(CANCER RESEARCH 50, 691-699, February 1, 1990]
Induction of Dorsolateral Prostate Adenocarcinomas and Other Accessory Sex
Gland Lesions in Male Wistar Rats by a Single Administration of /V-Methyl/V-nitrosourea, 7,12-Dimethylbenz(o)anthracene, and 3,2'-Dimethyl-4aminobiphenyl after Sequential Treatment with Cyproterone Acetate
and Testosterone Propionate1
Maarten C. Bosland2 and Menk K. Prinsen
Department of Biological Toxicology, TNO-CIVO Toxicology and Nutrition Institute, P. O. Box 360, 3700 AJ Zeist. The Netherlands
ABSTRACT
Groups of 20-25 male Wistar rats (Cpb:WU), nine groups of 4-weekold rats, and nine groups of 8-week-old rats, were given cyproterone
acetate (CA) s.c. or by gavage daily for 18 days at a dose of 50 mg/kg/
day. Directly following CA treatment, the rats received 3 daily s.c.
injections with testosterone propionate (TP) at a dose of 100 mg/kg/day.
On the day after the last TP administration, a single dose of one of the
following carcinogens was given to 3 groups: /V-methyl-/V-nitrosourea
(MNU), 50 mg/kg i.V.; 7,12-dimethylbenz(a)anthracene, 30 mg/kg i.V.;
3,2'-dimethyl-4-aminobiphenyl, 250 mg/kg s.c. Three other groups re
ceived the same carcinogen treatments after 7 days of recovery from the
CA administration. The last 3 groups received carcinogen without TP
treatment, but immediately after CA pretreatment was stopped. A 25%
incidence of invasively growing, metastasizing adenocarcinomas was
found in the dorsolateral prostate region of 8-week-old rats that had
received MNU after treatment with CA plus TP. In addition, this group
had a 5% incidence of carcinoma in situ and a 5% incidence of atypical
hyperplasia in the dorsolateral prostate. Lower incidences of adenocarcinoma of the dorsolateral prostate region and of carcinoma in situ and
atypical hyperplasia of the dorsolateral prostate were found in other
groups that were treated with MNU or 7,12-dimethylben7.(a)anthracene
after pretreatment with CA, followed by TP or recovery, but never in rats
that had been treated with CA only. In the groups treated with 3,2'dimethyl-4-aminobiphenyl, which is slowly metabolized, these lesions
were also found in groups that were pretreated with only CA. The
carcinomas seemed to originate from the dorsolateral prostate and their
average latency time was approximately 61 weeks. The 8-week-old rat
given a MNU injection after sequential treatment with CA and TP may
provide a relevant animal model for human prostatic cancer.
INTRODUCTION
Prostate cancer is one of the main human cancers in the
Western world. The etiology of this neoplastic disease is essen
tially unknown (1-3). Research on prostatic carcinogenesis has
been impeded by a lack of adequate and reliable animal models.
Growth of prostatic carcinomas beyond the phase of progres
sion to invasively growing carcinoma can be studied in a few
transplantable systems (4-6). However, certain factors influ
encing the transition of early noninvasive latent carcinomas
into invasive adenocarcinomas are thought to be decisive for
prostatic cancer risk in humans (1,7, 8); specifically a role for
diet and sexual factors has been suggested in this respect (1-3,
9. 10). Therefore, it is essential that appropriate animal models
Received 2/16/89; revised 6/9/89. 9/25/89; accepted 10/26/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by Grants CIVO 80-3 and CIVO 84-3 from The Nether
lands Cancer Foundation. Koningin Wilhelmina Fonds.
2 Present address: Institute of Environmental Medicine, New York University
Medical Center, 550 First Avenue. New York, NY 10016. To whom requests for
reprints should be addressed. This work was submitted as part of a dissertation
to the State University of Utrecht. The Netherlands, in partial fulfillment of the
requirements for the Doctorate Degree.
of prostatic carcinogenesis possess identifiable early neoplastic
stages that allow the study of such factors.
Spontaneous prostatic adenocarcinomas have been reported
in some rat strains in aging animals (11-14). In most strains,
these lesions develop in the ventral prostate lobes (11-13), for
which there is no human homologue (15, 16). Furthermore,
they occur only at a very old age (12-14) and sometimes require
germ-free conditions (14). These features, site, long latency,
and germ-free status, are not desirable for a practical and
appropriate animal model for prostatic carcinogenesis. Hor
monal induction of prostatic cancer reported in some rat strains
(5, 17) involves lasting, profound changes in the endocrine
milieu, notably long-term treatment with androgens at high
doses. Since at least some modifying factors of prostatic carci
nogenesis are believed to act by affecting relevant hormonal
systems (1-3), hormonal induction of prostatic cancer is not a
very appropriate model for studying prostatic carcinogenesis
and modification thereof.
Fingerhut and Veenema (18) reported carcinomas induced
by DMBA1 in gonadectomized animals having atrophied pros
tates, but this has not been reproducible (19).4 Recently, hyperplastic epithelial changes of the rat prostate were described after
repeated systemic administration of /V-nitrosobis(2-oxopropyl)amine (20). However, these hyperplasias occurred in the
ventral prostate and developed into squamous cell carcinomas
and not adenocarcinomas. Repeated parenteral injection of
DMABP resulted in epithelial proliferative lesions of the ven
tral prostate classified as adenocarcinoma in situ in a 33%
incidence (19, 21).
