Stress during puberty facilitates precancerous prostate lesions in adult rats
Puberty can be a critical period for the long-term development of diseases, especially for stress-related disorders that depend on neuroendocrine and immune responses. Some organs like the prostate are prone to diseases that result from neuroendocrine or immune challenges, such as cancer. Aim: In th...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Zitieren: | Stress during puberty facilitates precancerous prostate lesions in adult rats / D. Herrera-Covarrubias, G.A. Coria-Avila, M.E. Hernandez, N. Ismail // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 269–275. — Бібліогр.: 49 назв. — англ. |
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| author | Herrera-Covarrubias, D. Coria-Avila, G.A. Hernandez, M.E. Ismail, N. |
| author_facet | Herrera-Covarrubias, D. Coria-Avila, G.A. Hernandez, M.E. Ismail, N. |
| citation_txt | Stress during puberty facilitates precancerous prostate lesions in adult rats / D. Herrera-Covarrubias, G.A. Coria-Avila, M.E. Hernandez, N. Ismail // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 269–275. — Бібліогр.: 49 назв. — англ. |
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| description | Puberty can be a critical period for the long-term development of diseases, especially for stress-related disorders that depend on neuroendocrine and immune responses. Some organs like the prostate are prone to diseases that result from neuroendocrine or immune challenges, such as cancer. Aim: In the present study, we assessed the long-term effects of an acute pubertal stressor (immune-challenge) on the development of precancerous lesions in adult rats, and compared them with testosterone-induced prostatic lesions. Materials and Methods: Pubertal male rats received a single injection of lipopolysaccharide (LPS) or saline during puberty (5 weeks old). At adulthood (8 weeks old) males were subcutaneously implanted with either an empty capsule or filled with testosterone propionate (100 mg/kg). This resulted in a total of five groups: 1) intact untreated, 2) saline-treated and implanted with a blank capsule, 3) saline-treated and implanted with a testosterone capsule, 4) LPS-treated and implanted with a blank capsule, 5) LPS-treated and implanted with a testosterone capsule. Four weeks later, the rats were sacrified and their prostates processed for histology (hematoxylin and eosin stain) and blood serum processed for hormone analysis (testosterone and corticosterone). Results: Males treated with LPS (stressed during puberty via immune challenge) expressed epithelium dysplasia (specially in the ventral prostate), anisocytosis, presence of mononuclear cells, anisokariosis, non-basal polarity, abnormal nucleus-cytoplasm ratio, proplastic myoepithelium, and granular content in the lumen. These histological alterations were similar, but less severe than those observed in males implanted with testosterone during adulthood. Conclusion: These results indicate that pubertal exposure to an immune challenge (stress) facilitates the long-term development of prostatic lesions in adult male rats.
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Experimental Oncology 39, 269–275, 2017 (December) 269
STRESS DURING PUBERTY FACILITATES PRECANCEROUS
PROSTATE LESIONS IN ADULT RATS
D. Herrera-Covarrubias1, 2, *, G.A. Coria-Avila2, M.E. Hernandez2, N. Ismail1
1School of Psychology, University of Ottawa, Ottawa K1N 6N5, ON, Canada
2Centro de Investigaciones Cerebrales, Universidad Veracruzana, Xalapa 91193, VER, Mexico
Puberty can be a critical period for the long-term development of diseases, especially for stress-related disorders that depend
on neuroendocrine and immune responses. Some organs like the prostate are prone to diseases that result from neuroendocrine
or immune challenges, such as cancer. Aim: In the present study, we assessed the long-term effects of an acute pubertal stressor
(immune-challenge) on the development of precancerous lesions in adult rats, and compared them with testosterone-induced pros-
tatic lesions. Materials and Methods: Pubertal male rats received a single injection of lipopolysaccharide (LPS) or saline during
puberty (5 weeks old). At adulthood (8 weeks old) males were subcutaneously implanted with either an empty capsule or filled with
testosterone propionate (100 mg/kg). This resulted in a total of five groups: 1) intact untreated, 2) saline-treated and implanted with
a blank capsule, 3) saline-treated and implanted with a testosterone capsule, 4) LPS-treated and implanted with a blank capsule,
5) LPS-treated and implanted with a testosterone capsule. Four weeks later, the rats were sacrified and their prostates processed
for histology (hematoxylin and eosin stain) and blood serum processed for hormone analysis (testosterone and corticosterone).
