block and were serially sectioned sagittally for 10 sections animals had fewer folds, and those that were present were less starting at the sagittal midline. If a microscopic lesion was observed, convoluted and shallower than were those in control tissues. In additional serial sections were made to include the entire area of some animals, the uterine and cervical lumen appeared as a pathological change. In addition to reproductive tract, representative simple tube. Squamous metaplasia in the endocervical canal in sections of liver, lung, kidney, adrenal, and spleen were stained with hematoxylin and eosin. conjunction with excessive keratinization in some cases was Neonatally DES-treated Animal Experiments. Eighteen untreated observed in 45% of the 1-month-old and 80% of the older pregnant female mice, housed individually, delivered their young on animals. Day 19 of gestation. All litter sizes were adjusted to 8 at birth. Ten Table 1 litters were untreated (control), and 8 litters were given DES dissolved Abnormalities of cervicovaginal area in female mice exposed prenatally to DES in corn oil as a s.c. injection on Days 1 through 5 of neonatal life (2 /ig/pup/day in 20 /il corn oil). These animals were maintained and abnormalitiesControlAbnormalityStructural No. of mice with weaned by the same schedule described for the prenatally exposed animals. At 35 days of age, 10 control and 8 DES-exposed females, each from a different litter, were sacrificed by cervical dislocation. mo.0/200/200/200/200/200/200/2018mos.0/100/100/101/100/102/100/10DES"1mo.17/20C15/20C9/20d16/20C0/2089/201mos.9/1 Oc8/1 Reproductive tract tissues were processed by the method described alterationsCervical Od8/1 enlargementSquamous by Plapinger and Bern (30). 0"9/1 inendocervicalmetaplasia canalExcessive Od4/102/10Â°3/10" RESULTS inectocervixkeratinization vaginaBasal and upper vaginawithcell hyperplasia in Cervicovaginal Changes in Female Mice Exposed Prena ofepitheliumLeukocyticirregular rete pegs tally to DES. The genital tract of prenatally DES exposed infiltrationProminent females had numerous cervicovaginal abnormalities in 1- and 1/20C18 Wolffian remnants1 18-month-old animals. The prevalence of abnormalities of the a Females were the offspring of CD-1 mice treated on Days 9 through 16 of vagina ranged from 0 among control mice at 1 month and 20% gestation with DES. 100 ^g/kg/day. Tissues were processed by the method of at 18 months to 85% at 1 month and 90% at 18 months of age Plapinger and Bern (30). Defects in MÃ¼llerianduct fusion and vaginal fornix formation. in the prenatally DES-100 exposed females (Table 1). The Statistical significance of DES-exposed animals to corresponding age- abnormalities in control animals at 18 months were consistent matched controls by Fisher exact test. with usual age-related changes (26) whereas the changes in c p < 0.0001. 0 p < 0.001. the DES-100 animals at 1 and 18 months were not. These e Not significant. DES-related changes included prominent Wolffian duct rem ' p = 0.04.

2004 CANCER RESEARCH VOL. 42

1a -^

Fig. 1. a, cross-section through the common cervical canal of a 1-month-old mouse exposed prenatally to DES. Lining epithelium is cornified and stroma is mildly hyperplastic. WD, the lumen of prominent remnants of the Wolffian duct. DES-100. H & E, x 15. o, higher magnification of the field shown in a. WD, Wolffian duct remnant. DES-100. H S E. x 75.