A well-known phenomenon in chemical carcinogenesis is the
enhancing effect of cell proliferation in the target tissue during
treatment with carcinogens leading to fixation of promutagenic
DNA lesions (22, 23). In the rodent ventral prostate, but also
in other accessory sex glands in males, cell proliferation can be
induced by daily administration of androgens to animals that
are castrated 7-30 days previously (24, 25). In this system, the
rate of cell proliferation usually reaches a peak on the fourth
day of androgen administration (24). We applied temporary
chemical castration rather than surgical castration and gave a
single carcinogen administration after three daily testosterone
injections following cessation of the chemical castration. This
ensured an intact status of the animals during the promotion
and progression stages of prostatic carcinogenesis. In addition,
the effects of this treatment on rats that were prepubertal at the
start of the CA treatment and that had juvenile prostates were
compared with those on young adult rats with actively secreting
prostates. The carcinogens arbitrarily selected for this study are
'The abbreviations used are: DMBA, 7.12-dimethylbenz(a(anthracene;
CA,
cyproterone acetate; DMABP. 3.2'-dimcthyl-4-aminobiphenyl; MNU. iV-methylA'-nitrosourea; TP. testosterone propionate.
4 M. C. Bosland, unpublished observations.
691
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CHEMICAL INDUCTION OF RAT PROSTATE ADENOCARCINOMAS
Table I Summary of experimental protocols in Experiments A, B, and C
Group012
at start of
experiment
(wk)4
19-21: TP
1-18:CA
22: Carcinogen
Days 1-18: CA
Days 19-21: TP
8
Day 22: Carcinogen
Days
19-25:
RCC
Days 1-18: CA
4
Day 26: Carcinogen
34
Days 19-25:RC
Days 1-18: CA
Day 26: Carcinogen
8
Days 1-18: CA
Day 19: Carcinogen
4
5
Days 1-18: CATreatment*Days Day 19: CarcinogenDay
8Days
6Age
•¿
N = 20 (Experiments A and B). or 25 (Experiment C).
*CA, 50 mg/kg/day, by s.c. injection, dissolved in oil (Experiments A and B). or by gavage, suspended in water (Experiment C): TP, 100 mg/kg/day, by s.c.
injection, dissolved in oil; carcinogen: Experiment A, MNU, 50 mg/kg i.V., in saline (pH 5); Experiment B, DMBA, 30 mg/kg i.V., in fat emulsion; Experiment C,
DMABP. 250 mg/kg s.c., in oil.
c RC, recovery (no treatment) after 18 days of CA exposure.
The dosages of CA and TP and the duration of the treatment and
the recovery period for Groups 3 and 4 of each experiment were chosen
on the basis of data from pilot experiments on the effects of CA and
TP on accessory sex gland and body weights (see "Results" and Ref.
known pluripotent carcinogens in rats, belonging to three cat
egories of chemical carcinogens: MNU, DMBA, and DMABP.
DMABP has also been claimed to induce in situ adenocarcinomas of the ventral prostate in F344 rats (19). The effects of
MNU in 8-week-old rats have been published previously (26);
the data from that paper have been reevaluated and are incor
porated in the present report.
MATERIALS
AND METHODS
Animals. Male random-bred specific-pathogen-free Wistar rats
(Cpb:WU) were obtained from the Central Institute for the Breeding
of Laboratory Animals (CPB-TNO), Zeist, The Netherlands, and were
used after at least 1 week of adaptation. They were housed under
conventional conditions, 5 to a cage, in stainless steel wire mesh cages,
in a well-ventilated room, at an ambient temperature of 23 ±1°Cand
50 ±10% (range) relative humidity, lighted 12 h/day. The rats had ad
libitum access to an in-house prepared natural ingredient diet and to
tap water, both regularly screened for a large number of contaminants.
At 4 or 8 weeks of age, the animals were randomly assigned to the
various experimental groups as indicated below.
Chemicals. CA (CAS 427-51-0; Schering, Berlin, Federal Republic
of Germany) was either used as the pure compound, dissolved in benzyl
benzoate and Ricinus oil (1:5, v/v) at a concentration of 85 mg/ml, or
as Androcur tablets (50 mg CA/tablet) dispersed in tap water. A TP
(CAS 57-85-2) solution in oil (50 mg/ml; Neohombreol) was obtained
from Organon, Oss, The Netherlands. A 15% fat emulsion containing
5 mg/ml DMBA (CAS 57-97-6; Upjohn, Kalamazoo, MI) as described
by Schurr (27) was stored at 4°Cin the dark until used. MNU, wetted
with 3% acetic acid (CAS 684-93-5; Sigma, St. Louis, MO), stored at
-20°Cuntil used, was freshly dissolved in saline, 10 mg/ml, yielding a
pH of approximately 5.5. DMABP (CAS 13394-86-0) was freshly
prepared from DMABP-HC1 (Ash Stevens, Inc., Detroit, MI) by dis
solving in ether and treatment with a saturated sodium bicarbonate
solution. After dehydration with magnesium sulfate and evaporation of
the ether, DMABP was immediately dissolved in corn oil at a concen
tration of 120 mg/ml.
