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[CANCER RESEARCH 43, 4879-4884,
October 1983]
Transplacental Action of Diethylstilbestrol on Mammary
Carcinogenesis in Female Rats Given One or Two
Doses of 7,12-Dimethylbenz(a)anthracene1
Elizabeth S. Boylan2 and Robert E. Calhoon
Department of Biology of Queens College and the Graduate School of the City University oÃ-New York, Flushing, New York 11367
ABSTRACT
Aspects of the development, morphology, and estrogen bind
ing capacity of mammary tumors in rats exposed prenatally to
the synthetic estrogen, diethylstilbestrol (DES), and treated postnatally with 7,12-dimethylbenz(s)anthracene
(DMBA) were ana
lyzed as part of a project aimed at understanding the effects of
transplacental exposure to DES on estrogen-sensitive tissues.
Pregnant Sprague-Dawley rats were given injections of DES
(total dose, 1.2 ¿¿g)
or vehicle alone on Days 15 and 18 of
gestation. All female offspring were given gastric intubations of
DMBA, either a single 10-mg dose on Day 50 or two doses (10
mg each) on Days 50 and 57. Among rats treated postnatally
with 10 mg of DMBA, the DES-exposed group had a significantly
greater incidence of palpable mammary tumors than did the
vehicle-exposed controls. In addition, there was an earlier time
of appearance of palpable tumors in the DES-exposed group.
When the data from rats treated postnatally with two 10-mg
doses of DMBA were analyzed, there were no significant differ
ences in palpable mammary tumor incidence or tumor latency
between the DES-exposed and vehicle-exposed groups. When
the pathology of the mammary tumors produced in rats treated
with 10 mg of DMBA was analyzed, the DES-exposed group
had a significantly higher proportion of benign tumors (fibroad
enoma, adenoma, lobular hyperplasia) than adenocarcinomata
compared to vehicle-exposed controls. Both exposure groups
had similar numbers of nonpalpable mammary lesions discovered
at necropsy. Estrogen binding capacities of representative ad
enocarcinomata did not differ significantly between the two pre
natal exposure groups treated postnatally with 10 mg of DMBA.
These results demonstrate the importance of the dose of the
challenge carcinogen in revealing the effects of transplacental
drug exposure and may have special significance for women
who were exposed to DES in utero.
INTRODUCTION
The anatomical development of the mammary gland and the
incidence of mammary tumors in rodents are known to be
influenced by exogenous estrogenic hormones administered in
the perinatal period. We have shown previously that prenatal
exposure to the synthetic estrogen DES3 had teratogenic effects
1This work was supported in part by Grant CA-18458 from the National Cancer
Institute (USPHS), Grant RR-07064 from the NIH BiomédicalResearch Program,
and Grant 11626 from the PSC-CUNY Research Award Program of the City
University of New York. Grants from the City University Research Project were
used for computer analysis of the data.
2To whom requests for reprints should be addressed, at Department of Biology,
Queens College of City University of New York, 65-30 Kissena Blvd., Flushing,
N. Y. 11367.
3 The abbreviations used are: DES, diethylstilbestrol; DMBA, 7,12-dimethylbenz(a)anthracene.
Received October 5,1982; accepted June 30,1983.
on nipple structure in neonatal rats (3) and altered incorporation
of [3H]uridine in the primary mammary duct epithelium of 5-dayold pups (2). Others have observed nipple malformations in
fetuses or newborn rats exposed to large doses of estradici
dipropionate (10, 16) or DES (9). Using mice, Jean (14) found
persistent nipple and mammary gland abnormalities in DESexposed offspring examined at 80 days of age; Raynaud (25)
described teratogenic effects on nipple structure of mouse fe
tuses when estradici dipropionate was injected directly into 12to 15-day embryos; and Yasuda ef al. (37) demonstrated a
hypertrophie effect of ethinyl estradiol (2 mg/kg) on the nipples
of female offspring. Mammary gland branching was inhibited by
prenatal exposure to estradiol dipropionate in mice examined as
neonates; this effect was overcome with time, since no effect
was observed when similarly treated mice were examined at
older ages (15). The effect of prenatal exposure to DES on
mammary tumorigenesis has been studied in several rodent
systems. Exposure of rat fetuses to DES (1 mg/kg) resulted in
an incidence of mammary fibroadenomata of 4 of 18 DESexposed rats versus 3 of 34 control rats at an average age of
approximately 2 years (23). Increased mammary tumor multiplic
ity was reported in hamster offspring exposed prenatally to DES
and treated postnatally with DMBA (28). However, mammary
tumorigenesis in the mouse was suppressed by prenatal expo
sure to DES on Day 12 of gestation; exposure on Day 17 had
no effect (21).