UTERINE HORN

CONTROL PRENATAL DES NEONATAL DES Chart 2. Schematic drawing of female reproductive tracts demonstrating cer Chart 1. Schematic drawing of reproductive tracts from 1-month-old female vicovaginal structural malformations. Control, untreated females (left). Prenatal CD-1 mice. Control, untreated females. Prenatal DES, females which were DES, females which were offspring derived from CD-1 mice treated with DES, offspring derived from CD-1 mice treated with DES. 100 ^g/kg/day. on Days 9 100 jig/kg/day, on days 9 to 16 of gestation. Treatment resulted in a shallow to 16 of gestation. Treatment resulted in general enlargement in the area of the vaginal fornix and a short (craniocaudal) cervical region. In the most extreme cervix. Oviductal malformations and abnormal urethral openings in the vagina case, there was lack of fusion of the MÃ¼llerianduct to form the common cervical were other abnormalities commonly observed. Neonatal DES, females which canal (right). were offspring from control CD-1 mice treated neonatally on Days 1 to 5 with DES, 2 (ig/kg/day/pup. Treatment resulted in a hypotrophic uterus which was much smaller than were any unstimulated control tissues. The vagina was single layer in the lining epithelium or forming a gland-like enlarged, but there were no cervicovaginal or oviductal malformations or abnor structure within the vaginal stroma. Columnar epithelium within mal urethral openings. the vaginal fornices (adenosis) was observed in the post-pub- ertal (1 month) as well as the older prenatally exposed animals A more striking structural abnormality was observed in serial (18 months), but in no controls. The number of animals in cross-sections through the cervicovaginal region of female volved, however, was low, and the region of involvement in mice exposed prenatally to DES. In the most severely affected each animal extended through only a few serial sections. Three cases, the MÃ¼llerian ducts did not fuse to form a common of 20 (15%) 1-month-old and one of 10 (10%) 18-month-old cervical canal. In other less severe cases, the cervix was short exposed animals had columnar cells abnormally located in the craniocaudally but still had a relatively large diameter, both vaginal fornices (Table 2). In one of the 1-month-old mice, a internal and external, with increased stromal elements. In ad layer of high columnar cells formed a cyst-like structure im dition, the vaginal fornix was always shallower than were con mediately basal to the lining epithelium (Fig. 4). The opening of trols and, in one 35-day-old animal, it was not present (Chart this gland into the vaginal lumen was observed in another 2). section. The second case of adenosis similarly demonstrated It was extremely difficult to distinguish changes in the loca columnar cells lining a subepithelial gland but with no demon tion of the squamocolumnar junction between control and strable opening into the vaginal lumen. In the third 1-month-old prenatally DES-exposed animals. Normally in the cycling con DES female, a patch of columnar cells was found in the region trol female, the squamocolumnar junction varied between ani of the vaginal fornix that is normally lined by squamous epithe mals, and the border was more or less distinct depending on lium (Fig. 5). There appeared to be early gland formation, but the stage of the estrous cycle (22). the abnormality was only observed in two 6-/Â¿mserial sections. The columnar epithelium described as adenosis in this report Vaginal adenosis that was observed in the one 18-month-old did not refer to tall mucus-producing cells present in the female resembled those described in the younger animals. superficial layer of stratified epithelium in the normal cycling Again, columnar cells formed a cyst in the stroma under the mouse vagina but rather to columnar cells arranged either in a epithelial layers. The lumen of this ' 'gland-like" cystic structure

MAY 1982 2005

Table 2 folds than did controls but more than did the prenatally DES- Incidence of adenosis in female mice exposed to DES exposed animals. In addition, vaginal keratinization was not No. of animals Mice with adenosis seen. Prenatal treatment Adenosis was observed in the neonatally exposed animals in 1-mo.-old offspring 6 of 8 DES-treated animals while it was not apparent in any of 20 0/20 (0) Control 3/20 (15)c DES- 100a 20 the controls. Adenosis occurred as columnar cells lining the 18-mo.-old offspring vaginal fornix and in some cases formed folds toward the 0/10 (0) Control 10 1/10 (10)c DES-100" 10 underlying stroma resembling glands (Fig. 11).

