BIOLOGY OF REPRODUCTION 61, 91–100 (1999)
Differential Activity of Diethylstilbestrol versus Estradiol As Neonatal Endocrine
Disruptors in the Female Hamster (Mesocricetus auratus) Reproductive Tract 1
William J. Hendry III,2,4 Brian L. DeBrot,4 Xinglong Zheng,3,4 William S. Branham,5 and Daniel M. Sheehan5
Department of Biological Sciences,4 Wichita State University, Wichita, Kansas 67260-0026
Division of Reproductive and Developmental Toxicology,5 National Center for Toxicological Research,
U.S. Food and Drug Administration, Jefferson, Arkansas 72079
ABSTRACT
The synthetic estrogen diethylstilbestrol (DES) is a potent
neonatal endocrine disruptor in the hamster. To test the speci-
ficity of this phenomenon, newborn animals were treated with
100 mg of either DES or the natural estrogen, estradiol-17b (E2).
Of the two, neonatal DES exposure caused greater morpholog-
ical disruption throughout the female reproductive tract in pre-
pubertal animals and in adults that either retained their ovaries
or were ovariectomized and then given the same levels of chron-
ic E2 stimulation. In the uterus, a characteristic histopathological
profile, including enhancement of both hyperplastic and apo-
ptotic activity, was initiated prepubertally and exclusively in the
endometrial epithelial cell compartment from the neonatally
DES-treated animals and then was promoted by E2 stimulation
during adulthood. Interestingly, apoptotic activity was not de-
tected in an area of endometrial epithelium that progressed to
the neoplastic state in a DES-exposed animal. Lastly, chronic
estrogen induction of lactoferrin was also restricted to the DES-
exposed endometrium. We conclude that 1) DES is more active
than E2 as a perinatal endocrine disruptor in the hamster and 2)
this experimental system should be generally useful as a means
to screen compounds for such activity and then probe their
mechanism of action.
INTRODUCTION
From the 1940s to the 1960s, the synthetic estrogen di-
ethylstilbestrol (DES) was often prescribed for the support
of high-risk pregnancies [1]. The negative consequences of
this practice began to emerge in 1971 when Herbst et al.
[2] described the very early occurrence of a rare cancer,
vaginal clear cell adenocarcinoma, in eight young women
who had been exposed to DES in utero. Numerous clinical
and experimental animal studies have since demonstrated
that perinatal DES exposure results in fertility deficits plus
teratogenesis and neoplasia throughout the male and female
reproductive tract [3, 4]. Thus, DES is an original and po-
tent member of the expanding list of chemicals known as
‘‘endocrine disruptors’’ [5]. There is now intense scientific
and public interest in the concept that inadvertent and un-
timely exposure to such synthetic agents (xenoestrogens),
or even to natural substances that possess estrogenic activ-
ity (phytoestrogens), may adversely affect the reproductive
Accepted February 9, 1999.
Received October 21, 1998.
1
This work was supported by the National Cancer Institute (CA60250),
the Flossie West Memorial Trust Foundation, and the United States Food
and Drug Administration.
2
Correspondence: William J. Hendry III, Department of Biological Sci-
ences, Wichita State University, 1845 Fairmount, Wichita, KS 67260–
0026. FAX: 316 978 3772; e-mail: hendry@wsuhub.uc.twsu.edu
3
Current address: Howard Hughes Medical Institute, Division of He-
matology and Oncology, Washington University School of Medicine, 660
South Euclid Avenue, Campus Box 8125, St. Louis, MO 63110–1093.
91
and general health of both human and wildlife populations
[6, 7].
Our analyses of the endocrine-disruptive activity of peri-
natal DES exposure have been performed in the hamster
because its gestation period is very predictable and short
(16 days) compared to that (18 to 21 days) of other rodents.
Thus we can treat neonates rather than fetuses but still tar-
get a very early stage of reproductive tract development.
We also reasoned that treating neonates rather than fetuses
would avoid uncertainties associated with maternal metab-
olism and placental transfer of the drug. After a single in-
jection of DES on the day of birth, abnormalities developed
throughout the female reproductive tract [8]. In our previ-
ous studies focusing on the uterus, we observed rapid and
profound morphogenetic alterations during the prepubertal
period [9] and a very high incidence of endometrial hyper-
plasia and neoplasia (adenocarcinoma) in adult animals [8].