Compared to the results when estrogens were used prenatally,
opposite effects on mammary tumorigenesis have been reported
when rats or mice were treated neonatally with estrogens. Thus,
in rats where prenatal exposure to DES increased mammary
tumor incidence, 17/3-estradiol or estradiol benzoate on Days 2
or 5 after birth resulted in suppression of mammary tumors
induced with DMBA (30,38). Furthermore, treatment of rats with
estradiol by injections on Days 1 to 30 or by an intrahypothalamic
implant inhibited or delayed mammary tumor appearance (13,
22). In mice, where early prenatal exposure to DES suppressed
mammary tumorigenesis, neonatal treatment with 170-estradiol
on Days 1 to 5 generally resulted in the stimulation of mammary
gland growth, differentiation and hormonal sensitivity, and an
increase in mammary dysplasias in older animals (17, 18, 3236); however, neonatal DES treatment inhibited duct branching
in 6- and 33-day-old mice (32). Thus, mice and rats appear to
differ in the period when perinatal hormone exposure influences
the later development of proliferative abnormalities in the mam
mary gland. In addition, some of the apparently contradictory
reports in the literature may have resulted from the use of both
natural and synthetic estrogens which differ in their affinity for
serum binding proteins, such as a-fetoprotein (8,29). For further
review of the literature on perinatal hormone treatment and
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£.S. Boylan and R. E. Calhoon
mammary tumorigenesis in rodents, see Mori ef al. (20).
Previously, we reported increased mammary tumor multiplicity
in rats exposed prenatally to DES and treated postnatally with 2
doses of 10 mg of DMBA (4). However, a subsequent
experiment4 where offspring were treated with a single dose of
15 mg of DMBA failed to confirm these results, with the DBSexposed and vehicle-exposed groups showing indistinguishable
tumor incidences (3.5 versus 3.6 tumors per rat) and percentages
of tumor-bearing rats (68 versus 60%). Here, we present results
of an experiment which directly tested the effect of the dose of
carcinogen used after transplacental exposure to DES on various
aspects of mammary tumor development. Data reported here
support the position that prenatal exposure to DES has a stim
ulatory effect on mammary tumorigenesis when a low dose of
DMBA is used. Use of the high dose of DMBA masked the effect
of DES. Further analysis of these data in relation to our earlier
paper is presented in the "Discussion." Animals whose mammary
tumors are described here were subjected to necropsy; data on
their reproductive and endocrine organs and serum hormone
levels are presented in a companion paper (6).
MATERIALS AND METHODS
Prenatal Exposure. Pregnant CD rats (Charles River Breeding Labo
ratories, Inc., Wilmington, Mass.) were assigned randomly to 2 treatment
groups: 18 rats were given injections s.c. of DES (Sigma Chemical Co.,
St. Louis, Mo.) dissolved in sesame oil on Days 15 and 18 (2 injections
of 0.6 Mg in 0.3 ml of sesame oil = 1.2 ^g of DES, total dose); 10 rats
received 2 injections of vehicle only. Animals were allowed to deliver and
raise their offspring to weaning. Large litters were reduced to no more
than 10 pups by removing male offspring. After weaning, female offspring
were housed 5 per cage. Details regarding animal care can be found in
a companion paper (6).
