Keywords
testosterone - proliferation - apoptosis - breast - ovariectomy
Palavras-chave
testosterona - proliferação - apoptose - mama - ovariectomia
Introduction
Testosterone (T) has been used by postmenopausal women, with a positive effect on
desire and sexual function.[1] Through cohort studies in worldwide populations, researchers have reported a small
but significant association between T, androstenedione, dehydroepiandrosterone (DHEA)
and dehydroepiandrosterone Sulfate (DHEAS), and breast cancer in postmenopausal women.[2]
[3]
[4] Most of the studies using T last from 1 month to 2 years, hence the information
that assures its long-term use is unknown, which is a concern for medical professionals
as well as for climacteric women regarding the risk and its impact on breast tissue.[5]
[6] Thus, considering the increasing use of T treatments by menopausal women, the lack
of in vivo controlled studies using T for hormone replacement, and the need for further
investigation of its biological impact on the breast tissue, it is relevant to study
such aspects in an animal model and the effect of this therapy on the breast. The
objective of the present study was to investigate the action of T, isolated or associated
with estradiol benzoate (EB), on proliferation markers and apoptosis of mammary tissue
in ovariectomized rats.
Methods
We randomly selected 48 castrated female Wistar rats at 250 days of age. All of them
underwent bilateral oophorectomy under anesthesia with ketamine and xylazine intraperitoneally.
A longitudinal ventral approach was used for the identification and ligation of the
ovarian pedicles and subsequent removal of the gonads. Three weeks later, microscopic
examinations of vaginal smears of all rats were performed to confirm hypoestrogenism.
Next, the animals were randomly divided into 6 groups of 8 each; the groups received
the corresponding daily hormonal dose in a volume of 0.1 ml by subcutaneous injections
in the dorsal region of each animal, during 5 consecutive weeks. The daily treatments
were as follows: group 1: control, sesame oil 0.1 ml/day (n = 8); group 2: EB 50 mcg + T 50 mcg/day (n = 8); group 3: T 50 mcg/day (n = 8); group 4: EB 50 mcg + T 300 mcg/day (n = 8); group 5: T 300 mcg/day (n = 8); group 6: EB 50 mcg/day (n = 8). The animals were kept in a quiet environment with a constant temperature of
23°C, lighting periods of 12 hours per day, in addition to water and feed ad libitum.
Five weeks later, after the end of the treatment, the rats were anesthetized with
ketamine 80 mg/kg, and sacrificed by thiopental overdose. The second right mammary
gland of each animal was resected and fixed in 10% buffered formalin and subsequently
prepared for histological and immunohistochemical evaluation.
Semiquantitative Histological Analysis
The mammary glands obtained were embedded in paraffin, cut into to 3 μm sections,
stained with hematoxylin-eosin (HE), and analyzed under high magnification microscopy
(400x). A histological analysis with HE staining was performed, using a semiquantitative
method (zero as absent, + as mild, ++ as moderate, and +++ as intense), and the samples
were observed using a Nikon (Minato, Tokyo, Japan) double-head microscope. This procedure
was performed by the pathologist (A.M.M.) and by the main researcher (J.C.C.O.), and
both of them reached a consensus about the presence or absence of atrophy, microcalcification
in the glandular lumen, and degree of glandular secretory activity. The quantification
of secretory activity was reported as follows:
-
+: minimal secretion in the form of cytoplasmic vacuolization without accumulation
or dilation of light.
-
++: secretion in the cytoplasm and light, with minimal architectural distortion of
the mammary lobes.
-
++ + : exuberant secretion.
-
0: absent secretory activity.
Histological Analysis: Histomorphometry
The histomorphometric quantitative analysis was performed under HE staining by the
pathologist (A.M.M.), using a Zeiss (Berlin, Germany) trinocular microscope with the
Axiovision 4 (Zeiss) image analysis software, under medium magnification (100x). The
images obtained were captured by a digital camera and transferred to a computer with
an image scanner. After calibration to measure in mm2, the entire mammary gland (glandular tissue, fibroadipous stroma and striated muscle
tissue) was delineated with the computer mouse, obtaining the total area. Then, only
the glandular acini were delineated, adding their area, also expressed in mm2. Subsequently, the ratio between the glandular area and the total breast area was
calculated.