Interestingly, the hyperplastic endometrial epithelium in
such adult animals is also a site of massive cell death by
apoptosis [10]. These lesions (endometrial hyperplasia, ap-
optosis, and adenocarcinoma) are not due merely to altered
function of the hypothalamo-pituitary-ovarian axis, because
they occurred in the DES-exposed group but not in the
control group of animals when both groups were ovariec-
tomized prior to puberty and then chronically stimulated
with replacement levels of estradiol-17b (E2) [8, 10]. To
further investigate the mechanistic basis of this phenome-
non, neonatal uteri from control or DES-treated donors
were transplanted into the cheek pouches (another advan-
tage of the hamster) of control or neonatally DES-treated
adult hosts that were ovariectomized and stimulated with
E2. The characteristic histopathological profile (endometrial
hyperplasia/dysplasia and apoptosis) occurred only in uter-
ine transplants derived from DES-exposed donors, and this
outcome was not influenced by the neonatal treatment his-
tory of the host [10]. Thus, atypical estrogen responsiveness
is an inherent property of the neonatally DES-exposed ham-
ster uterus rather than an indirect effect mediated by DES
acting peripherally within the host. However, this change
in estrogen responsiveness does not appear to be due to any
major alteration in the physicochemical or functional prop-
erties of the uterine estrogen receptor system [11]. We have
also determined that neonatal DES treatment induces per-
sistent and epithelial cell-specific imbalances in the estro-
gen-regulated uterine expression of certain oncogenes that
have been implicated in the control of cell proliferation (c-
jun, c-fos, c-myc) and apoptosis (bax, bcl-2, bcl-x) [12].
Together, these results support the hypothesis [10, 12] that
the neonatal DES insult directly initiates a permanent de-
velopmental change and that subsequent exposure promotes
the generation of uterine lesions that can ultimately pro-
gress to endometrial cancer.
92 HENDRY III ET AL.
An important unanswered question is whether estrogen-
icity alone determines the endocrine-disruptive activity of
a given agent. The estrogenicity of DES is important to its
activity as both a developmental toxicant and a carcinogen,
since clinical and experimental data show that perinatal
DES-induced teratogenesis and neoplasia are confined to
estrogen target organs [3, 4]. However, few direct compar-
isons of perinatal endocrine-disruptive activity have been
made between the synthetic agent (DES) and the primary
natural estrogen (E2). Those performed in rodents have sug-
gested that E2 is less disruptive than DES [13, 14] or have
led to differing conclusions about whether the long-term
consequences of perinatal DES exposure involves factors
in addition to its estrogenicity [15, 16]. Complicating such
studies is the fact that, in many perinatal rodents, E2 is
much less potent than DES as an estrogen due to high se-
rum concentrations of alpha-fetoprotein (AFP), which binds
E2 but not DES with high affinity [17]. In contrast, the
hamster is free of such complications since its AFP does
not bind E2 [18, 19]. Thus the hamster is an excellent sys-
tem for comparing the perinatal endocrine-disruptive activ-
ity of DES and E2. The comparison we have performed
includes a comprehensive chronicle of the immediate (pre-
pubertal) and delayed (adult) consequences of the two neo-
natal treatment regimens. Also, we employed prepubertal
ovariectomy plus chronic E2 supplementation to 1) control
for the possibility that function of the hypothalamo-pitui-
tary-ovarian axis status in adult animals might be altered
differently by the two neonatal treatment regimens and 2)
test the relative ability of the two neonatal treatment regi-
mens to directly and permanently disrupt estrogen respon-
siveness in the hamster uterus.
MATERIALS AND METHODS
Animal Treatment
Animal treatments, ovariectomy, chronic E2 stimulation,
anesthesia, and killing by decapitation were performed as
described previously [8, 9, 11] and in an AAALAC-accred-
ited facility according to the Guiding Principles for the Care
and Use of Research Animals promulgated by the Society
for the Study of Reproduction. Briefly, timed-pregnant Syr-
ian golden hamsters (Mesocricetus auratus) from Charles
River Breeding Laboratories (Wilmington, MA) or Harlan
Sprague Dawley (Indianapolis, IN) were caged singly under
a 14L:10D photoperiod and with food and water provided
ad libitum. Within 6 h of birth (Day 0), litter size was
adjusted to eight neonates per litter by eliminating males,
and all litter mates received a single s.c. injection of 50 ml
corn oil vehicle either alone (control) or containing 100 mg
(;33 mg/kg BW) of either E2 or DES. This dose level is
high but not unreasonable considering that DES ingestion
by pregnant women was as much as 150 mg daily and 18.2
g total during pregnancy [1]. Ovariectomy and placement
of E2 pellets were performed at 21 days of age under pen-
tobarbital-induced anesthesia. The pellets, inserted s.c. be-
tween the shoulder blades, consisted of a plugged Silastic
(Dow Corning, Midland, MI) tube (1.0-cm open lumen
length; 1.57-mm i.d.; 2.41-mm o.d.) filled with crystalline
E2. According to previous determinations [8, 10, 20], this
procedure maintains serum E2 levels at ;200 pg/ml for at
least 5 mo. At all the ages studied, at least three animals
from each treatment group were anesthetized with CO2,
weighed, killed, and immediately processed as described
below.