Postnatal Treatment. One-half of the 64 DES-exposed females and
one-half of the 59 vehicle-exposed females received one dose of DMBA
(Eastman Organic Chemicals, Rochester, N. Y.; 10 mg in 1 ml of sesame
oil) at 50 days of age, while the other half of each exposure group
received 2 doses of DMBA (10 mg each) on Days 50 and 57. DMBA
was dissolved in oil, stirred in the dark overnight to achieve complete
solubilization, and used the following day. A repeating syringe fitted with
an 18-gauge intubation needle was used to perform the gastric intuba
tions. The average weight of the rats was approximately 175 g; therefore,
each 10-mg dose of DMBA was the equivalent of about 60 mg of DMBA
per kg of body weight. Beginning 5 weeks after the first intubation with
DMBA and continuing until the animals reached 9 months of age,
palpations were performed weekly to determine the time of appearance
of each mammary tumor. Animals which were clearly moribund before
the end of the experiment were sacrificed so that tissues could be
obtained. At sacrifice, all palpable and nonpalpable mammary tumors
were removed and weighed. Where possible, a portion of each tumor
was frozen in liquid nitrogen and placed in a Reveo -80° freezer (Reveo,
Inc., West Columbia, S. C.) until assayed for estrogen binding capacity.
The remaining tumor tissue was fixed in neutral buffered formalin,
dehydrated in Cellosolve (ethylene glycol monoethyl ether) and toluene,
embedded in paraffin, and stained with hematoxylin and eosin.
Tumor Pathology. Palpable and nonpalpable mammary tumors were
categorized according to the guidelines of Young and Hallowes (39).
Tumors the morphology of which could not be readily classified were
examined by a veterinary pathologist.
Assay for Estrogen Binding Capacity. Cytosols were prepared from
thawed mammary tumors using a Brinkmann (Westbury, N. Y.) polytron
homogenizer and a Beckman (Palo Alto, Calif.) L5-65 ultracentrifuge with
a type 40 rotor. Binding capacity was measured using the dextran4 E. Boylan and R. Calhoon, unpublished data.
4880
coated charcoal procedure exactly according to the directions and with
the reagents provided in the New England Nuclear (Boston, Mass.)
estrogen receptor assay kit.
Statistical Methods. In all cases, the experimental unit was the litter.
All statistical tests, where litters were nested in treatments, were con
ducted using a modified version of the methods proposed by Peto (24)
for nonincidental tumors. These methods permitted the calculation of
expected numbers of tumors per litter, which compensated for unequal
numbers of rats per litter and also for rats which died during the course
of the experiments. The first modification involved the assumption of
independence of tumors within litters. It has been reported (1, 19) that
métastases have not been observed in rats bearing DMBA-induced
mammary tumors. Therefore, we made the statistical assumption that,
within litters, mammary tumors occurred randomly and independently,
and hence, all palpable mammary tumors were included in the analysis.
The second modification was to substitute the log likelihood ratio test
for x2. This statistic is distributed as x2 and appears to follow the
probability density function more closely than other computational pro
cedures (31). The cumulative incidence of tumors in the DES-exposed
litters was compared to that for litters exposed to vehicle only; rats
receiving 10 mg of DMBA and two 10-mg doses of DMBA were analyzed
separately. The cumulative tumor incidences for the intervals of 1 to 10,
11 to 20, and 21 to 29 weeks were partitioned and analyzed independ
ently.
Analysis of time of appearance of mammary tumors required a further
modification of the analytical methods. Here, the observed and expected
numbers of tumors were reduced to two 29 x 2 matrices and used to
test for the independence of time of appearance and prenatal treatment.
In any week in which no new tumors were detected, 1 d.f. was subtracted
from the total; i.e., weeks with expected values of less than one were
pooled.
Both the incidence of death and the time of death were analyzed using
the Peto method for incidental events. This permitted testing hypotheses
for differences in relative number of deaths between DES and vehicleexposed litters, as well as the independence of time of death and prenatal
treatment.
Tumors which had been sectioned were classified as either adenocarcinoma or fibroadenoma and adenoma. Expected values for adenocarcinoma and fibroadenoma and adenoma were calculated independently;
the log likelihood ratio statistics were calculated separately and then
pooled. Therefore, the independence of pathology versus prenatal treat
ment could be tested with 2 d.f.