Neonatal treatment DISCUSSION 1-mo.-old offspring Control 10 0/10 (0) Previous reports from this laboratory described a mouse DES" 6/8 (75)" 8 model for the transplacental effects of DES on the developing Females were offspring derived from CD-1 mice treated with DES, 100 fig/ fetal female reproductive tract and the subsequent associated kg/day, on Days 9 through 16 of gestation. " Numbers in parentheses, percentage. abnormalities (21, 23, 25, 26). Vaginal adenocarcinoma was c Not significant. observed in low incidence (1 of 140 DES-treated mice, all d Females were offspring from control CD-1 mice treated neonatally on Days doses) in these experiments (26); likewise, vaginal adenosis 1 through 5 with DES. 2 /ig/day/pup, or averaging approximately 1 mg/kg/day. Tissues were embedded and processed by the method of Plapinger and Bern was a rare histological observation (1 of 140 DES-treated (30). mice). This latter observation was surprising since Forsberg ep<0.01. demonstrated a high incidence of vaginal adenosis in NMRI mice after neonatal treatment with estrogen (11); more re was more dilated than were those seen in younger animals cently, Plapinger and Bern (30) reported adenosis after peri (Fig. 6). The cases of adenosis that were described occurred natal estrogenization. in animals with only slight structural abnormalities. The number of cases of adenosis observed in this prenatally In an attempt to study the relationship between adenosis and DES-exposed animal model may have been conservative be adenocarcinoma in the vagina of mice, the second study in cause excessive cornification in the cervix and vagina, inter- cluded a larger group of animals treated in utero with various animal variability in the squamocolumnar junction in the control doses of DES. In this series of animals, the incidence of vaginal as well as in the treated animals, and other structural abnor adenocarcinoma was as follows: controls, 0 of 158; DES-5, 1 malities in the DES-exposed animals made further determina of 29; DES-10, 0 of 16; and DES-100, 1 of 46. One DES-5 tions inconclusive. Considering these problems in identifying female became moribund and therefore was sacrificed at 17 adenosis in the mouse, the differences in incidence between months of age. Observations at the time of necropsy included prenatal and neonatal exposure are still apparent. Some pos ovarian cysts and mild cervical enlargement, but no other sibilities which may explain the lower incidence of adenosis in readily apparent abnormalities. Microscopic evaluation of the the CD-1 mouse following prenatal DES treatment rather than genital tract showed gland-like structures in the submucosal neonatal DES exposure were: (a) route of exposure and dose area of the vaginal fornix. These glands were lined by well- of DES; (Ã³) differences in sectioning techniques; (c) age of differentiated mucus-secreting columnar cells with a low de mouse; (d) strain of mouse; (e) stage of genital tract develop gree of nuclear pleomorphism. Invasion into surrounding tis ment during DES exposure. In order to determine the actual sues resulted in this lesion being diagnosed as vaginal adeno occurrence of adenosis in the prenatal mouse model and its carcinoma (Fig. 7). relationship to the pathogenesis of adenocarcinoma, this report Another 17-month-old animal exposed to DES-100 had a investigated each of these possibilities. large stone in the vagina. Serial longitudinal sections through The route of exposure to DES in studies which reported the entire reproductive tract of this mouse showed an abnormal vaginal adenosis (11, 30) was direct s.c. administration to the urethral opening with urethral glands in the lower vagina. The neonatal mouse whereas the prenatal reports (26) described surface epithelium of the vagina was heavily keratinized, and s.c. injections to the mother. Thus, the direct neonatal dose of in some areas there were small squamous papillomas. These DES, averaging approximately 1 mg/kg/day, was higher than alterations and focal inflammatory changes were probably re the indirect doses (5 to 100 ^g/kg/day) used in the prenatal lated to the presence of the stone. In addition to urethral studies. Higher prenatal doses were severely fetotoxic, making glands, a gland-like structure lined by tall mucus-secreting further investigation of the relationship of adenosis to increas cells (adenosis) opened into the lumen of the upper vagina. In ing amounts of prenatally administered DES impossible. It is the area of the opening of this gland, mild focal atypia in the interesting to note, however, that 2 of 3 cases of vaginal luminal epithelium was observed. An area directly adjacent to adenocarcinoma reported in the prenatal mouse model [one in this showed changes consistent with well-differentiated squa the present report and one reported previously (26)] occurred mous cell carcinoma (Fig. 8). Additional submucosal glands in in offspring exposed to lower doses, not higher doses, of DES. the vaginal fornix, not associated with the abnormal urethral The progression to neoplasia, or at least its expression, may opening, contained areas of squamous metaplasia (Fig. 9) and be more obvious in the absence of the extensive tissue changes adenocarcinoma (Fig. 10). associated with the higher doses of DES. For example, in an Cervicovaginal Changes in Female Mice Exposed Neo animal with adenosis, columnar cells in this lesion were partially natally to DES. At 35 days of age, the uteri and cervix of replaced by squamous cells (squamous metaplasia). Clinical neonatally DES-exposed mice were hypotrophic (Charts 1 and reports have suggested that squamous metaplasia is a healing 2). There was little gland formation in the uteri resulting in process of adenosis, and in some cases adenosis was replaced slender tubular structures. The cervical mucosa had fewer completely by squamous cells (36). Differentiation of squamous