Tissue Harvesting, Processing, and Histological Analysis
Prepubertal animals were eviscerated, and the lower tor-
sos were immersed in ice-cold fixative for at least 24 h
before reproductive tracts were excised, trimmed of adher-
ing fat and mesentery under a dissecting microscope,
weighed, and placed back in fixative. The fixative consisted
of 4% paraformaldehyde in Dulbecco’s PBS, pH 7.4. For
the adult animals, reproductive tracts were immediately ex-
cised and then also placed in fixative for at least 24 h before
trimming, weighing, and placement back in fixative. The
harvested tracts were ultimately divided into cervix, uterine
horns, and oviduct/ovary (when present) regions and em-
bedded in paraffin; 4- to 5-mm cross sections were pro-
cessed for light microscopy using standard hematoxylin and
eosin staining. At least 4 sections cut from midregions of
the uterine horns from all the animals per time point and
treatment group were analyzed. The photomicrographs
shown are representative of the histomorphological condi-
tion observed within that group of at least three animals.
Comparisons of endometrial epithelial height (basal-to-api-
cal cell dimensions) relied on manual measurements per-
formed on the original 2.5’’ by 3.5’’ photomicrographs that
correspond to those shown in the indicated figures.
Lactoferrin (LTF) Immunohistochemistry
Tissues harvested as described above from adult animals
were fixed by immersion in Kryofix (EM Diagnostic Sys-
tems Inc., Gibbstown, NJ) at 48C for 18 h and embedded
in paraffin. After dewaxing, 4- to 5-mm uterine cross sec-
tions were quenched of endogenous peroxidase activity by
incubation with 0.6% H2O2 in methanol at 378C for 10 min
and blocked with PBS 1 1.5% goat serum at 378C for 20
min. The rabbit anti-mouse LTF antiserum (from Dr. C.
Teng; NIEHS, Research Triangle Park, NC) was diluted (1:
1000) in PBS 1 1.5% goat serum and incubated with the
tissue sections at 48C for 18 h. A kit based on the avidin:
biotinylated enzyme complex method (Vectastain ABC;
Vector Laboratories Inc., Burlingame, CA) was used to de-
tect immunocomplexes. The diluent and washing media for
all steps was PBS 1 0.05% Tween 20. Incubations were
performed in a moist chamber at 378C with biotinylated
anti-rabbit IgG (1:100) for 1 h and then with avidin:bioti-
nylated peroxidase complex (1:100) for 30 min. Bound per-
oxidase was visualized with Sigma Fast (Sigma Chemical
Co., St. Louis, MO) substrate solution (diaminobenzidine:
H2O2:urea in 0.06 M Tris-HCl, pH 7.8) at 258C for 2 min.
Lastly, sections were counterstained with 0.2% methyl
green in 0.1 M ammonium acetate (pH 4.6) for 10 min.
Statistics
For the data on animal weight, absolute tissue weight,
and normalized tissue weight, a Statistical Analysis Sys-
tems statistical package [21] was used to calculate the
mean, SE, and the significance levels of differences among
the three neonatal treatment groups at all the time points.
Factorial ANOVA was followed by Tukey’s honestly sig-
nificant differences test for multiple comparisons [22], and
means were considered significantly different at p , 0.05.
RESULTS
Gross Reproductive Tract Development In Intact Animals
The objective of this study was to compare DES with
E2 as perinatal endocrine disruptors in the female hamster.
 DES VS. E 2 AS NEONATAL ENDOCRINE DISRUPTORS 93

We began by studying reproductive tract development prior

to the onset of puberty, which occurs normally at Week 4
of life in the hamster [23]. According to body weight mea-
surements, growth of the prepubertal animals was not af-
fected by neonatal treatment with either DES or E2 (not
shown). The differential ability of the two neonatal treat-
ment regimens to disrupt early female reproductive tract
development was emphasized by normalizing reproductive
tract tissue weights to body weights (Fig. 1A). In control
animals, reproductive tract growth matched body growth
during the first 3 wk of life. Both neonatal estrogen treat-
ment regimens altered this balance by causing an early
surge in tract growth. In E2-treated animals, the surge was
relatively modest, peaking at Day 3 and then subsiding to
control levels by Day 9. In DES-exposed animals, the early
surge in tract weight was much more dramatic, peaking at
Day 5 and then only partially declining so that relative tract
weight remained double that in both the control and E2-
treated groups. The differences in reproductive tract weight
profiles shown in Figure 1A were due almost entirely to
changes in mass of the uterine horns (not shown).