RESULTS
Tumor Incidence and Time of Appearance. Chart ÃŒA
pre
sents data on the cumulative incidence of palpable mammary
tumors from rats exposed prenatally to DES or vehicle, then
treated postnatally with 10 mg of DMBA, while Chart 1S shows
data on comparable groups of rats treated postnatally with two
10-mg doses of DMBA. It is evident that differences associated
with prenatal exposure to DES could be observed only when the
lower dose of DMBA was used. In Table 1, the increments in
palpable mammary tumor incidence from 1 to 10, 11 to 20, and
21 to 29 weeks following DMBA treatment are presented and
analyzed. When 10 mg of DMBA were used, statistically signifi
cant differences in the observed number of tumors between
DES-exposed and vehicle-exposed animals were found in the
intervals of 1 to 10 and 11 to 20 weeks post-DMBA. No signifi
cant difference was found when tumors appearing during the
21- to 29-week interval were analyzed. At sacrifice or death, the
mean number of palpable mammary tumors per rat was 4.3 ±
2.9 (S.D.) in the DES-exposed group, while the controls had only
2.8 ±2.2 tumors/rat. The distribution of the number of tumors
CANCER
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VOL. 43
Mammary Tumors in DES-exposed, DMBA-treated Rats
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15
WEEKS AFTER
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20
25
30
INTUBATION
WEEKS AFTER
INTUBATION
Chart 1. Cumulative incidence of palpable mammary tumors in rats exposed prenatally to DES (•)
or vehicle (O) and treated postnatally once (A) or twice (8) with
DMBA. Calculation maintains incidenceof tumors in animals which died prior to the end of the experiment. For incidenceof new tumors by time intervals and statistical
analysis, see Table 1.
5
Table 1
Incidence of new palpable mammary tumors following DMBA treatment in rats exposed prenatally to DESor
vehicle
Incidence of new palpable mammary tumors in time intervals (wk)
following DMBA intubation
Wk 1-10
Prenatal exposure, postnatal
treatment8
Wk 21-29*
Observed
Expected
Observed
Expected
Observed
Expected
29
10
20.1
18.9
70
42
55.7
56.3
20
23
21.2
21.8
DES, 10 mg of DMBA (nc = 32)
Vehicle, 10 mg of DMBA
(n = 30)
Statistic0
Wk 11-20
G = 8.55e (1 d.f.)
DES,
ofDMBA
two 10-mg doses
64.351
G = 7.43e (1 d.f.)
82.280
G = 0.13' (1 d.f.)
22.325
32)Vehicle,(n =
ofDMBA
two 10-mg doses
29)Statistic73
(n =
59.7G
84.8G
= 2.48' (1 d.f.)87
= 0.55' (1 d.f.)23
25.7G
= 0.04' (1 d.f.)
DES (total dose, 1.2 ng) or vehicle given to pregnant rats on Days 15 and 18 of gestation. Offspring
¡ntubatedwith DMBA as a single 10-mg dose or two 10-mg doses 1 week apart. DMBA treatments begun
on "Day
50. palpableonly after sacrifice in Week 30 were not included ¡n
Tumors
the analysis.
0 n, group size at time of intubation.
''G statistic is distributed as x2 and exhibits better continuity than other calculating procedures. See
"Statistical Methods."
" Significantly different from vehicle-exposedcontrol, p £0.01.
' Not significantly different from vehicle-exposedcontrol.
per rat for the DES-exposed
versus vehicle-exposed
groups
treated with 10 mg of DMBA is illustrated in Chart 2. The shift
in distribution from predominantly 0 and 1 tumor per rat in the
controls to 4 and 5 tumors per rat in the DES-exposed group is
evident and is reflected in the difference between mean number
of tumors per rat described above. Although the DES-exposed
group contained more tumor-bearing rats (30 of 32, 94%) than
did the vehicle-exposed group (22 of 30, 73%), the difference
was not statistically significant when the analysis compensated
for the nesting relationship of rats within litters.
When rats were treated with 2 doses of DMBA (10 mg each,
1 week apart), the cumulative incidence of tumors was substan-
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4881
E. S. Boy Ian and R. E. Calhoon
tially greater than after a single dose of DMBA (cf. Chart 1, A
and B); and the total tumor incidence in Weeks 1 to 29 postDMBA was elevated significantly in groups which had received
two 10-mg doses of DMBA compared to the single-dose groups
(G = 79.41, p «0.01). The mean number of tumors per rat at
sacrifice was 5.7 ±3.9 for the DES-exposed rats and 5.9 ±3.7
for the vehicle-exposed rats, and all rats in both groups had at
least one tumor. When the increments in tumor incidence were
analyzed for both exposure groups treated with two 10-mg
doses of DMBA (Table 1), no significant differences were found
in the 1- to 10-, 11- to 20-, or 21- to 29-week intervals.