Experimental Design. A summary of the experimental protocol is
given in Table 1. At 4 or 8 weeks of age, the rats were divided into 6
groups of 20 (Experiments A and B) or 25 animals (Experiment C)
each. They were given CA daily for 18 days, at a dose of 50 mg/kg. CA
was administered by s.c. injection in oil in Experiments A and B. In
Experiment C, CA was administered by gavaging a suspension of CAcontaining tablets dispersed in water. The suspension was constantly
stirred and the gavaging syringe was swirled until just before gavaging.
The reason for using CA in tablet form was the unavailability of the
pure compound in sufficiently large amounts at the time of Experiment
C. Directly after CA treatment. Groups 1 and 2 of each experiment
were given TP, 100 mg/kg, daily, on days 19, 20, and 21 of the
experiment between 8 and 12 a.m. On day 22, carcinogen was admin
istered as detailed below. Groups 3 and 4 were allowed to recover from
CA treatment from Day 19 till Day 25 and received carcinogen on day
26. Groups 5 and 6 were given carcinogen directly after the CA
pretreatment, on Day 19.
28). The time of carcinogen administration was based on studies by
Coffey et al. (24), who showed that on Day 4 of androgen treatment of
surgically castrated rats there is a maximum in cell proliferation in the
prostate.
Animals were checked daily and killed when moribund. Surviving
rats were killed at 81 (Experiment A), 79 (Experiment B), or 78 weeks
(Experiment C) after carcinogen treatment.
Carcinogen Treatment. On Day 21 (Groups 1 and 2), Day 26 (Groups
3 and 4) or Day 19 (Groups 5 and 6). the rats were transferred to a
carcinogen containment area in the same animal facility and housed, 5
to cage, in disposable plastic cages with filter tops (Scanbur, Koge,
Denmark) containing sterilized sawdust bedding. In Experiment A, the
animals received a single i.v. injection in the tail vein of MNU at a
dose of 50 mg/kg, immediately after the MNU solution was prepared.
In Experiment B. a single tail vein injection of DMBA, 30 mg/kg, was
given. In Experiment C, a single s.c. DMABP injection was given at a
dose of 250 mg/kg. Carcinogen was given between 10 a.m. and 1 p.m.
in all experiments. The dosages used were based on the lowest lethal
dose described in the literature (MNU, Ref. 29) or found in preliminary
experiments (DMBA and DMABP) and were aimed at a maximal
(sub)acute mortality of 5% or less. Extensive safety measures were
taken during carcinogen exposure and a period thereafter to protect
workers (Tyvek jumpsuits plus hoods/shoe covers; double latex gloves;
NIOSH-approved toxic particle respirators; working in pairs; carcino
gen injection in anesthetized (ether) animals; showering after disposal
of protective gear) and to prevent contamination of the environment
(carcinogen administration and animal housing in an area at negative
pressure, fitted with airlocks and HEPA-filtered air exhaust; doublebagged waste stored in airtight closed, incinerable plastic barrels; incin
eration of all waste at 1200°C).After a period of 2 weeks (MNU), 4
weeks (DMBA), or 8 weeks (DMABP), considered safe with regard to
excretion of the carcinogen, MNU (30), DMBA,5 or DMABP (31), the
animals were returned to a conventional room and wire mesh cages.
Pathology. The animals were killed by exsanguination via cannulation of the abdominal aorta while under ether anesthesia. Each rat was
subjected to a complete autopsy, and all observed gross lesions were
recorded. The following tissues were preserved in a 4% aqueous, neutral,
phosphate buffered formaldehyde solution: accessory sex glands (dorsolateral and ventral prostate lobes, ampullary glands, coagulating
glands, and seminal vesicles) and urinary bladder in foto; pituitary;
thyroids plus parathyroids; adrenals; lungs; liver; spleen; and all gross
lesions. Tissues were embedded in a paraffin wax. and 5-^m sections
were prepared and stained with hematoxylin and eosin. Sections of
pituitary, thyroids, and adrenals, made at 3 levels, and of liver, spleen,
and all gross tumor-like lesions were examined microscopically. Lungs
were histologically examined to detect métastases.In the pilot experi
ments, ventral and dorsolateral prostate were quickly dissected,
weighed, and fixed. In the tumor induction experiments, the accessory
sex glands were dissected after fixation as follows (see also Ref. 32).
5 M. C. Bosland and A. Schouten. unpublished observation.
692
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CHEMICAL INDUCTION OF RAT PROSTATE ADENOCARCINOMAS
Urinary bladder was trimmed away, leaving the prostatic part of the
urethra in place. Then, the ventral prostate lobes and seminal vesicle/
coagulating gland structures were cut away from the dorsolateral pros
tate complex, and embedded separately. The dorsolateral prostate
complex (with ampullary glands and prostatic urethra) was cut in
halves, transversely at a right angle to the urethra. A detailed histological examination of all accessory sex glands was carried out to search
for preneoplastic lesions and clues for the site of origin of the prostatic
carcinomas found. Twelve- to 15-step sections were made approxi
mately 200 urn distance of the ventral prostate lobes, of the dorsolateral
prostate lobes, including the ampullary glands, and of seminal vesicles
and coagulating glands. All tissues were examined by the same pathol
ogist (M. C. B.). Some accessory sex gland tumors were sampled for
electron microscopy (see Ref. 33).