Although both neonatal estrogen treatment regimens
slightly depressed animal growth after puberty (average
body weight reductions compared to controls were 9% in
the E2-treated group and 17% in the DES-treated group),
this effect was insufficient to explain the dramatic treat-
ment-dependent differences in reproductive tract weights
that occurred (Fig. 1B). In control animals, the increase in
normalized tract weight was relatively modest from 1 to 5
mo of age. In neonatally E2-treated animals, a slight addi-
tional increase occurred between 1 and 2 mo of age but
then remained parallel to the control profile at the later FIG. 1. Effect of neonatal treatment with DES vs. E2 on normalized re-
ages. In contrast, normalized reproductive tract weights in productive tract weight in prepubertal (A) and adult (B) female hamsters.
the neonatally DES-treated animals averaged at least 5.7- Animals were injected on the day of birth with 50 ml of corn oil vehicle
fold greater than in the control animals and at least 2.7-fold either alone (control, CON) or containing 100 mg of either E2 or DES. At
greater than in the E2-treated animals at every age. the indicated ages, animals were weighed and reproductive tracts (cervix,
uterine horns, oviducts, and ovaries) were removed and weighed. Repro-
ductive tract results were expressed by normalizing tissue weight to the
Uterine Histology In Intact Animals animal’s body weight. Values represent the mean 6 SE, n 5 3 (error bars
not shown where variability is so small as to be masked by the data point).
The differential ability of the neonatal estrogen treatment Indicated are means significantly different ( p , 0.05) from that of the
control group (*) or from those of both the control and the E2-treated group
regimens to disrupt endometrial histomorphology during (**) at that time point.
early development is shown in Figure 2, A–C. The low-
magnification photographs confirm the tissue mass trends
demonstrated in Figure 1A. They show that uterine cross- tological analysis at low magnification confirmed that
sectional area was sometimes modestly increased in E2-ex- cross-sectional dimensions were increased to a much great-
posed animals and always dramatically increased in the er extent by neonatal treatment with DES than with E2.
DES-exposed animals compared to controls. By Day 5 (Fig. Even at low magnification, the endometrial epithelium in
2A), the DES-exposed uteri exhibited endometrial epithelial DES-exposed uteri was clearly hypertrophic and contained
cell pseudostratification and hypertrophy (almost double the cavities. At higher magnification, the endometrial epithelial
height of that in the control and E2-exposed uteri). This cells in the neonatally E2-exposed uteri were about 50%
was also the earliest age at which the endometrial epithelial taller but were organized similarly to those in the control
cell compartment in DES-exposed uteri harbored cavities uteri. For neonatally DES-exposed uteri, disruption of the
that contained degenerating cells with the histomorphology endometrial cell compartment was more dramatic in adult
of apoptotic bodies [24]. By Day 9 (Fig. 2B), endometrial than in prepubertal animals (compare Fig. 2, D and E, with
glands had developed only in the DES-exposed uteri, but Fig. 2, A–C). In addition to being extremely hypertrophic
endometrial epithelial cell height in those uteri had re- (more than 3-fold taller than control), the epithelial cells
gressed to that seen in both the control and E2-exposed exhibited a very complex pseudostratified organization in-
uteri. By Day 15, developing endometrial glands were pres- dicative of a hyperplastic state. Additional distortion came
ent in all three groups (not shown). Also, DES-specific dis- from cavities that frequently harbored apoptotic cells. As
ruption of the endometrial epithelial compartment (cellular the animals aged, cystic glandular structures developed oc-
hypertrophy, pseudostratification, and cavities with apo- casionally in the endometrium of neonatally E2-treated an-
ptotic cells) intensified between Day 15 (not shown) and imals (Fig. 2E). Even in such cases, the histomorphology
Day 21 (Fig. 2C). of the epithelium lining the cysts resembled the epithelium
Further DES-specific disruption of endometrial histo- lining the lumen of control uteri (low columnar cells with
morphology during adulthood is shown in Figure 2, D and orderly and basally located nuclei) rather than that in neo-
E. In uterine horns from 1-mo-old animals (Fig. 2D), his- natally DES-exposed uteri (hypertrophic/hyperplastic cells
94 HENDRY III ET AL.

FIG. 2. Effect of neonatal treatment with DES vs. E2 on uterine histology in prepubertal and adult hamsters. Animals were injected on the day of birth
with 50 ml of corn oil vehicle either alone (control, CON) or containing 100 mg of either E2 or DES. Representative cross sections of uterine horns from
animals killed on Day 5 (A), Day 9 (B), Day 21 (C), 1 mo (D), and 4 mo (E) of age were photographed at 340 (left) and 3400 (right) (reproduced at
45%). Indicated in the high-magnification panels are the presence in neonatally DES-exposed uteri of pseudostratified endometrial epithelium (asterisks)
with cavities that often contained apoptotic cells (arrows) and the presence in neonatally E2-exposed uteri of cystic glandular structures (stars).
 DES VS. E 2 AS NEONATAL ENDOCRINE DISRUPTORS
FIG. 2. Continued.
in a pseudostratified organization that is riddled with cav-
ities containing apoptotic cells). In fact, the neonatally
DES-exposed epithelium at this age contained so many cav-
ities that it assumed a ‘‘foamy’’ appearance.