Results: Males treated with LPS (stressed during puberty via immune challenge) expressed epithelium dysplasia (specially in the
ventral prostate), anisocytosis, presence of mononuclear cells, anisokariosis, non-basal polarity, abnormal nucleus-cytoplasm
ratio, proplastic myoepithelium, and granular content in the lumen. These histological alterations were similar, but less severe than
those observed in males implanted with testosterone during adulthood. Conclusion: These results indicate that pubertal exposure
to an immune challenge (stress) facilitates the long-term development of prostatic lesions in adult male rats.
Key Words: prostate, cancer, stress, puberty, testosterone, LPS.
The prostate is an exocrine reproductive gland prone
to different types of diseases such as inflammation
(prostatitis), progressive enlargement (benign prostatic
hyperplasia, BPH), and prostate cancer (CaP). The risk
of CaP involves many environmental and hereditary fac-
tors such as unhealthy diet, obesity, older age, African
ancestry and atypical sexual hormonal milieu [1–6]. Pre-
cancerous lesions (e.g. epithelium dysplasia, anisocy-
tosis, anisokaryosis, apolarity, etc.) have been reported
in individuals with a previous history of prostatic chronic
inflammation due to infections [7], and in experimental
animals that undergo constant copulation [8], or in those
treated with systemic testosterone [9, 10] or prolactin [6,
11]. Accordingly, the risk for development of CaP is higher
when the prostate is exposed to hormonal and immune
challenges, especially in individuals at susceptible age,
with genetic predisposition and unhealthy diets.
Some studies indicate that puberty should be con-
sidered a critical period for the long-term develop-
ment of diseases, especially for those that depend
on neuroendocrine and immune responses, perhaps
because during puberty the hypothalamic-pituitary-
adrenal (HPA) axis is more responsive to stressors than
in adulthood [12]. For instance, as compared to adults,
pubertal male rats exposed to acute restraint stress
(30 min) express longer peaks of adrenocorticotropic
hormone (ACTH) and corticosterone. In addition, after
chronic restraint stress (30 min daily) pubertal rats
express higher peaks that return faster to baseline
levels [13]. Similarly, the serum levels of testoste-
rone [14] and prolactin [15] increase more in pubertal
rats than in adults after chronic stress. Higher or longer
hormonal responses during pubertal stress may result
in enduring changes in hormone-sensitive organs, in-
creasing the susceptibility to severe diseases like CaP.
Some studies in laboratory mice have used the
bacterial endotoxin lipopolysaccharide (LPS) to in-
duce a stress-like and immune response that induces
the display of sickness symptoms for about two days
or less [16–18]. LPS is a component of the cellular
membrane of gram-negative bacteria. Treatment with
LPS results in the production of cytokines, cyclooxy-
genase 2 (COX-2), and prostaglandins (PGE2) among
other molecules [18] which can activate the HPA axis.
In mice, treatment with LPS at 6 weeks of age (puberty)
results in permanent neuroendocrine alterations. For
example, pubertal females that receive LPS display
reduced sexual receptivity in adulthood. This does not
occur if LPS is injected during the postnatal weeks 3,
7, 8 or 10 [19], indicating that the long-term effects
of LPS treatment occur exclusively when it is expe-
rienced during the pubertal stress sensitive period
Submitted: May 9, 2017.
*Correspondence: E-mail: dherrera.covarrubias@gmail.com
Abbreviations used: ACTH — adrenocorticotropic hormone;
ANOVA — analysis of variance; AR — androgen receptor; BPH —
benign prostatic hyperplasia; CaP — prostate cancer; COX2 —
cyclooxygenase 2; CR — corticosterone receptor; DLP — dorso-
lateral prostate; ELISA — enzime-linked immunosorbent assay;
H & E — hematoxylin and eosin; HPA — hypothalamic-pituitary-
adrenal; HPG — hypothalamic-pituitary-gonadal; IL — interleukin;
IFN — interferon; i.p. — intraperitoneal; LPS — lipopolysaccharide;
mRNA — messenger RNA; N:C — nucleus:cytoplasm; MNU —
N-nitroso-N-methylurea; NF-κB — nuclear factor kappa light chain
enhancer of activated B cells; PGE2 — prostaglandin E2; PRL —
prolactin; s.c. — subcutaneous; TNF — tumor necrosis factor; VP —
ventral prostate.
Exp Oncol 2017
39, 4, 269–275
270 Experimental Oncology 39, 269–275, 2017 (December)
(5–6 weeks old). Moreover, mice treated with LPS
at 6 weeks of age display altered behavioral respon-
siveness to estradiol for anxiety-like and depression-
like behaviors and cognitive functioning. These find-
ings suggest that pubertal treatment globally alters the
behavioral responsiveness to estradiol (and probably
to other hormones) by affecting both reproductive and
non-reproductive behaviors [20–22].