Immunohistochemistry
Immunohistochemistry (IHC) was performed to analyze proliferating cell nuclear antigen
(PCNA) and apoptosis (caspase-3) in 3-μm thick histological sections. All samples
were submitted to IHC reaction using the following primary antibodies: PCNA (PC10)
1:1,000 (Cell Signaling Technology, Danvers, MA, US) and caspase-3 1:350 (BioVision,
Milpitas, CA, US). The IHC reaction was developed using the peroxidase kit LSAB-peroxidase
(DakoCytomation, Copenhagen, Denmark), according to the manufacturer's protocol. Then,
they were processed with chromogen (3–3'-diaminobenzidine (DAB) 100 mg (Sigma-Aldrich,
St. Louis, MI, US) in 70ml of phosphate-buffered saline (PBS) + 3 ml hydrogen peroxide,
and Harris hematoxylin counterstain (Sigma-Aldrich). The slides were analyzed with
a Nikon Eclipse TS100 light microscope, under the same light intensity and the same
height as the condenser, to identify the areas that best represent the immunostaining
of the analyzed molecules (hot spot). The areas that best represented the immunostaining
were selected by an experienced pathologist, who was blinded to the treatment groups,
and analyzed under 400x magnification . Photomicrographs of consecutive mismatched
fields with a resolution of 640 × 480 pixels were obtained using a Nikon Coolpix 4300
digital camera with the same settings for all blades. In each case, the quantification
of the immunostaining was performed by digital computer analysis, and the values were
expressed by the expression index (IE). The images obtained were analyzed using the
ImageLab (Softium Informática, São Paulo, SP, Brazil) image processing and analysis
system, adjusted to the micrometer (μm) scale.
Statistical Method
The data were organized into Microsoft Excel 2007 (Microsoft Corp., Redmond, WA, US)
spreadsheets. The qualitative variables were presented as absolute and relative frequencies,
and comparisons between the groups were performed using the Chi-squared test.
The numerical data were presented as means and standard deviations, and their normal
distribution was analyzed using the Shapiro-Wilk test. As this distribution was not
confirmed, the groups were compared using the Kruskal-Wallis test, while comparisons
between pairs of groups were made using the Mann-Whitney U-test. The statistical package
XLStat (Addinsoft, New York, NY, US), version 17.01, for Excel was used for all analyzes.
The level of statistical significance was set at 5% (p < 0.05).
Ethical Aspects
All of the study procedures were approved by the Animal Research Ethics Committee
of the Faculdade de Medicina do ABC under number 01/2016.
Results
During the study there was no loss of animals. After dissection and preparation of
the slides, 7 samples had no glandular tissue, so 41 samples with mammary glandular
tissue on the slides were included in the analysis.
[Table 1] shows the analysis of semiquantitative atrophy parameters, but no statistically
significant difference was found among the groups. However, there is a tendency for
lower occurrence of atrophy in groups receiving EB and T versus those receiving T
alone. Therefore, we decided to group all of the cases that received EB associated
with T, as well as those that received T alone, and to perform a comparative analysis
of these larger groups, noting a higher prevalence of atrophy in the group treated
with isolated T, with statistical significance (p = 0.015) ([Table 2]). As for microcalcifications ([Table 1]), there was a statistically significant difference among the groups (p = 0.024), with a higher occurrence in groups receiving EB. Similarly, we grouped
the results of the EB + T groups, comparing them with the groups that received only
T ([Table 2)], and a higher prevalence of microcalcification in the groups that received EB (p = 0.022) was observed.
Table 1
Results of the semiquantitative parameters by therapy (values expressed in absolute
numbers and percentages in parentheses)
|
Semiquantitative parameters
|
Placebo
n = 8
|
|
|
Estradiol benzoate 50 mcg
n = 7
|
|
Estradiol benzoate 50 mcg +
testosterone
50 mcg
n = 6
|
Testosterone
50 mcg
n = 5
|
Estradiol benzoate 50 mcg +
testosterone
300 mcg
n = 8
|
Testosterone
300 mcg
n = 7
|
|
Atrophya
|
|
No
|
2 (25%)
|
3 (50%)
|
0 (0%)
|
4 (50%)
|
0 (0%)
|
2 (28.57%)
|
|
Yes
|
6 (75%)
|
3 (50%)
|
5(100%)
|
4(50%)
|
7 (100%)
|
5 (71.42%)
|
|
Microcalcificationb
|
|
Absent
|
7 (87.5%)
|
4 (66.6%)
|
4 (80%)
|
3 (37.5%)
|
7 (100%)
|
2 (28.5%)
|
|
Present
|
1 (12.5%)
|
2 (33.3%)
|
1 (20%)
|
5 (62.5%)
|
0 (0%)
|
5 (71.4%)
|
|
Secretory activityc
|
|
Absent
|
3 (37.5%)
|
1 (16.6%)
|
1 (20%)
|
1 (12.5%)
|
6 (85.7%)
|
0(0%)
|
|
Present
|
5 (62.5%)
|
5 (83.3%)
|
4 (80%)
|
7 (87.5%)
|
1 (14.2%)
|
7(100%)
|
|
Absent
|
3 (37.5%)
|
1 (16.6%)
|
1(20%)
|
1 (12.5%)
|
6 (85.7%)
|
0(0%)
|
|
+
|
2 (25%)
|
3 (50%)
|
4 (80%)
|
2 (25%)
|
0 (0%)
|
2 (28.5%)
|
|
+ +
|
2 (25%)
|
2 (33.3%)
|
0 (0%)
|
3 (37.5%)
|
1 (14.2%)
|
3 (42.8%)
|
|
+ ++
|
1 (12.5%)
|
0 (0%)
|
0 (0%)
|
2 (25%)
|
0 (0%)
|
2 (28.5%)
|
Notes: a
p = 0.143; b
p = 0.024; c
p = 0.008. +: mild secretion; ++: moderate secretion; ++ + : intense secretion.