Adult Uterine Responses to Estrogen Stimulation
Like other rodents, hamsters exposed perinatally to es-
trogens enter a ‘‘persistent estrous’’ state characterized by
anovulatory and cystic ovaries that continuously secrete
high levels of E2 but little or no progesterone [8, 25]. If
the severity of such an altered endocrine state was differ-
ent after neonatal exposure to E2 compared to DES, it
could explain the differences in uterine responses among
the three groups of adult hamsters. To evaluate this pos-
sibility, animals in the three neonatal treatment groups
were ovariectomized prior to puberty (Day 21) and then
stimulated exogenously with E2. Under such conditions,
overall animal growth from 1 mo to 5 mo of age was the
same among the three neonatal treatment groups (not
shown), and the general profiles were quite similar to that
for the DES-exposed group of intact adult animals dis-
cussed above. Thus, differences in uterine weight profiles
among the three treatment groups were quite similar
whether expressed on an absolute basis (not shown) or
normalized to body weight (Fig. 3). In the youngest ani-
mals (1 mo) that had been exposed to E2 for 9 days, nor-
malized uterine weights were the same in control and neo-
natally E2-exposed animals but were about 2-fold higher
in the DES-exposed animals. Thereafter, control uteri grew
at a linear rate that generally kept pace with their body
weight. Both neonatal estrogen treatment regimens in-
creased the slope of the absolute (not shown) and the nor-
malized uterine growth curves, but this effect was most
pronounced in the neonatally DES-treated animals. Com-
pared to control values, increased weight (both absolute
and normalized average values) of the E2-exposed uteri
barely exceeded 2-fold, while that of the neonatally DES-
exposed uteri was approximately 3-fold higher.
95
FIG. 3. Effect of neonatal treatment with DES vs. E2 on normalized uter-
ine weights in ovariectomized adult hamsters given estrogen replacement.
Animals were injected on the day of birth with 50 ml of corn oil vehicle
either alone (control, CON) or containing 100 mg of either E2 or DES. On
Day 21, they were ovariectomized and given a Silastic implant filled with
crystalline E2. At the indicated ages, uteri were removed and weighed.
Results were expressed by normalizing tissue weight to the animal’s body
weight. Values represent the mean 6 SE, n 5 3 or 4 (error bars not shown
where variability is so small as to be masked by the data point). Indicated
are means significantly different ( p , 0.05) from that of the control group
(*) or from those of both the control and the E2-treated group (**) at that
time point.
Histological analysis revealed that a uniform level of E2
stimulation during adulthood induced very different pat-
terns of endometrial disruption in the two neonatal estrogen
treatment groups (Fig. 4). This was evident even in 1-mo-
old animals exposed to E2 for only 9 days (Fig. 4A). For
each treatment group, the endometrial epithelium was about
50–100% taller and also was more pseudostratified than in
the same treatment group in the intact animals at this age
(compare Fig. 4A with Fig. 2D). This indicates that the E2
pellets produced a generally higher estrogenic environment
than in the intact adult animals. Figure 4A also demon-
strates that the increased uterine tissue mass in both neo-
natal treatment groups at this early stage of E2 stimulation
was primarily due to gains in the mesenchymal compart-
ment (stroma). Lastly, Figure 4A demonstrates that the neo-
natally DES-exposed endometrial epithelium, even at this
early stage of chronic E2 stimulation, was more severely
disrupted than in the neonatally E2-exposed animals or at
any stage in the intact adult animals (compare Fig. 4A with
Fig. 2, D and E). At low magnification, the neonatally DES-
exposed endometrial epithelium appeared very foamy. High
magnification revealed that it also consisted of extremely
hyperplastic cells in a very chaotic pseudostratified organ-
ization that was riddled with cavities containing apoptotic
cells. One month later (Fig. 4B), low magnification showed
that the endometrium in control and neonatally E2-exposed
uteri contained many large cystic glands, whereas the com-
plex folded epithelium in the neonatally DES-exposed en-
dometrium retained a foamy appearance. High magnifica-
tion revealed that the endometrial epithelial cells were only
about 20% taller than control in the neonatally E2-exposed
group, but more than 2-fold taller than control in the neo-
natally DES-exposed group, and still retained a severely
disrupted cellular organization. As chronic E2 exposure
continued (Fig. 4C), cystic glandular development began to
develop in DES-exposed uteri (low magnification). While
endometrial epithelial cells in the control and neonatally
E2-exposed uteri were the same height, those in the neo-
natally DES-exposed uteri were almost 3-fold taller (high
96 HENDRY III ET AL.

FIG. 4. Effect of neonatal treatment with DES vs. E2 on uterine histology

in ovariectomized adult hamsters given estrogen replacement. Animals
were injected on the day of birth with 50 ml of corn oil vehicle either alone
(control, CON) or containing 100 mg of either E2 or DES. On Day 21, they
were ovariectomized and given a Silastic implant filled with crystalline E2.