2006 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. Vaginal Abnormalities in DES-exposed Mice metaplasia into mature, stratified squamous epithelium oc by Walker (38), the higher was the incidence of adenosis. curred; this epithelium became impossible to distinguish from Although the numbers of animals in these reports were small the normal squamous epithelium of the vagina resulting in a and 2 of the strains studied by Plapinger and Bern (30) did not complete involution of adenosis (36). It is possible that high have the same temporal relationship for adenosis, these data, prenatal doses may amplify the process of squamous metapla plus observations in this current study, suggest that adenosis sia and therefore result in lower incidences of observable can be demonstrated after neonatal exposure to DES but adenosis. The development of squamous metaplasia in aden prenatal exposure results in a lower incidence of the lesion. osis has raised the possibility of increased incidence of dyspla- The exact effect of DES on genital tract development remains sia and squamous carcinoma to several investigators (15, 35). to be determined; however, studies on the differentiation of the This was not the finding, however, of clinicians following women vagina may be informative. Forsberg (12) has suggested that at Massachusetts General Hospital (33) or patients enrolled in exposure to DES during development probably interferes in the National Cooperative Diethylstilbestrol Adenosis project some manner with the replacement of the MÃ¼lleriancolumnar (32). Their data suggest that rates of dysplasia are quite low in epithelium by squamous epithelium (vaginal transformation) the DBS-exposed population. Although squamous cell carci resulting in the persistence of MÃ¼lleriancells similar to those of noma is a lesion reported in the present and other studies (5, the endocervix, endometrium, and fallopian tube in an abnor 6, 14, 37), more evidence is needed to prove it to be a mal area. Using the neonatal animal model, Forsberg (11) has significant health problem in women exposed prenatally to produced adenosis in a system where vaginal transformation DES. This lesion, however, does suggest additional reason for takes place after birth. Recently, Forsberg and Kalland (14) continued careful follow up of the exposed population. have reported development of squamous cell carcinomas and The second possibility for the lower incidence of adenosis in adenosis with squamous metaplasia in the vaginas of mice the prenatally DES-exposed mouse was the difference in sec treated neonatally with DES; in addition, these investigators tioning techniques. Data from reproductive tracts of the animals demonstrated apparent adenocarcinomas in regions of aden that were reported in the previous long-term prenatal study osis in the cervix, not the vagina, following such early postnatal (26) were cut in the sagittal midline with the cut surface exposure to DES. In our laboratory prenatal exposure, before embedded down in the paraffin block, and 10 serial sections vaginal epithelial transformation has occurred, did not produce were made. If an abnormality was observed, additional sections a high incidence of adenosis. In the 225 mice exposed to DES- were made. The possibility existed that the vaginal fornix may 5, -10, or -100 in this and a previous report (26), a total of 3 not have been reached in all of the animals; therefore, in the cases of vaginal adenocarcinoma were obtained. present study, serial sections through the entire block were A recent report from this laboratory showed that DES causes done to ensure the inclusion of the vaginal fornix. In addition, transformation in a culture system using Syrian hamster em serial cross-sections obtained by the method described by bryo cells (4). It was known that DES induced genetic damage Plapinger and Bern (30) were done in another group of pre in cells by chromosome loss since Rao and Engleberg (31) natally DES-exposed animals. The incidence of vaginal aden have shown chromosome nondisjunction in HeLa cells and osis in transplacentally treated mice determined by both tech Chrisman (7) has shown aneuploidy in 8-day-old mouse em niques was still much lower than were the reported cases of bryos after DES exposure. An important correlation can be adenosis observed after neonatal DES exposure and therefore made with these studies and a recent report which shows an does not appear to be a possible explanation. association of aneuploidy with vaginal cellular dysplasia in The age and strain of mice used were also considered as young women exposed prenatally to DES (15). possibilities attributing to the lower incidence of adenosis in There is little information about the ultimate fate of columnar the prenatal mouse model compared to the neonatal mouse epithelium in the mouse vagina other than the possibility of a model. The initial findings of Forsberg suggested that adenosis replacement of the abnormality with upgrowing squamous ep occurred only in NMRI mice (10, 11, 13). However, Plapinger ithelium (14, 25). An alternative fate of adenosis in the mouse and Bern (30) have demonstrated vaginal adenosis in perina- might be the rare development of vaginal adenocarcinoma. tally estrogenized mice in 4 strains (NCS, BALB/cCrgl, C3H/ Although various squamous epithelial changes have been ob Crgl, C57/Crgl) and at ages reported in this study. In addition, served, vaginal adenocarcinoma has not been reported follow adenosis was recently described for CD-1 mice exposed late ing neonatal or late prenatal treatments with DES (5, 9,13,14, in gestation to DES (38). Therefore, strain differences are 28, 30, 37, 38). Using this strain of mouse, if there is a unlikely to account for our observations of lower incidences of correlation between adenosis and adenocarcinoma, it would adenosis. be reasonable to expect a higher incidence of adenocarcinoma The last possibility, stage of genital tract development during at later ages in the neonatal DES-exposed animals. This merits DES exposure, seems to be the most likely explanation for the further investigation. differences observed. For example, Plapinger and Bern (30) However, the present results suggest that adenosis is a reported that the NCS mouse, with only neonatal exposure to lesion that may be inducible during the process of transfor estradiol benzoate, had an incidence of 68% adenosis in mation; adenocarcinoma may require an earlier time for induc comparison to 60% with neonatal exposure plus 1 day prenatal tion. Support for this theory can also be found in clinical studies exposure, 38% with neonatal plus 2 days prenatal exposure, because there are several reports which suggest that adenosis and 0 with neonatal plus 4 days prenatal exposure. Likewise, is not seen in women exposed to DES after the 20th week of neonatal treatment with DES resulted in 100% adenosis (30). gestation, a time when vaginal transformation has already On the other hand, prenatal treatment with ethinyl estradiol occurred in humans (18). Also, recent data from the National resulted in only 3% of adenosis in offspring (39); moreover, Cooperative Diethylstilbestrol Adenosis project indicate that the later in gestation DES was given in the small series reported vaginal epithelial changes (adenosis and squamous metaplasia)