Table 2
Results of the semiquantitative parameters (values expressed in absolute numbers and
percentages in parentheses) in the groups that received estradiol benzoate plus testosterone
and isolated testosterone
|
Semiquantitative Parameters
|
|
|
Estradiol benzoate + testosterone
|
Testosterone
|
|
Atrophya
|
|
No
|
7 (50%)
|
0 (0%)
|
|
Yes
|
7 (50%)
|
12 (100%)
|
|
Microcalcificationb
|
|
Absent
|
7 (50%)
|
11 (91.7%)
|
|
Present
|
7 (50%)
|
1 (8.3%)
|
|
Secretory activityc
|
|
Absent
|
2 (14.3%)
|
7 (58.3%)
|
|
Present
|
12 (85.7%)
|
5 (41.7%)
|
Notes: a
p = 0.015, b
p = 0.022; c
p = 0.019.
The secretory activity was also different among the groups (p = 0.008), being higher in the groups that received isolated EB or EB + T compared
with the groups that received only T. In [Table 2], in the analysis performed with two groups containing all of the cases who received
E + T and all of the cases that received isolated T, a higher occurrence of secretion
was confirmed in the groups that received EB, with statistical significance (p = 0.019). [Fig. 1] shows the result of the glandular tissue area. Although the glandular area seemed
larger in the groups receiving EB than in those receiving only T or the controls,
no statistical significance was found in the overall analysis among groups. In relation
to the breast area, no significance was found either.
Fig. 1 Graphical representation of the mammary gland area in the collected tissue per groups.
The graph in [Fig. 2] represents the area ratio of mammary acini to the total area of breast, with larger
values being observed in the groups treated with EB compared with those treated only
with T or the controls, but the overall analysis showed no statistical significance.
However, when we compared the cases that received isolated T with the isolated EB
group, the difference was statistically significant (p = 0.047).
Fig. 2 Graphical representation of the ratio between gland area and mammary area per groups.
The PCNA quantification, shown in [Fig. 3], presents a statistically significant difference among groups (p = 0.028). The analysis of pairs of groups evidenced a significant difference between
the EB 50 mcg + T 300 mcg group and the controls (p = 0.001), between the EB 50 mcg + T 300 mcg and EB 50 mcg + T 50 mcg (p = 0.015) groups, and between the EB 50 mcg + T 300 mcg and T 300 mcg (p = 0.027) groups.
Fig. 3 Graphical representation of quantification by immunohistochemistry of PCNA per groups.
[Fig. 4] graphically quantifies the immunohistochemistry of caspase-3. No statistically significant
difference was found among the groups (p = 0.236); however, in the analysis of pairs of groups, a statistically significant
difference was observed for the T 300 mcg group compared with the control group (p = 0.037), and between the T 300 mcg compared with the E + T 300 mcg group (p = 0.025).
Fig. 4 Graphical representation of the quantification by immunohistochemistry of caspase-3
per groups.
Discussion
Loss of sexual desire, musculoskeletal health impairment, and decreased cognitive
performance are some of the characteristic symptoms of the climacteric. The exogenous
use of T has been indicated for such complaints, especially for the decrease in sexual
desire, but it generates concern among the users. Few studies have been published
on the action and effects of T supplementation in women, as well as studies that clarify
the contribution of T to the risk of developing cardiovascular, musculoskeletal and
cognitive disorders and cancer.[7]
The variety of hormone-related information, specifically T in breast tissue, combined
with the divergence of outcomes from studies on the risk of breast cancer and hormone
replacement therapy users[8]
[9] was sufficient reason to develop this research; however, the difficulties in studying
the human breast made us opt for the analysis of the response of mammary tissue to
the hormones tested in an experimental model with rats.