Statistical Analysis. Differences in body »eights,relative weights of
prostate lobes, and mean survival were analyzed by means of Student's
/ test. Differences were considered statistically significant at the 95%
level (one-sided for weight data and two-sided for survival). Incidences
of tumors and other histopathological data were analyzed using the
Fisher exact probability test; differences were considered statistically
significant at the 95% level (two-sided). Within each experiment, com
parisons were made between the 4- and 8-week-old groups for each
treatment (Groups 1 and 2. Groups 3 and 4, and Groups 5 and 6). and
between the three treatments for the 4- and 8-week old groups separately
(i.e.. Groups 1, 3, and 5 and Groups 2, 4, and 6).
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RESULTS
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Effects of Pretreatment with CA and TP on Body Weight and
Accessory Sex Gland Weights and Morphology
In preliminary experiments (data not shown; see Ref. 28),
p.o. administration of 50 mg/kg/day of CA for 18 days ap
peared to be maximally effective in reducing body and prostatic
weights and about equally effective as the same dose adminis
tered by. s.c. injection. A 3-day period of treatment with TP
(100 mg/kg/day) following CA treatment caused drastic in
creases in relative prostate weights and a slight increase in body
weight. These increases were comparable to those observed
after a 7-day recovery following CA treatment. Morphologi
cally, CA treatment resulted in smaller alveoli and decreased
epithelial height. TP treatment for 3 days, following CA ad
ministration, greatly increased epithelial cell height. Mitotic
figures were distinctly more frequent in the prostate of TP
treated rats than in control prostates, in which mitotic activity
was rare, and no mitoses were present in the prostate of animals
treated with only CA.
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The average survival in the various treatment groups ranged
from 58 to 74 weeks but did not differ considerably among the
3 experiments (Tables 2, 4, and 6). Survival was similar in
Experiments A and B (average survival, 63 and 60 weeks,
respectively) and somewhat longer in DMABP treated rats
(Experiment C, average of 69 weeks). Within each experiment,
there were, with some exceptions, no significant differences in
survival among the 6 groups (Tables 2, 4, and 6).
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In all three experiments, adenocarcinomas located in the
dorsolateral region of the prostate were found in several groups
(Tables 2, 4, and 6). In the groups that were given injections of
MNU, following pretreatment with CA and TP, the combined
incidence of adenocarcinomas and carcinomas in situ of the
dorsolateral prostate was 30% in Group A-2 (rats treated from
8 weeks of age) and 10% in Group A-l (rats treated from 4
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693
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CHEMICAL INDUCTION
OF RAT PROSTATE ADENOCARCINOMAS
Table 3 Experiment A: incidence and types of hyperplastic and melaplaslic lesions of the accessory sex glands in rats given a single i.V.injection ofMNU, following
pretreatment with CA alone for 18 days, or with CA followed by TP for 3 days, or by a 7-day recovery
inGroupA-1A-2A-3A-4A-SA-6TreatmentCA
No. (%) of rats with lesions
prostateN192017172016Atypicalhyperplasia1
prostateN"192016161916Atypicalhyperplasia1(5)M5)1
VesiclesAtypicalhyperplasia—2(13)———Hypertr
startofat
experiment(wk)48484gDorsolateral
MNUCA
+ TP +
(5)3(15)1
MNUCA
+ TP +
MNUCA
+ RC +
(6)———Squamousmetaplasiaa2(10)————Ventral
(6)——1(6)N202015151816Seminal
MNUCA
+ RC +
MNUCA
+
+ MNUAge
* —¿,
no lesions observed. No hyperplastic or metaplastic lesions were observed in the coagulating glands and ampullary glands.
* yV,number of rats from which tissue was evaluated; only tissues without signs of autolysis are included.
c P < 0.001 for difference with Groups A-2 and A-4; P < 0.05 for difference with Group A-5.
(55)16(80)10(67)14(93)11
(61)3(19)'
Table 4 Experiment B: incidence and types of tumors of the accessor)' sex glands in rats given a single i.v. injection ofDMBA, following pretreatment with CA alone for
18 days, or with CA followed by TP for 3 days, or a 7-day recovery
inDorsolateral
No. (%) and survival of rats with primary tumors
regionAge
at start
experimentGroupB-lB-2B-3B-4H-5B-6TreatmentCA
of
prostate
no.
ofrats*202020202020Survival'
Adenocarcinoma61586158583
of
rats with
carcino
ma''
Sarcoma51
prostateSurvival
of Carcinoma
situsarcoma''
rats with
in
(CIS)69,
ratswith
of
CIS*42
—¿1(5)61—64
75
DMBACA
+ TP +
1(5)3
2(10)—M5)—1(5)3(15)Dorsolateral
°334
—¿
DMBACA
-1-TP +
DMBACA
+ RC +
DMBACA
+ RC +
—¿65
—¿60
DMBACA
+
4
—¿Survival
±4
—¿Survival
+ DMBA(wk)484848Effective
* —¿,
no tumors observed.
6 Initial number of rats was 20.
' Time of death (weeks) after carcinogen administration, mean ±SEM.
áTime of death with tumor: weeks after carcinogen administration: mean ±SEM or individual data (when W < 2).