MAY 1982 2007

rarely develop if the first exposure occurred well after the 20th REFERENCES week (29). 1. Antonioli, D. A. Glandular dysplasia in diethylstilbestrol-associated vaginal Although the time for induction may be different for these adenosis. Am. J. Clin. Pathol., 71: 715-721, 1979. lesions, it is apparent that the basic biological relationship 2. Antonioli. D., and Burke, L. Vaginal adenosis. Analysis of 325 biopsy between adenosis and adenocarcinoma of the vagina is not specimens from 100 patients. Am. J. Clin. Pathol., 64: 625-638. 1975. 3. Barber, H. R. K., and Sommers. S. C. Vaginal adenosis, dysplasia, and clear completely understood. cell adenocarcinoma after diethylstilbestrol treatment in pregnancy. Obstet. This could account for the epithelial abnormalities (basal cell Gynecol., 43: 645-652, 1975. hyperplasia with irregular rete pegs) as well as other anatomical 4. Barrett, J. C.. Wong, A., and McLachlan, J. A. Diethylstilbestrol induces neoplastic transformation without measurable gene mutation at two loci. abnormalities of connective tissue observed in this study (pap Science (Wash. D. C.), 212: 1402-1404, 1981. illary structures resembling transverse ridges in exposed 5. Bern, H. A., Jones, L. A., and Mills, K. T. Use of the neonatal mouse in studying long-term effects of early exposure to hormones and other agents. women). With abnormalities of the stroma, including stromal J. Toxicol. Environ. Health., ): 103-116, 1976. hyperplasia and edema (cervical enlargement) and neoplasia 6. Bern, H. A., and Talamantes, F. J. Neonatal mouse models and their relation in mice exposed prenatally to DES (26), the possibility of a to disease in the human female. In: A. L. Herbst and H. A. Bern (eds.), Developmental Effects of Diethylstilbestrol (DES) in Pregnancy, pp. primary stromal action of DES must be considered. 129-147. New York: Thieme-Stratton, 1981. The role of Wolffian duct-derived tissues in the abnormal 7. Chrisman, C. L. Aneuploidy in mouse embryos induced by diethylstilbestrol development of typically MÃ¼llerianstructures, such as cervi- diphosphate. Teratology, 9: 229-232, 1974. 8. Cunha, G. R. Stromal induction and specification of morphogenesis and covaginal tissues, is presently unclear. As long ago as 1959, cytodifferentiation of the epithelia of the MÃ¼llerianducts and urogenital sinus Gruenwald (16) suggested that Wolffian duct mesenchyme during development of the uterus and vagina in mice. J. Exp. Zool., 196: 361-370, 1976. contributed to the definitive stroma of the cervix and uterus 9. Dunn, T. B., and Green, A. W. Cysts of the epididymis, cancer of the cervix, (MÃ¼llerian-derived structures). The association of the hyper- granular cell myoblastoma, and other lesions after estrogen injection in plastic, retained Wolffian structures observed in the present newborn mice. J. Nati. Cancer Inst., 31: 425-454, 1963. 10. Forsberg, J.-G. The development of atypical epithelium in the mouse uterine study with the abnormally developing MÃ¼lleriantissue is an cervix and vaginal fornix after neonatal oestradiol treatment. Br. J. Exp. area of current investigation. In fact, we reported previously Pathol., 50: 187-195, 1969. 11. Forsberg, J.-G. Estrogen, vaginal cancer, and vaginal development. Am. J. (26) that cystic Wolffian duct remnants were associated with Obstet. Gynecol., 113: 83-87, 1972. vaginal sections in 63% of the 12-month-old DES-100-treated 12. Forsberg, J.-G. Cervicovaginal epithelium: its origin and development. Am. mice, while the corresponding control value was 9% (26). J. Obstet. Gynecol., 115: 1025-1043, 1973. A striking change in the cervicovaginal area of DES-treated 13. Forsberg, J.-G. Late effects in the vaginal and cervical epithelia after injections of diethylstilbestrol into neonatal mice. Am. J. Obstet. Gynecol., mice involved structural defects which included malfusion of 121: 101-104, 1975. the MÃ¼llerian ducts as well as structural alterations in the 14. Forsberg, J.-G., and Kailand, T. Neonatal estrogen treatment and epithelial abnormalities in the cervicovaginal epithelium of adult mice. Cancer Res., vaginal fornices (Chart 2). Whether these malformations play a 41: 721-734, 1981. role in the expression of epithelial lesions seen in the cervico 15. Fu, Y. S., Reagan, J. W., Richart, R. M., and Townsend, D. E. Nuclear DNA vaginal tissue of DES-exposed mice is currently being investi and histologie studies of genital lesions in diethylstilbestrol-exposed prog eny. Am. J. Clin. Pathol., 72. 