Thus, in the present study, we tested the effects
of pubertal stress on precancerous prostatic lesions
and levels of hormones known to modulate prostate
histology and stress. Our first hypothesis stated that
pubertal treatment with LPS would induce histological
prostatic lesions in adulthood comparable to the lesions
observed in males exposed to exogenous systemic tes-
tosterone (a positive control for precancerous lesions).
In addition, we hypothesized that the baseline blood le-
vels of testosterone and corticosterone would be higher
in adult males that received LPS during puberty.
MATERIALS AND METhODS
Animals. Forty Wistar male rats (Rattus norvegi-
cus albinus) were purchased and shipped at 4 weeks
of age from a certified laboratory animal supplier
in Mexico (Circulo ADN®). They were housed in groups
of five rats in large Plexiglas cages (50 × 30 × 20 cm)
and kept in a colony room at the Centro de Investiga-
ciones Cerebrales, Universidad Veracruzana, Mexico,
in a 12–12 h reverse Light-Dark cycle (lights off
at 8:00 h). Water and commercial rat chow (RismartÒ)
were provided ad libitum. All the experimental pro-
cedures were carried out according to the Official
Mexican Norm for use and care of laboratory animals
(NOM-062-ZOO-1999) [23] and the International
Guiding Principles for Biomedical Research [24].
Groups and treatments. The rats were randomly
assigned to one of the following five groups: 1) intact
(n = 8), 2) saline-blank (n = 8), 3) saline-testosterone
(n = 8), 4) LPS-blank (n = 8), 5) LPS-testosterone (n = 8).
Table 1 indicates treatment for each group and the age
(weeks) at the time of treatment. At 5 weeks of age,
rats from groups 4 and 5 received one intraperitoneal
(i.p.) injection of LPS (LPS from E. coli, Sigma-Aldrich)
at a dose of 1.5 mg/kg in a volume of 1 ml/kg of sterile
saline. Groups 2 and 3 received exclusively a saline
injection (i.p.) and group 1 received no treatment.
Rats were monitored at 2, 4, 8, 24 and 48 h following
the injection to detect the presence of five sickness
symptoms (ptosis, piloerection, lethar gy, huddling)
as previously reported [18], and the posture of kyphosis.
Such period is sufficient to detect whether or not rats
respond to LPS. At each time-point, rats were given
a score from 0 to 5, depending on the number of symp-
toms observed (Fig. 1). During the following three
weeks, the rats were handled daily, and their gene ral
health was monitored. At 8 weeks of age, groups 3 and
5 were implanted subcutaneously (s.c.) with a silastic
tube filled with testosterone (Sigma-Aldrich Mexico
catalogue 1649007), whereas groups 2 and 4 were
implanted with a blank (empty) capsule. Group 1 re-
ceived no treatment. The silastic tube (Dow Corning
CorpÒ 25 mm length, 1.57 mm I.D. × 3.18 mm O.D.)
contained powdered testosterone propionate (Sigma-
Aldrich química, Mexico). This resulted in approximately
100 mg/kg of body weight as previously used in other
studies [8, 11, 25]. Surgical implantation of the tube
was done under inhaled galothane anesthesia, and
took less than 3 min for each rat. A detailed description
of the procedure can be found in our previously pub-
lished paper [8]. After confirmation of deep anesthesia,
we performed a 10 mm skin incision on the lower back.
A surgical probe was used to separate the skin from the
muscle. The silastic tube was inserted under the skin
and pushed rostrally until placed s.c. in the upper back,
between the two scapulae. The lower back incision was
sutured and the rat was allowed to fully recover before
it was placed back into its home cage. Early studies
showed that these silastic capsules release testoste-
rone at a rate of ~30 µg/day/cm [26].
Table 1. Groups and treatments
Group
Treatment
5 weeks
single intrape-
ritoneal (i.p.)
injection
8 weeks
subcutane-
ous (s.c.)
implant
12 weeks
(1) Intact nothing nothing histopathology
(2) Saline-blank saline empty histopathology
(3) Saline-testosterone saline testosterone histopathology
(4) LPS-blank LPS empty histopathology
(5) LPS-testosterone LPS testosterone histopathology
Note: At 5 weeks of age (puberty) male rats received an i.p. injection of either
LPS (1.5 mg/kg/ml), saline (1 ml/kg) or nothing. At 8 weeks of age (adult-
hood) the same rats received a s.c. implant filled with testosterone, empty,
or remained untreated. At 12 weeks of age all the males were sacrificed and
their prostates processed for histology.