Cross sections of uterine horns from animals at 1 mo (A), 2 mo (B), and 4
mo (C) of age were photographed at 340 (left) and 3400 (right) (repro-
duced at 45%). Indicated are areas of hyperplastic endometrial epithelium
with a chaotic pseudostratified organization (asterisks) plus numerous cav-
ities often containing apoptotic cells (arrows), cystic glandular structures
(stars), and areas of adenomatous epithelium (arrowheads).

magnification). At this stage in the neonatally DES-exposed
uterus, some areas of the endometrial epithelium assumed
an adenomatous appearance (low magnification) that still
retained the characteristic profile of disrupted cellular or-
ganization (high magnification). Also note in Figure 4 that
the ratio of epithelial to stromal tissue in the DES-exposed
uteri appeared to increase with the duration of E2 stimula-
tion.
A noteworthy observation was made in a DES-exposed
animal at 5 mo of age. In a uterine cross section from this
animal (Fig. 5), most of the endometrium (top panels) ex-
hibited the characteristic disrupted state described above
(hypertrophic/hyperplastic epithelial cells and cavities con-
taining degenerating cells with the histopathology of apo-
ptotic bodies). However, the section also contained the pro-
file of a distinctive polypoid mass (bottom panels). It had
the same histomorphology (numerous back-to-back glan-
dular profiles consisting of atypical epithelial cells and little
intervening stromal tissue) as other tumors that have been
identified as endometrial adenocarcinomas in neonatally
 DES VS. E 2 AS NEONATAL ENDOCRINE DISRUPTORS 97

FIG. 5. Comparison of histopathology between nontumorous (upper

panels) and tumorous (lower panels) endometrial regions of a neonatally
DES-exposed uterus. The uterus was from a 5-mo-old animal that was
injected on the day of birth with 100 mg DES, ovariectomized on Day
21, and also given a Silastic implant filled with crystalline E2. Uterine
cross sections were photographed at 340 (left) and 3400 (right) (repro-
duced at 47%). Apoptotic cells (arrows) were present in the hyperplastic
endometrial epithelium but not in the adjacent tumor mass.

DES-treated hamsters [8]. Importantly, the tumor mass was

devoid of the cavities and apoptotic bodies that lent a
foamy appearance to the adjacent nontumor regions of the
disrupted endometrium.

Immunohistochemical Analysis of LTF Expression

To extend our histomorphological observations with bio-
chemical analyses, we chose the estrogen-regulated glyco-
protein LTF. Shown in Figure 6 for the three neonatal treat-
ment groups are typical immunohistochemical results from
adult animals chronically stimulated with E2 in adulthood.
In uteri from both the control and neonatally E2-treated
animals, similar low levels of LTF-specific reaction product
were localized within the endometrial epithelial compart-
ment. Some apparently secreted immunoreactive material
also appeared within the cystic glandular structures that de-
veloped in the uterus from the neonatally E2-treated animal.
This phenomenon was occasionally observed in both the
control and neonatally E2-treated groups (not shown). In
contrast, the disrupted endometrial epithelium in neonatally
DES-treated animals was always densely labeled with LTF-
specific reaction product.
FIG. 6. Effect of neonatal treatment with DES vs. E2 on expression of LTF
DISCUSSION in the uterus of ovariectomized adult animals given replacement estrogen.
Animals were injected on the day of birth with 50 ml of corn oil vehicle
We found that DES is more active than E2 as a neonatal either alone (control, A) or containing 100 mg of either E2 (B) or DES (C).
endocrine-disrupting agent in the female hamster. While the On Day 21, they were ovariectomized and given a Silastic implant filled
uterus is the focus of this report, the ovary, oviduct, and with crystalline E2. Uteri were harvested from 5-mo-old animals, and
cervix also exhibited much greater disruption in neonatally cross sections were incubated with a polyclonal antibody against mouse
DES-treated than in neonatally E2-treated animals (not LTF. Immunocomplexes were detected using the ABC/peroxidase method
shown). Preliminary descriptions of our ovarian, oviductal, and visualized by reaction with a H2O2:diaminobenzidine substrate so-
lution that deposits an insoluble, dark brown product. Sections were also
and cervical observations have been reported [26, 27] and counterstained with methyl green and then photographed at 340.
generally resemble those described in other rodents follow-
ing perinatal DES treatment [13, 14].