515-520, 1979. gated. 16. Gruenwald. P. Growth and development of the uterus: the relationship of Although a recent study suggests that DES-related abnor epithelium to mesenchyme. Ann. N. Y. Acad. Sci., 75. 436-440, 1959. malities diminish with age (1 ), the potential for in utero trans 17. Herbst, A. L., Kurman. R. J., Scully, R. E., and Poskanzer. D. C. Clear cell adenocarcinoma of the genital tract in young females. N. Engl. J. Med., 287. formation of actively differentiated cells as well as environmen 1259-1264, 1972. tal factors later in life affecting normal cells in an abnormal 18. Herbst, A. L., Poskanzer, D. C., and Robboy, S. J. Prenatal exposure to stllbestrol: a prospective comparison of exposed female offspring with location (adenosis) resulting in adenocarcinoma are possibili unexposed controls. N. Engl. J. Med., 292: 334-339, 1975. ties that must be considered. Forsberg (10) observed that 19. Herbst. A. L., Ulfelder, H.. and Poskanzer. D. C. Adenocarcinoma of the ovariectomy at puberty prevented development of adenosis in vagina: association of maternal stilbestrol therapy with tumor appearance in young women. N. Engl. J. Med., 284: 878-881, 1971. the cervicovaginal region of female mice treated neonatally 20. Hill, E. C. Clear cell carcinoma of the cervix and vagina in young women. with estrogen or DES. These observations, in conjunction with Am. J. Obstet. Gynecol., ) 16: 470-484, 1973. 21. Kurman, R. J., and Scully, R. E. The incidence and histogenesis of vaginal the fact that adenosis and adenocarcinoma were observed in adenosis. Hum. Pathol., 5. 265-276, 1974. prenatally exposed women more often at puberty, suggest a 22. Lamb, J. C., IV, Stumpf, W. E., Newbold, R. R., and McLachlan, J. A. possible role of estrogen as a promoter in the pathogenesis of Transitional changes in the surface epithelium of the cycling mouse vagina, cervix, and uterus: scanning electron microscopic studies. Biol. Reprod., these lesions at puberty. 19: 701-711, 1978. In summary, the results presented in this report combined 23. McLachlan, J. A. Prenatal exposure to diethylstilbestrol in mice: toxicological with additional knowledge of the effects of DES in other sys studies. J. Toxicol. Environ. Health, 2: 527-537, 1977. 24. McLachlan, J. A. Rodent models for perinatal exposure to diethylstilbestrol tems suggest that the stage of cellular differentiation at the and their relation to human disease in the male. In: A. L. Herbst and H. A. time of DES exposure may be critical in the final expression of Bern (eds.), Developmental Effects of Diethylstilbestrol (DES) in Pregnancy, a particular abnormality. pp. 148-157. New York: Thieme-Stratton, 1981. 25. McLachlan, J. A., and Dixon, R. L. Transplacental toxicity of diethylstilbes trol: a special problem in safety evaluation. In: M. A. Mehlman, R. E. Shapiro, and H. Blumenthal (eds.), Advances in Modern Toxicology, Vol. 1, Part 1, pp. 423-448. New York: Halsted Press, 1976. ACKNOWLEDGMENTS 26. McLachlan, J. A., Newbold, R. R.. and Bullock, B. C. Long-term effects on the female genital tract after prenatal exposure to diethylstilbestrol. Cancer The authors wish to thank Mindy Chambers for technical assistance, James Res.. 40: 3988-3999, 1980. Mangum for handling and care of the mice, and the National Institute of Environ 27. Metzler. M., and McLachlan, J. A. Oxidative metabolites of diethylstilbestrol mental Health Sciences Histology Laboratory for their assistance in preparing in fetal, neonatal, and adult mouse. Biochem. Pharmacol., 27. 1087-1094, slides. Also, the authors are indebted to Dr. Beth Gladen for statistical analysis; 1978. Dr. Bill Bullock. Bowman-Gray School of Medicine. Winston-Salem, N. C., for 28. Nomura, T., and Kanzaki, T. Induction of urogenital anomalies and some consultation in pathology and valuable advice; and Dr. Stanley Robboy, Massa tumors in the progeny of mice receiving diethylstilbestrol during pregnancy. chusetts General Hospital, Boston, Mass., for helpful comments on the manu Cancer Res., 37 1099-1104, 1977. 29. O'Brien, P. C., Noller, K., Robboy, S. J., Barnes, A. B., Kaufman, R., Tilley, script.