The studies by Russo et al[10] and Russo and Russo[11] contributed with mammary tissue studies with rats and considered an adequate and
representative model of human breast, which supported the decision to choose the murine
model in the present study.
The present study did not find a statistically significant difference among the groups
for the semiquantitative parameters of atrophy, but when comparing the isolated T
groups to the EB + T groups, there was a lower occurrence of atrophy in the groups
treated with EB. Thus, we can conclude that, in the absence of estrogen, the mammary
lobes atrophy, leading to a decrease in the proliferation of the epithelium, with
a decrease in the number of lobes in the breast tissue, as shown by Clarke,[12] who stated that estrogen is responsible for mitogenesis in the breast during menstruation,
even in the presence of T. The novelty is that isolated T in our study did not lead
to proliferation; on the contrary the rates of atrophy were higher in the groups treated
with isolated T.
The results found in this study are compatible with those of the study by Grynberg
et al,[13] who state that this increase in atrophy of the glandular tissue and fibrosis of
the stromal tissues is caused in the postmenopausal period, because of the absence
of estrogenic hormonal stimulation in the mammary tissue.
Regarding secretory activity, a statistically significant difference was found in
the overall analysis and the analysis among groups, with higher secretory activity
in the groups treated with EB + T. This shows that isolated T exerted little stimulus
in the proliferative activity of breast tissue, a stimulus that was evidenced whenever
EB was added.
Our study showed a statistically significant difference among treatment groups in
the proliferative activity measured by PCNA, especially the comparison of groups treated
with higher doses of T combined with EB compared with the groups treated with lower
doses of T combined with EB, isolated T, or the controls.
Through graphic analysis and based on multiple comparisons, a clear difference in
the effect of T/EB on the epithelial proliferation expressed by PCNA is evident. In
contrast, a lower rate of proliferation was observed with T alone.
Our study converges with the results of the study by Somboonporn and Davis,[14] which describes that androgens act in a direct protective way on cell proliferation
by controlling the mitogenic effect of estrogen on breast tissue. In addition, one
of the few human studies evaluating the effect of T on breast-cell proliferation,
conducted by Hofling et al,[15] found that postmenopausal women who received T (300 mcg/day) associated with hormone-replacement
therapy (2 mg of estradiol and 1 mg of norethisterone acetate) showed no increase
in cell proliferation; on the other hand, there was a more than five-time increase
in cell proliferation in women receiving estrogen therapy associated to progestogen.
This finding suggests that although the histological study has not shown proliferative
mammary T activity, there may be a dose-dependent effect when T is associated with
EB.
The analysis of caspase-3 showed a higher apoptosis in the group that received EB + T
in high doses compared with the group that received the same dose of T, but without
EB. This is evidence that the increased proliferation with higher doses of T associated
with EB led to increased cell apoptosis.
The present study reinforces the findings of the study by Zhou et al,[16] who investigated ovariectomized rhesus macaques regarding the sexual steroid effects
on mammary epithelial proliferation, with isolated estradiol increasing breast epithelial
proliferation by ∼ 50%, and T reducing the induced estradiol proliferation by 40%,
suggesting that it is a positive androgen replacement therapy in menopausal women.
Although not always statistically significant, our study shows a lower proliferation
in the presence of T in the graphic analysis, but we observed a dose-dependent effect
of T, which may mean that, in larger doses, T may not suppress the proliferation induced
by estrogen.
Some limitations of our study can be mentioned, such as the fact that we have evaluated
proliferation and apoptosis with only one marker for each one, PCNA and caspase-3
respectively, although these have shown results and have already been used in previous
studies such as the one by Pompei et al.[17] Another limiting factor was our relatively small number of animals in the different
groups. A larger sample would probably show statistical significance in other parameters
that in the graphic analysis seem to be different among groups. As a strong point,
the double-blinded design was highlighted, in which the researchers and the pathologist
who analyzed the slides were not aware of the treatment in each group, eliminating
a source of bias. In addition, comparing different doses of T in combination or not
with estrogen helps to understand the possible dose-dependent effect and to support
new studies addressing these issues.
Conclusion
Isolated T did not have a proliferative effect on mammary tissue, contrary to EB.
The combination of T and EB may or may not decrease the proliferation, depending on
the dose of T.