Table 5 Experiment B: incidence and types of hyperplastic lesions of the accessor)- sex glands in rats given a single i.v. injection ofDMBA, following pretreatment with
CA alone for 18 days, or with CA followed by TP for 3 days, or by a 7-day recovery
inGroupB-l
No. (%) of rats with lesions
prosat start
of experiment
(wk)4
vesiclesAtypical
talew16
hyperplasia
N—°
hyperplasia1
glandsW16
hyperplasia14(82)
hyperplasia1(5)Hypcrtrophy-
18
+ TP -1-DMBA
(6)2(10)
B-2
16
17
CA + TP + DMBA
19
8
B-3
20
1919
CA + RC + DMBA
4
20
—¿
19
B-4
3(16)3(16)N17
CA + RC + DMBA
8
1919
B-5
IX
19
4
CA + DMBA
19Atypical
B-6TreatmentCA
18Seminal
CA + DMBAAge
8Dorsolateral18Atypical —¿
* —¿,
no lesions observed. No hyperplastic lesions were observed in the ampullary gland.
*yv, number of rats from which tissue was evaluated; only tissues without signs of autolysis are included.
c/><0.05 for difference with Group B-l.
d P< 0.01 for difference with Group B-2.
hyperplasia1(5)
14(82)
16
17(89)
19
1 1 (58)
19
8 (44)f
18
6 (33)"Coagulating
18Hypertrophy-
tumors involved more than one structure, and thus their exact
site of origin was not clear. However, lesions classified as
carcinomas in situ were found only in the dorsal or lateral lobes
of the prostate, and not in any of the other accessory sex glands
(Tables 2, 4, and 6). In a separate paper (33), several character
istics of these carcinomas (in situ), including their morphology
and metastasizing capacity, are described in detail.
The survival of animals with adenocarcinomas of the dorso
lateral prostate region was similar to the mean survival of other
rats in the same groups (Tables 2, 4, and 6). It ranged from 46
to 80 weeks after carcinogen treatment in the various groups
and experiments (mean, 61 weeks). Carcinomas in situ of the
dorsolateral prostate were observed only in animals that were
sacrificed at the end of the experiment, or close to that moment
(Tables 2, 4, and 6). An exception was an animal in Group B2 that was killed at 42 weeks after DMBA injection following
recovery from CA, while moribund due to a generalized lym-
weeks of age). In other groups and experiments, the incidence
of carcinomas in the dorsolateral prostate was low (approxi
mately 5%).
In Experiments A and B, carcinomas and carcinomas in situ
were found only in rats that had been treated with TP or that
were allowed to recover after CA administration before carcin
ogen was given (Tables 2 and 4). No carcinomas were found in
animals that were given carcinogen directly after pretreatment
with CA only in these experiments. In Experiment C, however,
carcinomas were not found in rats pretreated with CA and TP,
but only in animals given CA with or without recovery (Table
6).
The adenocarcinomas always involved the dorsolateral pros
tate, ampullary glands, and the base of seminal vesicles and
coagulating glands, whereas the ventral prostate lobes and the
apical parts of the seminal vesicle-coagulating gland complex
were always free of these malignant tumors. Even the smaller
694
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CHEMICAL INDUCTION OF RAT PROSTATE ADENOCARCINOMAS
phoma, and showed a carcinoma in situ in the dorsolateral
prostate.
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Sarcomas
Sy..OBiCB-oCMn
2
In Experiment A, two sarcomas located in the dorsolateral
prostate region were found (versus 9 carcinomas; Table 2). In
Experiment C (DMABP; Table 6), and particularly Experiment
B (DMBA; Table 4), there were more sarcomas in the dorso
lateral prostate region: respectively, 3 (versus 3 carcinomas)
and 7 (versus 2 carcinomas). The sarcomas occurred randomly
in the various groups (Tables 2, 4, and 6). A few tumors had
light and electron microscopic characteristics of neurogenic
sarcomas or leiomyosarcomas. The great majority of the tumors
were diagnosed as histiocytic sarcomas, based on their light and
electron microscopic characteristics (34-36), which are de
scribed elsewhere (28).
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Tumors of Other Accessory Sex Glands (Tables 2, 4, and 6)
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In Experiment A, a benign tumor of the ventral prostate was
found in Group 2. This microscopic tumor was classified as a
cystadenoma; its morphological appearance, which differed
from ventral prostate tumors described by others (11-13, 19,
21), is described elsewhere (28, 37).
In Experiment B, no tumors were observed in accessory sex
glands other than the dorsolateral prostate. However, in Ex
periment C (DMABP), two adenocarcinomas were found in
Group C-6 that clearly originated from the seminal vesicle, and
one distinct coagulating gland adenocarcinoma was seen in a
rat from Group C-4 (see Ref. 38 for description of their mor
phology). These tumors were all found at the terminal kill and
were of microscopic size.
regD."esc53S2S«
Nonneoplastic Proliferative Lesions of the Accessory Sex Glands
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Dorsolateral Prostate. Lesions, classified as focal atypical
hyperplasia, were found in Experiment A (MNU) in Groups 1,
2, and 3 in a low incidence (Table 3). No such lesions were
observed in Experiment B (DMBA; Table 5), while a few
atypical hyperplasias occurred in Groups 1 and 5 of Experiment
C (DMABP; Table 7). The morphology of these lesions is
described in detail in a separate paper (33).