0
1
2
3
4
5
1 h 2 h 4 h 8 h 24 h 48 h
M
ea
n
+/
- S
EM
Sickness score
Saline
LPS
Fig. 1. Sickness scores in male rats treated either with saline
or LPS during puberty (5 week old)
Prostate samples and histology. At 12 weeks
of age the rats were deeply anesthetized with sodium
pentobarbital (60 mg/kg i.p.). Then, 3 ml of blood were
obtained by cardiac puncture for hormone analysis (see
Hormone measurements for further details). After blood
sampling rats were sacrificed with an overdose of sodium
pentobarbital (120 mg/kg i.p.). An abdominal incision
was performed and the accessory sexual organs were
carefully removed and placed into a container with 0.9%
Experimental Oncology 39, 269–275, 2017 (December) 271
saline solution. The prostate was identified under a dis-
secting microscope (MEJI, EMZ-TRÒ) and divided into
ventral (VP) and dorsolateral (DLP) prostate. As in our
previous studies [8, 11] the VP and DLP were soaked
in 10% formalin for 24 h, then dehydrated in 70% and
80% alcohol (1 h each), and 95% (3x2 h each), and 100%
ethanol overnight, plus two more changes (1 h each),
the following day. Then xylene (3x1 h each), always
in constant shaking. Tissue was embedded in paraffin
wax (2x2 h each), sliced (5 µm thick) with a microtome
(RM 2125RT Leica, Germany), mounted on slides
in a bath at 52 °C (containing pork skin-based gelatin
2.5 mg/100 ml) and then processed for hematoxylin
and eosin (H & E) dye technique as follows: 1 h at 57 °C,
deparaffinization in xylene (3x5 min each), rehydrated
in alcohol/xylene (1:1) 5 min, ethanol 96% 3 min, he-
matoxylin (10 min), water (30 s), acid alcohol (quick
immersion), water (10 s), lithium carbonate (30 s), water
(10 s), eosin (4 quick immersions). Dehydration in etha-
nol 96% (3 min), ethanol 100% (2 min), ethanol/xylene
1:1 (2 min), and xylene (5 min). Then, the slides were
coverslipped with Permount (SP15-500 Fisher chemi-
cals), air dried, and observed under a light microscope
(Olympus Ax70, Japan). Photomicrographs were taken
at 40× and analyzed by the same experimenters. As for-
merly reported, we assessed prostate histology by taking
into consideration 12 histological features (Table 2) [8,
11]. Experimenters were blind to the treatment at the
time of diagnosis.
Hormones measurement. Concentrations of tes-
tosterone and corticosterone in blood serum were mea-
sured at one single time point at 12 weeks of age, at the
time of prostate extraction. Blood was collected in vacu-
tainer tubes containing no anticoagulant and incubated
in upright position at room temperature for 30 min to allow
clotting. Tubes were centrifuged for 15 min at 1000 rpm.
Supernatant was aspirated at room temperature and se-
rum was kept in 500 ml aliquots and frozen at −20 °C for
a few days until processing. Hormones levels were quan-
tified using enzyme-linked immunosorbent assay (ELISA)
and commercial kits for testosterone (ALPCO, USA) and
corticosterone (ALPCO, USA). The procedure was car-
ried out as instructed by the supplier. The assays were
read in an IMARK microplate reader with the software
microplate manager from Bio-Rad.
Variables and statistical analysis. Sickness
score: We examined the intensity (0–5) and duration
(0–48 h) of sickness after treatment with LPS during
puberty. The number of rats expressing symptoms
at each time point were recorded. Histology: Twelve
histological features were analized in each male in adult-
hood (see Table 2). Tables 3 and 4 indicate histological
results observed in at least 6 out of 8 males for group.
Hormones: Levels of testosterone and corticosterone
(ng/ml) in adulthood were analyzed with a one-way
analysis of variance (ANOVA), followed by a Fisher
LSD post hoc test to compare individual differences.
All statistical analyses were performed using GraphPad
Prism version 6.00 for Mac, GraphPad Software, La Jolla
California USA, www.graphpad.com and the alpha level
was set at p < 0.05.
RESULTS
Sickness score during puberty. Of the 16 rats
that received LPS during puberty, only two failed
to express any sickness symptom. The most common
symptom was lethargy (68%), followed by kyphosis
(50%), ptosis (38%), huddling (38%) and piloerec-
tion (13%). None of the males from the saline group
expressed symptoms after injection. Fig. 1 depicts
the sickness score, indicating that a maximum peak
response occurred 2 h after injection and lasted for
less than 24 h.