Prenatal exposure to DES accelerated the time of appearance
of palpable mammary tumors in rats treated with 10 mg of DMBA
but not in rats treated with two 10-mg doses of DMBA (Table
2). The difference in mean time of appearance between the DESexposed and control groups was 2.3 weeks when 10 mg of
DMBA were used and 1.1 weeks when two 10-mg doses of
DMBA were given. Overall, tumors appeared 2.1 weeks earlier
in groups treated with two 10-mg doses of DMBA than in groups
treated with 10 mg of DMBA.
Some rats from all groups died before the end of the experi
ment. Irrespective of the dose of DMBA, DES-exposed groups
exhibited greater mortality prior to termination of the experiment,
but the differences were not statistically significant. Treatment
with two 10-mg doses of DMBA more than doubled the death
VEHICLE-EXPOSED
.ll.l
4
6
8
IO
I2+-
NUMBER OF PALPABLE MAMMARY TUMORS PER RAT
Chart 2. Distribution of the numbers of palpable mammary tumors in rats
exposed prenatally to DES or vehicle and treated postnatally with 10 mg of DMBA.
Table 2
Timeof appearance of palpable mammary tumors
Prenatal exposure, postnatal treatment"
Wk of appearance
DES, 10 mg of DMBA (n°= 32)
Vehicle, 10 mg of DMBA (n = 30)
Statistic0
14.7 ±5.9°
17.0 + 6.4
G = 39.78e (23 d.f.)
DES, two 10-mg doses of DMBA
13.2 ±5.7
(n = 32)
Vehicle, two 10-mg doses of DMBA
14.3 ±5.8
G = 33.02' (24 d.f.)
Statistic
" DES (total dose, 1.2 ^g) or vehicle given to pregnant rats on Days 15 and 18
of gestation. Offspring were intubated with DMBA as a single 10-mg dose or two
10-mg doses 1 week apart. DMBA treatments begun on Day 50.
" n, group size at time of intubation.
0 Mean ±S.D.
"G statistic is distributed as x2 and exhibits better continuity than other
calculating procedures. See "Statistical Methods."
8 Significantly different from vehicle-exposedcontrol, p < 0.05.
' Not significantly different from vehicle-exposedcontrols.
4882
tumors, both exposure groups had similar numbers of these
small lesions, and the proportion of tumors of adenocarcinomata
to fibroadenomata plus adenomata did not differ (data not
shown).
Specific binding of 17/ì-[3H]estradiolin the cytosol of repre
sentative mammary tumors did not differ significantly between
exposure groups. The mean binding capacity of 15 adenocarci
nomata from 7 DES-exposed litters was 36.2 ±3.2 fmol/mg
cytosol protein (range, 7.8 to 138.2), while in the 4 vehicleexposed litters, specific estrogen binding in 16 adenocarcinom
ata was 28.9 ±3.0 fmol/mg cytosol protein (range, 8.8 to 102.7).
Dissociation constants varied between 1.0 x 10~10 to 6.7 x
DES-EXPOSE
•lililÃ-..!.
2
cinomata to fibroadenomata plus adenomata compared to the
vehicle-exposed group. In terms of the nonpalpable mammary
DISCUSSION
• mm
0
rate compared to treatment with a single dose (for DES, 10 of
32 or 31% at 10 mg of DMBA versus 21 of 32 or 66% at two
10-mg doses of DMBA; for vehicle, 4 of 30 or 13% at 10 mg of
DMBA versus 14 of 29 or 48% at two 10-mg doses of DMBA).