Reactive hyperplasia associated with acute and chronic in
flammatory processes was common in the dorsolateral prostate
in all groups in all experiments; there was no association
between the occurrence of this lesion and the treatments (data
not shown). This type of hyperplasia was distinctly different
from atypical hyperplasia (see Ref. 32). The diagnosis of atyp
ical hyperplasia was never attached to a lesion with a distinct
inflammatory component.
Focal squamous metaplasia was occasionally observed. It was
never associated with atypical or reactive hyperplasia (Tables
3, 5, and 7). There was no apparent relationship between the
occurrence of this change and the various treatments.
Ventral Prostate. Atypical hyperplasia of the ventral prostate
was observed in low incidence (0-16%) in all experiments.
There was no association between the occurrence of this lesion
and any of the treatments (Tables 3, 5, and 7). The morphology
of this lesion is described in detail elsewhere (32) and was very
similar to that described by others for spontaneous and chemi
cally induced atypical hyperplasia of the rat ventral prostate
(11-13,19,21).
Reactive hyperplasia occurred in lower frequency than in the
dorsolateral prostate; there was no apparent relationship be
tween the incidence of this lesion and the various treatments
(data not shown).
695
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CHEMICAL
INDUCTION
OF RAT PROSTATE ADENOCARCINOMAS
Table 7 Experiment C: incidence and types of hyperplastic and metaplastic lesions of the accessory sex glands in rats given a single s.c. injection ofDMABP, following
pretreatment with CA alone for IS days, or with CA followed by TP for 3 days, or by a 7-day recovery
inDorsolateral
No. (%) of rats with lesions
prostateN202024242525Atypicalhyperplasia1(5)——1
vesiclesN181923232324Hypertrophy-hype
glandsN151924232324Atypicalhyperplas
prostateof
experimentGroupC-lC-2C-3C-4C-5C-6TreatmentCA
rplasia7(39)10(53)13(57)15(65)9
"—1
DMABPCA
+ TP +
DMABPCA
+ TP +
(4)—1(4)—Ventral
DMABPCA
+ RC +
(4)1(4)1
DMABPCA
+ RC +
DMABPCA
+
(4)Seminal
+ DMABP(wk)484g4gA/"191723212424Atypicalhyperplasia2(11)———1(4)—Squamousmetaplasia—
*—¿,
no lesions observed. No hyperplastic or metaplastic lesions were observed in the coagulating glands.
*JV,number of rats from which tissue was evaluated; only tissues without signs of autolysis are included.
' P < 0.01 for difference with Group C-4.
Seminal Vesicles. The most common proliferative lesion in
these glands was a focal change, best characterized as a com
bination of focal hyperplasia and cellular hypertrophy. Increase
of cell size was generally much more pronounced than the
apparent increase in the number of cells (Fig. 1). There was no
distinct cellular atypia and the lesion was usually well demar
cated (Fig. 2), while it occasionally seemed to compress sur
rounding normal epithelium. The lesion was highly variable in
size and seemed capable of secretory activity. Focal dysplastic
areas were occasionally present within these lesions (see below).
There was no structural association between the areas of hypertrophy-hyperplasia or dysplasia and the two seminal vesicle
adenocarcinomas observed in Group 6 of Experiment C. The
incidence of hypertrophy-hyperplasia was variable (Tables 3, 5,
and 7). The lesion occurred less frequently in Experiment C
than in Experiments A and B. Its incidence was distinctly lower
than average in Group 6 in all experiments. In the other groups,
there was no apparent association with treatment.
In a total of three animals in Experiment A (Group 3) and
(39)5(21)fAmpullary
S<P£ .-.^T^
C'
¿x.
(4)—
^
£•. «i* »' j
;
£.•£
JrtgSWB
Fig. 2. Detail of a small hypertrophic-hyperplastic lesion in the seminal vesicle.
Note the abrupt change from normal epithelium (top) and hypertrophic-hyper
plastic epithelium and the distinctly larger size of the cells in the lesion compared
to those in the normal epithelium. H & E, x 120.
m
3fÃ
$$s&
•¿v
?Aî.iv,-.-.»VT
, ^:-v\y ..'»V.
Fig. 1. Large hypertrophic-hyperplastic
X60.
lesion in the seminal vesicle. H & E,
Experiment B (Group 4) a focal lesion was seen that was
classified as atypical hyperplasia (Tables 3 and 5). These dys
plastic lesions consisted of focal hyperplastic areas with cellular
atypia and loss of cellular polarity (Fig. 3). The lesion occurred
both inside and outside areas of hypertrophy/hyperplasia.
Occasionally, reactive hyperplasia was encountered, usually
in association with severe inflammation in the other accessory
sex glands; there was no correlation between the occurrence of
this lesion and the various treatments (data not shown).
Coagulating Glands. Reactive hyperplasia was observed in a
few rats in each of the three experiments (data not shown). One
animal in Group A-3 showed a focal hypertrophy/hyperplasia
that was very similar to that found in the seminal vesicles (Table
3).
Ampullary Glands. Focal reactive hyperplasia (Fig. 4) oc
curred in variable frequency. The lesion was almost invariably
associated with the presence of sperm in the ampullary gland
696
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CHEMICAL INDUCTION OF RAT PROSTATE ADENOCARCINOMAS
r/» t^v>'i'--SLri-cÃ-' "i/ '/
•¿
•¿Â£
JÖöäägfex.