In addition to the comparative data presented, this study Thus it complements previous findings made at different
provides a comprehensive histomorphological chronicle of developmental stages and under various experimental con-
1) the appearance and progression of neonatal DES-induced ditions [8–10]. Those aspects that differ from results of
disruption in the hamster uterus and 2) the way in which a DES-related studies in other experimental systems are not-
directly induced and permanent form of altered estrogen ed below.
responsiveness contributes to the disruption phenomenon. In prepubertal hamsters, the pattern and degree of dis-
98 HENDRY III ET AL.
rupted uterine development at the gross level was much
greater in hamsters treated neonatally with DES than with
E2. This result cannot be due to differences in the onset of
ovarian steroidogenesis among the neonatal treatment
groups because measurements of serum E2 levels during
this period detected no significant difference between con-
trol and neonatally DES-treated animals, nor were these
levels significantly altered in animals ovariectomized on
Day 3 of life [8]. On the other hand, ovariectomy on Day
3 of life partially reduced the abnormal uterine growth that
occurred between Days 15 and 21 in the DES-treated ani-
mals [8]. Thus, the prepubertal ovary appears to modulate
the ability of neonatal DES exposure to disrupt early uterine
development, but the mechanism remains to be determined.
In the endometrial epithelial cell compartment of pre-
pubertal hamsters, neonatal E2 treatment also failed to in-
duce the disruption pattern that was induced by neonatal
DES treatment. The DES-specific disruption pattern, sepa-
rate aspects of which were noted in previous studies [8–
10], included a hypertrophic and hyperplastic response that
resulted in cellular crowding and disorganization, the ap-
pearance of cavities that harbored degenerating (apoptotic)
cells, and the precocious development of endometrial
glands. The latter response contrasts with the situation in
the rat, where endometrial gland development was inhibited
by neonatal DES exposure [28].
As the ovary-intact animals reached adulthood, the dif-
ference in degree of uterine disruption at the gross and his-
tological level continued to increase between the groups
treated neonatally with DES or E2. Interestingly, this and
previous studies [8–10] show that acute perinatal exposure
to DES elicits a consistent hypertrophic/hyperplastic re-
sponse in the hamster uterus, whereas it elicits a hypotro-
phic/hypoplastic response in the rat [28, 29] and either a
hypotrophic/hypoplastic or a hypertrophic/hyperplastic re-
sponse in the mouse [13, 30, 31].
When animals from both neonatal estrogen treatment
groups were ovariectomized and exposed chronically dur-
ing adulthood to the same level of E2, uterine disruption at
the gross and histological level remained most severe in the
DES-treated group. Thus the differences in uterine disrup-
tion are not due to different degrees of altered neuroendo-
crine function in the two treatment groups. The results also
confirm previous evidence [10] that neonatal DES treatment
permanently alters estrogen responsiveness in the hamster
uterus via a direct mechanism. However, the increased es-
trogen responsiveness in the prenatally DES-treated ham-
ster contrasts with evidence of reduced estrogen respon-
siveness in the prenatally DES-treated mouse [31, 32].
The scope and design of this comparative study provide
some new insight into the morphogenesis of neonatal DES-
induced uterine disruption in the hamster. For instance, the
dysplastic changes in the endometrial epithelial cell com-
partment of neonatally DES-treated animals followed a pat-
tern of progression with noteworthy architectural and cy-
tological features. Architecturally, the ratio of epithelial to
stromal tissue increased until, by 4 mo of age, the epithe-
lium was often crowded into complex adenomatous struc-
tures with little intervening stroma. Evaluated according to
the clinical description and staging of endometrial hyper-
plasia and carcinoma, this pathological situation in the ham-
ster is reminiscent of the condition originally termed ade-
nomatous hyperplasia [33] and now referred to as complex
hyperplasia with cellular atypia [34]. We also captured an
interesting example of the neoplastic outcome often pro-
moted by E2 stimulation of the neonatally DES-exposed
hamster endometrium [8]. It was characterized as an en-
dometrial adenocarcinoma (distinct from complex hyper-
plasia) based on the greater degree of cytological atypia
(larger nuclei of variable size and shape that have lost po-
larity, increased nuclear-to-cytoplasmic ratios, prominent
nucleoli, and irregularly clumped chromatin) plus greater
general architectural disruption (numerous areas of back-
to-back glands with few or no intervening stromal cells)
[34].
The tendency for chronic E2 stimulation to induce the
formation of endometrial cysts in all three neonatal treat-
ment groups is a phenomenon that we observe consistently
in our experimental system. We assume that it somehow
represents a distortion of the normal process of endometrial
gland formation. However, the actual mechanism respon-
sible for this architectural anomaly remains unknown.