Tumor Pathology and Estrogen Binding Capacity. In both
exposure groups treated postnatally with 10 mg of DMBA,
palpable and nonpalpable mammary tumors were examined
microscopically. When the effect of prenatal exposure to DES
on the pathology of palpable mammary tumors was analyzed,
significant differences were found (Table 3). The number of
palpable adenocarcinomata in the DES-exposed group was sig
nificantly greater than in the vehicle-exposed group (G = 6.78, p
< 0.01), as was the number of palpable fibroadenomata and
adenomata (G = 14.57, p < 0.01 ). Among the palpable mammary
tumors, the DES-exposed group had a lower ratio of adenocar
When the 10-mg dose of DMBA was administered to 50-dayold rats, effects of transplacental exposure to DES were ex
pressed in the offspring as an increased incidence of palpable
mammary tumors, detectable by the end of the tenth week
following carcinogen treatment. During the following 10 weeks,
the new tumors discovered in the DES-exposed group also
exceeded those found in the control group. However, for the
last 9 weeks of palpation, there was no detectable difference in
the rate at which palpable tumors appeared in the 2 exposure
groups. Thus, the effect of prenatal exposure to DES is to
accelerate the appearance of mammary tumors; rats which
escape the effect of DES on this early tumor induction phase
develop palpable mammary tumors at a similar rate and com
parable incidence to vehicle-exposed rats treated with 10 mg of
DMBA.
This sensitizing effect of DES on the induction of tumors by
DMBA was not observed when the dose of carcinogen was
doubled. Although the higher dose was more efficient in tumor
induction, use of this dose prevented the increase in tumor
incidence related to DES exposure from being detected. There
fore, to elicit a response of an increased incidence of palpable
mammary tumors in DES-exposed rats, the amount of DMBA
used must be below the level which results in 100% tumorbearing animals. Our earlier report (4) describing an increased
tumor multiplicity in DES-exposed, DMBA-treated rats used a 2dose, 10-mg regimen of DMBA. However, there were several
groups of animals involved which were treated with DMBA over
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VOL. 43
Mammary Tumors in DES-exposed, DMBA-treated Rats
Tables
Pathology of palpable mammary tumors in rats exposed prenatally to DES or vehicle and treated postnatally
with WmgofDMBA
tumors8Prenatal
No. of palpable mammary
and
adenoma"Observed38
exposureDES
Expected99
(n = 32)
Vehicle (n = 30)
of
observed
adenocarcnoma
to observed
fibroadenoma
and
adenoma2.6
82.3
67 83.7G
Statistic0AdenocarcinomaObserved = 6.78
12G
25.2
5.6
= 14.57Ratio
G = 21 .35"Fibroadenoma
e(2d.f.)Expected24.8
'' Includes all palpable tumors found prior to and after sacrifice at 30 weeks post-OMBA.
" Also includes lobular hyperplasia. hyperplasia with duct ectasia.
c G statistic is distributed as x2 and exhibits better continuity than other calculating procedures.
"Statistical Methods."
" Sum of the independently derived statistics for the 2 pathology categories (G = 6.78 + 14.57).
" Significantly different from vehicle-exposed control, p s 0.01.
a period of weeks, and the DMBA was not made up just prior to
each intubation, as in the present experiment. (In both cases,
however, the solution was kept in a foil-wrapped container to
prevent photodegradation.) Thus, we propose that the apparent
discrepancy in results between the earlier published report and
data presented here is related to a lower effective dose of DMBA
present in the solution used in the first experiment. The experi
ment using a single 15-mg dose of DMBA which resulted in no
difference in tumor incidence between the DES-exposed and
vehicle-exposed groups (see "Introduction") can also be viewed
as support for the necessity of lowering the dose of carcinogen
used to challenge animals exposed transplacentally to drugs
previously. It should also be noted here that the 2 studies on
rats which combined neonatal treatment with sex steroids and
subsequent DMBA intubations used a single 20-mg dose of
DMBA (30, 38). Since these experiments showed a reduction in
the percentage of tumor-bearing rats (30, 38) and number of
mammary tumors per rat (38) after neonatal treatment with
estradiol benzoate or 17/3-estradiol, it would be of interest to
determine whether a lower dose of DMBA confirms the inhibitory
action of neonatal estradiol treatment on chemically induced
mammary tumorigenesis.
When the histological type of the palpable mammary tumors
was considered, prenatal exposure to DES altered the ratio of
adenocarcinomata to benign mammary tumors. While, in abso
lute terms, more adenocarcinomata were induced in the DESexposed group, they comprised only 68% of all palpable mam
mary tumors. The higher proportion of benign lesions in the DESexposed group may be due, in part, to the fact that more DESexposed rats died prior to termination of the experiment. If some
of the tumors classified as adenomata in the DES-exposed group
would have progressed to become adenocarcinomata given
additional time, the opportunity for these tumors to develop into
adenocarcinomata was lost when the host died before the pro
gression was complete. It is interesting that Shellabarger and
Soo (30) also found a similar disturbed proportion of adenocar
cinomata to fibroadenomata in rats treated neonatally with estra
diol benzoate followed by DMBA compared to the proportion in
the DMBA-treated controls; this is in spite of the fact that the
absolute numbers of mammary tumors per group did not differ
significantly in this experiment.