2008 CANCER RESEARCH VOL. 42

B., and Townsend, D. E. Vaginal epithelial changes in young women enrolled 1978. in the National Cooperative Diethylstilbestrol Adenosis (DESAD) project. 34. Scully, R. E., Robboy, S., and Welch. W. R. Pathology and pathogenesis of Obstet. Gynecol., 53. 300-308, 1979. diethylstilbestrol-related disorders of the female genital tract. In: A. L. Herbst 30. Plapinger, L., and Bern. H. Adenosis-like lesions and other cervicovaginal (ed.). Intrauterine Exposure to Diethylstilbestrol in the Human, pp. 8-22. abnormalities in mice treated perinatally with estrogen. J. Nati. Cancer Inst., Chicago: American College of Obstetricians Gynecologists. 1978. 63: 507-517, 1979. 35. Stati, A., and Mattingly, R. F. Vaginal adenosis: a precancerous lesion? Am. 31. Rao, P. N., and Engleberg, J. Structural specificity of estrogens in the J. Obstet. Gynecol., 120: 666-673, 1974. induction of mitotic chromatid non-disjunction in HeLa cells. Exp. Cell Res.. 36. StaÂ«, A., Mattingly, R. F., Foley, D. V., and Fetherston, W. C. Clinical 48: 71-81, 1967. diagnosis of vaginal adenosis. Obstet. Gynecol.. 43: 118-128, 1974. 32. Robboy, S. J., Kaufman, R. H., Prat, J., Welch, W. R., Gaffey, T., Scully. R. 37. Takasugi. N. Cytological basis for permanent vaginal changes in mice treated E., Richart, R., Fenoglio. C. M., Virata, R., and Tilley, B. C. Pathologic neonatally with steroid hormones. Int. Rev. Cytol.. 44: 193-224, 1976. findings in young women enrolled in the National Cooperative Diethylstilbes 38. Walker, B. E. Reproductive tract anomalies in mice after prenatal exposure trol Adenosis (DESAO) Project. Obstet. Gynecol., 53. 309-317, 1979. to DES. Teratology, 21: 313-321, 1980. 33. Robboy. S. J., Keh, P. C., Nickerson, R. J., Helmanis, E. K.. Prat, J.. 39. Yasuda, Y., Kihara, T., and Nishimura, H. Transplacental effect of ethinyl Szyfelbein, W. M., Taft, P. D.. Barnes, A. B., Scully, R. E., and Welch, W. R. estradici on mouse vaginal epithelium. Dev. Growth Differ., 79. 241-247, Squamous cell dysplasia and carcinoma in situ of the cervix and vagina after 1977. prenatal exposure to diethylstilbestrol. Obstet. Gynecol., 51: 528-535.