-Cv> ¿'»3Ê
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—¿^-.'x'.'h'AiJS
Fig. 3. Dysplasia of the seminal vesicle epithelium (L. lumen). H & E, x 120.
Fig. 4. Reactive hyperplasia of the ampullary gland. Note the normal epithe
lium in the upper left-hand corner and the presence of sperm in the lumen of the
hyperplastic gland. H & E. x 90.
alveoli involved, which may have been the cause of the reaction.
There was no apparent relationship between incidence and
treatment in the various groups and experiments (data not
shown).
young adults at the start of the experiment.
The results of Experiment B and particularly those of Exper
iment A point to a decisive role of cell proliferation during the
initiation phase of prostatic carcinogenesis in the rat. Invasive
adenocarcinomas, carcinomas in situ, and atypical hyperplasias
of the dorsolateral prostate were found only in groups that
received TP or recovery following CA, and not in groups treated
with only CA, without TP treatment or recovery. Cell prolifer
ation in the prostate can be enhanced effectively only by androgen treatment in castrated animals and not in intact ones (24,
25). Thus, the chemical castration caused by treatment with
CA preceding the TP injections is an essential step in enhancing
cell proliferation in the prostate in the approach that was
applied in this study. A 7-day recovery after CA treatment is
probably also accompanied by enhanced cell proliferation in
the prostate. There may well be differences between the various
accessory sex gland structures in the timing of the cell prolif
eration response to sequential treatment with CA and TP. This
might explain why (pre)neoplastic lesions were only found in
the dorsolateral prostate in the experiments with MNU and
DMBA, and not in the ventral prostate, seminal vesicle, or
coagulating gland.
The carcinogen DMABP (Experiment C) yielded different
results. Prostatic carcinomas and atypical hyperplasias were not
found in Groups 1 and 2, as was the case in Experiments A and
B, but only in Groups 3 and 4 and Groups 5 and 6. In the latter
two groups, cell proliferation was strictly inhibited by CA at
the time of carcinogen administration. Furthermore, neoplastic
epithelial lesions were found in the seminal vesicles and coag
ulating glands, unlike results in Experiments A and B. DMABP
is slowly metabolized to its putative active form (31). Therefore,
activated DMABP may have "missed" the transient phase of
maximally stimulated cell proliferation in the dorsolateral pros-
Tumors in Organs Other Than the Accessory Sex Glands
In all three experiments a wide spectrum of tumor types and
sites was found. The incidence and latency time of these tumors
are described elsewhere (28). Tumors of the ear duct and/or
Zymbal's gland were the most common tumors in Experiment
A (MNU), while both ear duct/Zymbal's gland tumors and s.c.
sarcomas occurred very frequently in Experiment B (DMBA).
These tumors did not result in early mortality in comparison
with the mean survival of each experimental group (data not
shown). Generalized lymphomas, on the other hand, which
occurred in an incidence between 0 and 20% in all three
experiments, often caused early mortality (data not shown).
DISCUSSION
The major observation in this study is the 30% incidence of
carcinomas of the dorsolateral prostate in a group of rats that
received a single injection with the direct-acting nitrosamide
MNU. Adenocarcinomas were found in 25% of these rats, while
5% had adenocarcinomas in situ. The carcinomas were found
some 63 weeks after MNU injection, i.e., when the rats were
about 75 weeks old. This is a highly significant finding, because
the strain of rats used in this study has a known very low
incidence of spontaneous prostate cancer, i.e., 0.03% in rats of
over 2 years of age (39). Full development of the prostate gland
prior to carcinogen exposure was not an absolute prerequisite
for the CA plus TP plus MNU protocol to be carcinogenic to
the dorsolateral prostate, inasmuch as carcinomas were induced
in both rats with a juvenile prostate and in animals that were
697
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CHEMICAL
INDUCTION OF RAT PROSTATE ADENOCARCINOMAS
tate in Groups 1 and 2 and "hit" maximal cell proliferation in
other accessory sex glands instead. In Groups 5 and 6 enhance
ment of cell turnover in the dorsolateral prostate perhaps
occurred later than in Groups 1-4, coinciding with the forma
tion of activated DMABP.
To our knowledge, this is the first report of a substantial
incidence of invasively growing adenocarcinomas of the pros
tate induced in experimental animals by a single carcinogen
administration without further treatment. Recently, Pollard
and Luckert (40) reported that a single MNU injection without
concomitant stimulation of prostatic cell turnover greatly en
hanced testosterone-induced carcinoma incidence in the dor
solateral prostate of the Lobund-Wistar rat from 10-15% to
65-70%. The mechanism of this reported effect of MNU is
puzzling. Pour and Stepan (41) similarly produced prostatic
carcinomas by A'-nitrosobis(2-oxopropyl)amine
administered
during androgen-enhanced cell proliferation in the prostate, but
only when carcinogen injection was followed by long-term
testosterone treatment. The incidence was 15-21%, and most
tumors were adenocarcinomas occurring in the dorsolateral
prostate; some were found in the ventral lobes, where squamous
cell carcinomas also were found in a 19-29% incidence.