The phenomenon of altered estrogen responsiveness in
the neonatally DES-exposed hamster uterus includes in-
creases in both cell growth and apoptosis in the endometrial
epithelial cell compartment [10]; it appears to be driven by
imbalances in the expression of certain oncogenes that have
been implicated in the control of cell proliferation (c-jun,
c-fos, c-myc) and apoptosis (bax, bcl-2, bcl-x) [12]. How-
ever, the tumor mass shown in this study was devoid of
cavities with apoptotic cells even though adjacent regions
of hyperplastic endometrial epithelium were riddled with
such elements. Thus our experimental system may repre-
sent a situation in which apoptosis serves to eliminate mu-
tated cells that develop as a result of abnormal proliferative
activity and in which frank neoplasms erupt at sites where
apoptotic activity is either lost or overwhelmed [35]. Two
related processes may influence apoptosis in the endome-
trial epithelial compartment of neonatally DES-treated
hamsters. One possibility is that inhibition of adhesion be-
tween epithelial cells induces them to undergo apoptosis
[36]; the other is that disruption of interactions between
epithelial cells and the basement membrane induces a form
of apoptosis known as anoikis [37]. In fact, preliminary
studies have provided 1) ultrastructural evidence that the
basement membrane is disrupted under the severely dys-
plastic and apoptotic endometrial epithelium in neonatally
DES-treated hamsters [38] and 2) biochemical evidence for
an altered association between specific proteins (cadherins
and b-catenin) [39] that mediate adhesion between epithe-
lial cells [40] and also appear to play a central role in some
epithelial neoplasms [41].
Since neonatal DES treatment caused epithelial cell-spe-
cific imbalances in the estrogen-regulated expression of
several genes in the hamster uterus [12], we tested whether
neonatal E2 treatment could induce the same phenomenon.
As a new target, we chose the glycoprotein product of the
LTF gene. This iron-binding molecule is expressed in a
wide variety of tissues, but it is a particularly sensitive and
directly up-regulated marker of estrogen stimulation in the
endometrial epithelium [42, 43]. Although the function of
LTF is largely unknown, a recent study suggests that it is
associated with malignant transformation of the human en-
dometrium [44]. In the mouse, perinatal DES exposure per-
manently disrupts LTF expression [42, 45], apparently by a
gene demethylation mechanism [46]. Our immunohisto-
chemical analysis showed that neonatal treatment with DES
but not with E2 resulted in up-regulated expression of the
LTF protein throughout the endometrial epithelial cell com-
partment of E2-stimulated adult hamsters. This is strong
biochemical evidence that DES is more potent or that it
 DES VS. E 2 AS NEONATAL ENDOCRINE DISRUPTORS
acts differently than E2 as a neonatal endocrine disruptor
in the hamster uterus.
The plasma protein AFP is present at high concentration
during fetal and neonatal development [47]. The fact that,
in some rodents, AFP is a high-affinity and high-capacity
binder of E2 but not DES has been suggested as an expla-
nation for the striking transplacental and neonatal toxic ef-
fects of DES [17]. The hamster provides a good means to
test this hypothesis because, like human AFP [48], hamster
AFP does not bind E2 [18, 19]. Thus unequal availability
of the two estrogens to target tissues because of differential
binding to AFP in the neonatal hamster is unlikely to ex-
plain the observed differences in activity between the two
estrogens as neonatal endocrine disruptors. On the other
hand, albumin does bind DES preferentially in rats and
hamsters [49, 50], which could influence the duration of
exposure and thus duration of response. In fact, this phe-
nomenon has been cited as a possible explanation [50] for
our observation in the hamster uterus of a major discrep-
ancy in the binding affinity of cytosol and nuclear forms
of the estrogen receptor [11]. Another hypothetical scheme
to explain the perinatal endocrine-disruptive action is based
on the fact that DES can be metabolized to reactive inter-
mediates that can form adducts with DNA [51] but retain
considerable affinity for the estrogen receptor [52]. Such
potentially mutagenic molecules could then be targeted by
the estrogen receptor in target cells to the promoters of
those regulatory genes that drive estrogen-dependent
growth. These possibilities need to be probed in future stud-
ies.
In summary, the synthetic estrogen DES was more active
than the natural estrogen E2 as a neonatal endocrine dis-
ruptor agent in the female hamster. The impact of this phe-
nomenon was very dramatic in the uterus, where the neo-
natal DES insult initiated alterations in early uterine devel-
opment that were strongly promoted by estrogen stimula-
tion in adulthood. The DES-dependent uterine lesions were
most striking in the endometrial epithelial cell compart-
ment. They consisted of hyperplasia and apoptotic activity
during the preneoplastic stage, whereas apoptosis appeared
to be lost at sites of neoplastic progression. These morpho-
logical abnormalities were accompanied by altered expres-
sion of the estrogen-responsive gene LTF. More recently,
we have determined that DES is also much more active
than E2 as a neonatal endocrine disruptor in the reproduc-
tive organs of male hamsters [53]. On the basis of these
and previous observations, the hamster appears to be a par-
ticularly valuable experimental system in which to screen
compounds for potential endocrine-disruptive activity in
both sexes and then probe their mechanism of action.
ACKNOWLEDGMENTS
We gratefully acknowledge the excellent processing of histology sam-
ples by Pathology Associates International, Jefferson, AR. We also thank
Dr. Christina T. Teng for the gift of the anti-LTF antiserum and Dr. Karen
L. Brown for valuable guidance in performing statistical analyses.
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