Table 2. Characterization of both normal (expected) and abnormal
(non-expected) histology in the prostate of adult rats
Histological feature Region Normal
(expected cases)
Abnormal
(non-expected
cases)
Epithelium form DLP
VP
cubic
columnar
metaplasia,
dysplasia
Epithelium size DLP
VP
even anisocytosis
Epithelium papillae DLP
VP
scarce plenty
Interstice space DLP
VP
even compressed
Interstice content DLP
VP
collagen mononuclear
Nucleus size DLP
VP
even anisokariosis
Nucleus location DLP
VP
basal cell polarity non-polar
Nucleus-cytoplasm ratio DLP
VP
1:3 < 1:3
Myoepithelium DLP
VP
euplasia proplasia
Pattern (at 4×) DLP
VP
tubular cribiform
Lumen content DLP
VP
amorphous granular
Chromatin DLP
VP
heterochromatin euchromatin
Note: Normal features were inferred from the number of cases observed
in groups 1 (intact) and 2 (saline-blank) of the present study.
Table 3. Histological features of the VP of males from the different groups.
Features were accepted when it was observed in at least 6 out of 8 males
Histology
Ventral prostate
Intact Saline
blank
Saline
testoste-
rone
LPS
blank
LPS
testoste-
rone
Epithelium
Form columnar columnar dysplasia dysplasia dysplasia
Size anisocy-
tosis
even anisocy-
tosis
anisocy-
tosis
anisocy-
tosis
Papillae plenty plenty plenty plenty plenty
Interstice
Space even even even even even
Content collagen collagen collagen collagen3 collagen
Nucleus
Size even1 even1 anisoka-
ryosis
anisokary-
osis
anisoka-
ryosis
Location polar polar no polar polar no polar
Nucleus:
cytoplasm
> 1:3 > 1:3 1:1 1:1 1:1
Myoepithelim euplasia euplasia proplasia euplasia proplasia
Pattern (4×) tubular tubular tubular tubular tubular
Lumen
content
amor-
phous
amorphous amorphous amour-
phous
amorphous
Chromatin hetero-
chrom2
hetero-
chrom2
hetero-
chrom2
hetero-
chrom2
hetero-
chrom2
Notes: 1some cases may express anisokaryosis; 2some cases may express
euchromatin; 3some cases may express mononuclear content.
272 Experimental Oncology 39, 269–275, 2017 (December)
Prostate histology in adulthood. The results
indicate that pubertal LPS treatment resulted in abnor-
mal prostatic histology in adulthood (see Tables 3, 4,
Fig. 2–4). For instance, there were more cases of epi-
thelium dysplasia, specially in the VP (see Fig. 2, 3),
but also anisocytosis, presence of mononuclear cells,
anisokariosis, non-basal polarity, abnormal nucleus-
cytoplasm ratio, proplastic myoepithelium, and granular
content in the lumen. These histological features are
abnormal and can be considered precancerous.
0
20
40
60
80
100
1 2 3 4 5
Pe
rc
en
ta
ge
o
f c
as
es
Groups
dysplasia
metaplasia
normal
Fig. 2. Epithelium features in the DLP of male rats: 1 — in-
tact untreated; 2 — saline-treated and implanted with a blank
capsule in adulthood; 3 — saline-treated and implanted with
a testosterone capsule; 4 — LPS-treated and implanted with
a blank capsule; 5 — LPS-treated and implanted with a testos-
terone capsule. Histology was assessed with H & E stain in order
to identify precancerous lesions
Serum levels of testosterone and corticoste-
rone. With regard to testosterone, the ANOVA revealed
significant differences F(4,35) = 3.5 (p < 0.01). The
posthoc test indicated that serum from LPS-testos-
terone rats contained significantly higher testosterone
levels (mean = 10.21 ng/ml) than LPS-blank controls
(mean = 7.3 ng/ml). LPS-blank rats (mean = 7.2 ng/ml)
contained lower testosterone levels than saline-testos-
terone rats (mean = 11.05 ng/ml). As expected, saline-
testosterone rats (mean = 11.05 ng/ml) contained higher
testosterone levels than intact rats (mean = 8.5 ng/ml)
(Fig. 5). With regard to corticosterone, the analysis did
not to detect significant differences between groups
F(4,35) = 0.37 (p > 0.05) (Fig. 6).
Table 4. Histological features of the DLP of males from the different groups.