.- '

Fig 2 Basal cell hyperplasia in the vagina of an 18-month-old female exposed prenatally to DES; irregular extension of epithelium into the underlying stromal tissue DES-100. H & E. x 75. Fig. 3. Vaginal epithelium forming papillary projections into the lumen of an 18-month-old animal. DES-100. H & E, x 75. Fig. 4. a, adenosis (Adi in a cross-section through the vaginal fornix of a 1-month-old mouse exposed prenatally to DES. Gland-like structure in stroma of fornix opens into the vaginal lumen in another section. Lining epithelium is keratinized, and there is stromal edema. DES-100. H & E, x 15. b, higher magnification of a. A layer of tall columnar cell lines gland-like structure. DES-100. H & E, x 75.

MAY 1982 2009

'.Â¿-Â»t?*!/Â«

2010 CÃ•NCER RESEARCH VOL. 42

.â€” â€¢â€¢â€¢

mj&\Ã¤.7*m&pviâ€¢ Ã¯Â£ j J**^ 4 :^^Ãˆ--

' 11 t. '#.'â€¢ â€¢ ,.

Fig. 5. a, portion of cross-section through vaginal fornix of a 1-month-old mouse exposed prenatally to DES. Immature squamous epithelium is interrupted by an area of columnar cells. Ad, Adenosis. DES-100. H & E, x 75. b. higher magnification of adenosis in a. Columnar cells approximate a gland-like structure in the stroma of the vaginal fornix. This abnormality was observed in only two 6-fim sections. DES-100. H & E, x 120. Fig. 6. Adenosis in the vaginal fornix of an 18-month-old animal prenatally exposed to DES. Columnar cells form a cyst in the stroma under the epithelial layers. The lumen of this gland-like structure is more dilated compared to the younger animals. DES-100. H & E, x 75. Fig. 7. Vaginal adenocarcinoma in a 17-month-old female exposed prenatally to DES. Gland-like structures in the submucosal area of the vaginal fornix are lined by mucus-secreting columnar cells with a low degree of nuclear pleomorphism. Invasion into the underlying tissue is consistent with well-differentiated adenocarcinoma of the vagina. DE3-100. H & E. x 75. Fig. 8. Squamous cell carcinoma in the vagina of a 17-month-old prenatal DES-treated mouse. There is cellular pleomorphism and a lack of orderly progression from basal to squamous cells. DES-100. H & E, x 75. Fig. 5. Submucosal glands in the vaginal fornix of the same 17-month-old DES-exposed female in Fig. 8. containing areas of squamous metaplasia (arrow). DES- 100. H&E, x 75. Fig. 10. Adenocarcinoma of the vagina lying adjacent to the area shown in Fig. 9. Cellular pleomorphism and invasion are characteristics of this tumor. DES-100. H&E, x 100. Fig. 11. Adenosis (arrow) in the vaginal fornix of a 35-day-old female exposed neonatally to DES. Columnar epithelium forms fold toward the underlying stroma. Neonatal DES. H & E, x 75.

MAY 1982 2011

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. Vaginal Adenosis and Adenocarcinoma in Mice Exposed Prenatally or Neonatally to Diethylstilbestrol

Retha R. Newbold and John A. McLachlan

Cancer Res 1982;42:2003-2011.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/42/5/2003

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/42/5/2003. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.