These dramatic effects associated with the transplacental
OCTOBER
See
action of DES on mammary tumorigenesis should be examined
in light of DES-related effects on other estrogen target tissues.
The morphology of the reproductive organs, the pituitary and
adrenal glands, and nontumorous mammary gland tissue was
examined thoroughly in these animals; these data are presented
in a separate paper (6). By comparison to effects observed on
mammary tumorigenesis, DES-associated alterations of the
structure and function of reproductive and endocrine organs
were relatively minor. The most unexpected finding was probably
that, at the age when DMBA was given, DES-exposed rats had
lower mean serum prolactin levels than did vehicle-exposed
controls. From reports which linked DMBA susceptibility of var
ious rat strains to the presence of elevated serum prolactin levels
(7, 12), one might have predicted that DES-exposed rats would
have increased levels of prolactin in their serum.
It was also surprising that significant differences in mammary
gland morphology could not be demonstrated between exposure
groups at 2 months of age, whether the tissue was prepared
and analyzed as whole mounts or in histological sections. Russo
and coworkers (26,27) demonstrated that DMBA has its greatest
tumor-initiating capacity when the mammary gland tissue still
has a high density of terminal end buds prior to differentiation
into alveolar buds and lobules, a state which normally occurs
when an animal is 7 to 8 weeks old. Since the DES-exposed
rats developed many more palpable mammary tumors after
treatment with 10 mg of DMBA than did the vehicle-exposed
controls, it seemed reasonable to expect that differences in
mammary gland morphology would be evident at the stage when
DMBA was administered, but this was not the case.
Thus, we can conclude that there is an effect on the mammary
gland associated with transplacental exposure to DES, which
results in the appearance of more mammary tumors when rats
are treated postnatally with 10 mg of DMBA; however, the
mechanism by which DES acts on the mammary gland is still to
be determined. It is essential to bear in mind that these DESexposed animals were normal with respect to fertility and ovar
ian-vaginal cyclicity (3, 6) and that DES exposure alone was not
associated with an increased incidence of mammary tumors in
rats up to 14 months of age (4). We also have demonstrated
that the growth of DMBA-induced tumors in DES-exposed rats
was as likely to be ovarian dependent as that in DMBA-treated
controls, although tumors in the DES-exposed group tended to
1983
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E. S. Boylan and R. E. Calhoon
enter a new phase of growth following
ovariectomy-induced
regression (5).
These findings may have special significance for women who
were exposed to DES in utero. Although there is no evidence to
date of an increased incidence of breast cancer among DBSexposed women (11), these young women have yet to reach the
age range when the risk for breast cancer among American
women is highest. American women in general have a chance of
1 of 11 of developing breast cancer and are exposed to many
known carcinogens and tumor promoters at low but persistent
levels in the American diet and environment. In light of such
epidemiological findings and these data on DES-exposed ro
dents, DES-exposed women may be at increased risk for breast
cancer.
ACKNOWLEDGMENTS
For her contributions to all aspects of this project, the authors express their
thanks to Jacqueline A. Doody. The assistance of Philip Romm and Chañaran
Shanmugam in the detection of tumors and in the preparation of tumor slides is
gratefully acknowledged. The cooperation of Dr. Edward H. Fowler in the analysis
of tumor pathology is also acknowledged with thanks. During preparation of the
manuscript, the authors were aided by helpful comments of Dr. Barbara K.
Vonderhaar and by the excellent secretarial skills of Sylvia Schatfel.
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VOL. 43
Transplacental Action of Diethylstilbestrol on Mammary
Carcinogenesis in Female Rats Given One or Two Doses of
7,12-Dimethylbenz(a)anthracene
Elizabeth S. Boylan and Robert E. Calhoon
Cancer Res 1983;43:4879-4884.
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