Features were accepted when it was observed in at least 6 out of 8 males
Histology
Dorsolateral prostate
Intact Saline
blank
Saline
testosterone
LPS
blank
LPS
testosterone
Epithelium
Form cubic cubic4 dysplasia cubic6 dysplasia
Size even even5 anisocytosis anisocytosis anisocytosis
Papillae scarce scarce moderate moderate plenty
Interstice
Space even even even even even
Content collagen collagen collagen mononu-
clear
collagen
Nucleus
Size even1 even1 anisokary-
osis
anisokary-
osis
anisokary-
osis
Location polar polar no polar no polar no polar
Nucleus:
cytoplasm
> 1:3 > 1:3 1:1 1:1 1:1
Myoepithe-
lium
euplasia2 euplasia2 proplasia proplasia proplasia
Pattern (4×) tubular tubular tubular tubular tubular
Lumen
content
amor-
phous
amor-
phous
amorphous granular granular
Chromatin hetero-
chrom3
hetero-
chrom3
hetero-
chrom3
hetero-
chrom3
hetero-
chrom3
Notes: 1some cases express anisokariosis; 2some cases express proplasia;
3some cases express euchromatin; 4some cases express metaplasia;
5some cases express anisocytosis; 6some cases express dysplasia.
0
20
40
60
80
100
1 2 3 4 5
Pe
rc
en
ta
ge
of
c
as
es
Groups
dysplasia
metaplasia
normal
Fig. 3. Epithelium features in the VP of male rats: 1 — intact untreated;
2 — saline-treated and implanted with a blank capsule in adulthood;
3 — saline-treated and implanted with a testosterone capsule; 4 —
LPS-treated and implanted with a blank capsule; 5 — LPS-treated
and implanted with a testosterone capsule. Histology was assessed
with H & E stain in order to identify precancerous lesions
Fig. 4. Photomicrographs of the DLP and VP (× 40). Histological
abnormalities are observed in treatments saline-testosterone,
LPS-blank and LPS-testosterone. See Table 2 for details
Experimental Oncology 39, 269–275, 2017 (December) 273
DISCUSSION
The results of the present study indicate that
acute LPS treatment to pubertal rats (5 week old)
results in long-term abnormal histological features
of the prostate, observable in adulthood (12 weeks
old). LPS-treated rats expressed more cases of epi-
thelium dysplasia specially in the VP (see Fig. 2, 3),
anisocytosis in DLP, presence of mononuclear cells
in DLP and VP, anisokariosis in DLP and VP, non-basal
polarity in DLP, abnormal nucleus-cytoplasm ratio
in DLP and VP, proplastic myoepithelium in DLP and
granular content in the lumen in DLP. Anisocytosis
(unequal abnormal size of epithelium cells) and ab-
normal nuclear shape (anisokaryosis) can be used
to identify cancerous cells [27, 28]. Anysokaryosis,
apolarity and abnormal nucleus-cytoplasm ratio de-
note changes in chromosome organization, and pre-
sumably affect gene expression [29] that may result
in metaplasia or dysplasia; the latter considered the
anteroom of CaP. When pubertal LPS was combined
with testosterone in adulthood (LPS-testosterone
group) the number of animals with prostatic lesions
did not increase. Lesions on these animals were similar
to those observed in the group that received testos-
terone only (saline-testosterone), suggesting that LPS
during puberty has no additional effect than those
caused by testosterone in adulthood, or vice versa.
However, two males of the LPS-testosterone group
failed to express sickness symptoms after the injec-
tion with LPS during puberty. Those males expressed
normal (cubic) epithelium in the DLP, but dysplasia and
stratified epithelium in the VP, respectively. They also
expressed anisokaryosis and non-basal polarity. This
might suggest a link between the susceptibility to ex-
press sickness after receiving LPS in pubertal animals
and the probability to develop precancerous prostate
lesions. Further research is needed to investigate this
possible relationship.
LPS and endocrine alterations. Previous
stu dies indicate that chronic stress (i.e. restraint)
during puberty results in higher levels of serum
testosterone [14], which is a hormone that can
induce cell division and spontaneous mutations
within the prostate [9, 10], and a confirmed cause
of epithelium dysplasia [11]. Accordingly, we first
hypothesized that acute immune stress during pu-
berty (LPS-induced) would result in higher levels
of testosterone in adulthood, with the correspond-
ing prostatic lesions. However, the levels of serum
testosterone increased exclusively in males with
a testosterone implant (as expected), but not
in animals that received one injection of LPS dur-
ing puberty (LPS-blank) (see Fig. 5). Some reports
indicate that the density of androgen receptors (AR)
is modified in CaP or following an infection [30, 31].
We speculate about the possibility that LPS may
alter the AR density, which would contribute to the
gland’s susceptibility to suffer spontaneous cell
division or mutations under the effects of normal
serum testosterone levels. AR gene can mutate
or be amplified and therefore may respond to lower
or equal levels of androgens in adulthood [6, 32–34].