In Experiments A and B, atypical hyperplasias and carcino
mas in situ were observed only in the dorsolateral prostate, and
they occurred only in those groups in which invasive carcinomas
were found. For reactive hyperplasia and squamous metaplasia
such associations were not present. This suggests that (a) the
dorsolateral prostate is the most likely site of origin of the
tumors found in the dorsolateral prostate region and (b) the
atypical hyperplasias and carcinomas in situ are precursor le
sions of the adenocarcinomas. These putative precursor lesions
have never been observed in 12-month-old control rats of the
same strain (N = 111).4 It is unlikely that any tumors have
arisen from the seminal vesicles, coagulating glands, or ampullary glands in Experiments A and B, because there were no
obvious preneoplastic lesions in these glands occurring in as
sociation with the treatments. In addition, none of the carci
nomas was exclusively located in these structures in Experi
ments A and B. The ventral prostate was clearly not the site of
origin of the carcinomas, because it was neither macroscopically
nor microscopically involved in any of the tumor processes.
Furthermore, no putative precursor lesions (atypical hyperpla
sia) occurred in association with the treatments. These lesions
are commonly found in rats over 1 year of age in a range of
strains (11, 12); in the Wistar strain that was used in the present
study the incidence was 4.5% in a group of 44 rats (32). The
papillary cystadenoma of the ventral prostate that was found in
Group A-l may have resulted from the treatment. This lesion
has, to our knowledge, not been described before. The reactive
hyperplasias in the various accessory sex glands and the focal
hypertrophic-hyperplastic
lesions in the seminal vesicles are
most likely not related to the development of the prostatic
tumors, since the incidence of these changes was comparable in
all experimental groups.
An important conclusion of this study is that a variety of
chemical carcinogens can, in combination with appropriate
stimulation of cell proliferation, produce prostatic cancer in
rats. The direct-acting carcinogen MNU was most effective,
while the carcinogens DMBA and DMABP, both requiring
metabolic activation, induced a lower tumor incidence. Inter
estingly, the occurrence of sarcomas in the dorsolateral prostate
region was apparently not related to the presence of a high level
of cell proliferation in the prostate. Rather, it was associated
with the type of carcinogen. DMBA, which induced many
sarcomas at other sites (see Ref. 28), was also the most effective
carcinogen in causing dorsolateral prostate sarcomas.
The approach presented in this paper offers a promising
animal model for human prostatic carcinogenesis. However,
this model must comprise a number of essential characteristics,
which are detailed in a companion paper (33). In this regard, it
is important that the presence of most other tumors did not
interfere with the risk to the animals of developing prostate
tumors, since they did not cause early death, as indicated by a
comparable survival of rats with and without prostatic carci
nomas. Only lymphomas caused a reduction of survival.
Four important features make the model stand out favorably
in comparison with other induction models: (a) the tumors are
adenocarcinomas, and not squamous cell carcinomas as in the
model described by Pour (20); (h) the tumors are invasively
growing and are capable of metastasizing (see Ref. 33), unlike
the lesions reported by Katayama et al. (19) and Shirai et al.
(21); (e) no long-term hormonal treatment is required. After
initial endocrine manipulation to stimulate cell proliferation,
the animals remain untreated, unlike prostate cancer models
that involve long-term, high dose androgen treatment (5, 17,
40, 41); and (d) the tumors do not originate from the ventral
prostate as they do in some of the other chemical induction
models mentioned (19-21), but from the dorsolateral lobes
which are embryologically homologous to the parts of the
prostate in which carcinomas develop in humans (8, 15, 16). In
addition, the fact that only a single carcinogen injection is
needed in the present model and not repeated carcinogen ad
ministration (19-21) or long-term hormonal treatment (5. 17,
40, 41) provides excellent opportunities for studying the pro
motion stage of prostatic carcinogenesis and its modification
by environmental factors.
ACKNOWLEDGMENTS
We are very grateful to Drs. C. F. M. Hendriksen and R. Kroes for
crucial discussions in the initial stages of this study and to B. J. Spit
for the electron microscopic studies. We want to thank Dr. J. A. Joles
for editorial help; M. Freitag for typing assistance; and G. Cook, J.
Gorczynski, B. Weir, and F. van Welie for help with microphotography.
The expert assistance of the animal house, histology, and secretarial
staff of the Department of Biological Toxicology of the TNO-CIVO
Toxicology and Nutrition Institute is greatly appreciated. We are very
grateful to Dr. P. E. Schurr (Upjohn) for his gift of DMBA emulsion,
to Organon B.V., Oss, The Netherlands, for donating Neohombreol;
to Schering AG, Berlin. Federal Republic of Germany, for providing
cyproterone acetate; and to A. Houba (Schering Nederland. B.V.,
Muiden, The Netherlands) for his help in obtaining Androcur tablets.
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Induction of Dorsolateral Prostate Adenocarcinomas and Other
Accessory Sex Gland Lesions in Male Wistar Rats by a Single
Administration of N-Methyl-N-nitrosourea, 7,12-Dimethylbenz(a
)anthracene, and 3,2′-Dimethyl-4-aminobiphenyl after Sequential
Treatment with Cyproterone Acetate and Testosterone
Propionate
Maarten C. Bosland and Menk K. Prinsen
Cancer Res 1990;50:691-699.
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