Further research is needed to understand the long-
term effects of pubertal LPS on the distribution and
presence of AR in the adult prostate.
Similarly, we hypothesized that corticosterone
baseline serum levels would be higher in those adult
males that received LPS during puberty as a con-
sequence of enduring HPA axis activity enhance-
ment. Indeed, other studies have shown that stress
in adulthood facilitates the progression of different
types of cancer [35–38]. For example, stressed rats
(i.e. isolated) that receive a carcinogenic drug such
as N-methyl-N-nitrosurea (NMU) express higher lev-
els of blood corticosterone and 30% more mammary
tumors that express more corticosterone receptors
(CR) as compared to rats that only receive NMU
without being stressed [35]. Our results, however,
indicated that males from all the groups expressed
similar serum levels of corticosterone in adulthood
(see Fig. 6). Therefore, we consider that further re-
search is needed to explore the effects of LPS on the
distribution and proportion of CR in the prostate.
1 2 3 4 5
Groups
0
5
10
15
a
a
b
b
a
Le
ve
l o
f s
er
um
te
st
os
te
ro
ne
,
ng
/m
l
Fig. 5. Mean ± S.E.M of baseline serum levels of testosterone
in the different groups: 1 — intact untreated; 2 — saline-treated
and implanted with a blank capsule in adulthood; 3 — saline-
treated and implanted with a testosterone capsule; 4 — LPS-
treated and implanted with a blank capsule; 5 — LPS-treated
and implanted with a testosterone capsule. Different letters
indicate p < 0.05
1 2 3 4 5
Groups
0
200
400
600
800
Le
ve
l o
f s
er
um
c
or
tic
os
te
ro
ne
,
ng
/m
l
Fig. 6. Mean ± S.E.M of baseline serum levels of corticosterone
in the different groups: 1 — intact untreated; 2 — saline-treated
and implanted with a blank capsule in adulthood; 3 — saline-
treated and implanted with a testosterone capsule; 4 — LPS-
treated and implanted with a blank capsule; 5 — LPS-treated
and implanted with a testosterone capsule
274 Experimental Oncology 39, 269–275, 2017 (December)
Some reports indicate that treatment with LPS
to neonatal rats results in immediate increase
of corticosterone that does not correspond to adult
response [39]. For example, in one study 5-day-old
males were treated with LPS (from S. enterica) and
4 h later their levels of corticosterone were higher
(20 ng/ml) than controls (17 ng/ml), but the levels
of testosterone were lower (0.1 ng/ml) than controls
(0.4 ng/ml). Interestingly, in adulthood, the levels
of corticosterone were lower (350 ng/ml) than con-
trols (550 ng/ml) following a stress challenge. LPS
rats also expressed lower levels of testosterone
(2 ng/ml) than controls (3 ng/ml) [39]. These al-
terations correlated with the presence of abnormal
testicular epithelium. Accordingly, LPS in neonatal
rats affects epithelia and can alter the levels of tes-
tosterone and corticosterone in adulthood, but such
alterations are observed only following a stress
challenge.
Stress, inflammation and CaP. LPS increases
the level of some cytokines such as IL-1b, IL-6,
IL-10, IL-12 [18, 36], and also IFN-γ, TNF-α [18], and
NF-kB [40] which results in subclinical inflammation.
Inflammation is very common within the adult human
prostate [7], and some studies have reported positive
correlations between prostatitis and higher probability
of developing CaP [41, 42]. In our study, LPS-treated
males expressed more mononuclear cells than con-
trol animals seven weeks later (indicating subclinical
and chronic inflammation). E. coli and Enterococcus
spp. are the most common microorganisms causing
prostatitis [43, 44], and mouse models of prostatitis
following injections of E. coli also express epithelial
proliferation and reactive hyperplasia, dysplasia and
oxidative DNA damage [45, 46]. Other organisms
such as Pseudomonas spp., Proteus mirabilis, Kleb-
siella spp. and Serratia spp. have also been identified
as a cause of prostatitis, all of them gram negative
bacteria (LPS-holders).
Differential effects of LPS on DLP and VP. The
two portions of the rat’s prostate (DLP and VP)
respond differently to experimental manipulations
[8, 11]. For instance, multiple trials of copulation
result in histological alterations in the DLP, and the
addition of exogenous testosterone results in even
greater alterations [8]. However, in the VP copulation
plus exogenous testosterone results in fewer cases
of dysplasia. The VP expresses different proportion
of AR and AR-mRNA [47] and such heterogene-
ity may account for the different effects observed
following exogenous testosterone with repetitive
copulation [8]. Interestingly, the results of the pres-
ent study showed that LPS resulted in more cases
of dysplasia in the VP.
CONCLUSIONS
Pubertal immune challenge results in histological
alterations in the two prostatic portions (DLP, VP) in adult
rats. These lesions can be considered precancerous,
but are not cancer per se, and are likely to be reversible.
In addition, pubertal LPS treatment does not affect se-
rum testosterone or corticosterone levels in adulthood,
suggesting that the observed histological alterations
are not a consequence of abnormal hypothalamic-
pituitary-gonadal or HPA axes activity. Further research
is needed to understand the specific role of pubertal
LPS treatment on the levels of AR in the prostate and the
development and maintenance of prostatic diseases,
including cancer [48, 49]. We conclude that pubertal
stress can influence the development of prostatic pre-
cancerous lesions in adulthood.
ACkNOwLEDGMENTS
This study was supported by Consejo Nacional
de Ciencia y Tecnología (CONACyT) from Mexico, with
a Repatriation grant (CVU-210442 to DHC) and the
Natural Sciences and Engineering Research Council
of Canada (211075-190799-2001 to NI).
CONFLICTS OF INTEREST
The authors declare that they have no conflict
of interest.
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Copyright © Experimental Oncology, 2017
|
| id | nasplib_isofts_kiev_ua-123456789-138584 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-11-24T10:20:45Z |
| publishDate | 2017 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Herrera-Covarrubias, D. Coria-Avila, G.A. Hernandez, M.E. Ismail, N. 2018-06-19T10:01:31Z 2018-06-19T10:01:31Z 2017 Stress during puberty facilitates precancerous prostate lesions in adult rats / D. Herrera-Covarrubias, G.A. Coria-Avila, M.E. Hernandez, N. Ismail // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 269–275. — Бібліогр.: 49 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138584 Puberty can be a critical period for the long-term development of diseases, especially for stress-related disorders that depend on neuroendocrine and immune responses. Some organs like the prostate are prone to diseases that result from neuroendocrine or immune challenges, such as cancer. Aim: In the present study, we assessed the long-term effects of an acute pubertal stressor (immune-challenge) on the development of precancerous lesions in adult rats, and compared them with testosterone-induced prostatic lesions. Materials and Methods: Pubertal male rats received a single injection of lipopolysaccharide (LPS) or saline during puberty (5 weeks old). At adulthood (8 weeks old) males were subcutaneously implanted with either an empty capsule or filled with testosterone propionate (100 mg/kg). This resulted in a total of five groups: 1) intact untreated, 2) saline-treated and implanted with a blank capsule, 3) saline-treated and implanted with a testosterone capsule, 4) LPS-treated and implanted with a blank capsule, 5) LPS-treated and implanted with a testosterone capsule. Four weeks later, the rats were sacrified and their prostates processed for histology (hematoxylin and eosin stain) and blood serum processed for hormone analysis (testosterone and corticosterone). Results: Males treated with LPS (stressed during puberty via immune challenge) expressed epithelium dysplasia (specially in the ventral prostate), anisocytosis, presence of mononuclear cells, anisokariosis, non-basal polarity, abnormal nucleus-cytoplasm ratio, proplastic myoepithelium, and granular content in the lumen. These histological alterations were similar, but less severe than those observed in males implanted with testosterone during adulthood. Conclusion: These results indicate that pubertal exposure to an immune challenge (stress) facilitates the long-term development of prostatic lesions in adult male rats. This study was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) from Mexico, with a Repatriation grant (CVU-210442 to DHC) and the Natural Sciences and Engineering Research Council of Canada (211075-190799-2001 to NI). en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Stress during puberty facilitates precancerous prostate lesions in adult rats Article published earlier |
| spellingShingle | Stress during puberty facilitates precancerous prostate lesions in adult rats Herrera-Covarrubias, D. Coria-Avila, G.A. Hernandez, M.E. Ismail, N. Original contributions |
| title | Stress during puberty facilitates precancerous prostate lesions in adult rats |
| title_full | Stress during puberty facilitates precancerous prostate lesions in adult rats |
| title_fullStr | Stress during puberty facilitates precancerous prostate lesions in adult rats |
| title_full_unstemmed | Stress during puberty facilitates precancerous prostate lesions in adult rats |
| title_short | Stress during puberty facilitates precancerous prostate lesions in adult rats |
| title_sort | stress during puberty facilitates precancerous prostate lesions in adult rats |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/138584 |
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