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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2019 May 26;100(3):192–201. doi: 10.1111/iep.12317

Short‐term exposure to chrysin promotes proliferative responses in the ventral male prostate and female prostate of adult gerbils

Mônica S Campos 1, João P A Silva 2, Danilo S Lima 2, Luis O Regasini 3, Mara Rúbia Marques 2, Manoel F Biancardi 2, Sebastião R Taboga 1, Fernanda C A Santos 2,✉
PMCID: PMC6658909  PMID: 31131507

Summary

Chrysin (5,7‐dihydroxyflavone) is a bioactive compound found in different fruits, vegetables, honey and propolis. This flavone has been suggested for the treatment of reproductive dysfunction, mainly because of its antioxidant and hormonal properties. However, the effects of this polyphenol on the prostate are still poorly understood. The purpose of this study was to evaluate the effects of short‐term chrysin exposure on the ventral male and female prostates of adult gerbils. To evaluate the androgenic potential of chrysin, gerbils were also exposed to testosterone. Male and female gerbils were exposed to chrysin (50 mg/kg/day, orally) or testosterone cypionate (1 mg/kg/week, subcutaneously) for 3, 7 and 21 days. Prostates were dissected for morphological, stereological and immunohistochemical analyses. Serum levels of testosterone and 17β‐estradiol were measured by ELISA. Serum testosterone levels were not increased by chrysin supplementation in males or females. However, only females treated with chrysin for 21 days showed an increase in estradiol levels. Increased androgen receptor immunoreactivity, higher proliferation rates and glandular hyperplasia were observed in male and female prostates for all chrysin treatment times. Additionally, increased oestrogen receptor alpha immunoreactivity was observed in all chrysin‐treated females. Although chrysin and testosterone promoted similar morphological changes in the gerbil prostate, chrysin supplementation was less deleterious to prostate health, since it resulted in lower incidence of hyperplasia and an absence of neoplastic foci.

Keywords: androgen receptor, oestrogen receptor, flavonoids, histopathology, immunohistochemistry

Abbreviations

AR

androgen receptor

Chr

chrysin

DAB

diaminobenzidine

ERα

oestrogen receptor alpha

PCNA

proliferating cell nuclear antigen

T

testosterone

1. INTRODUCTION

Flavonoids are polyphenols synthesized by many plants and considered important components of the human diet.1 Chrysin (5,7‐dihydroxyflavone) is a naturally occurring compound of the flavone class found in passion blue flowers (Passiflora caerulea),2 on the geranium leaf surface (Pelargonium crispum),3 and in honey and propolis.4 This flavone has shown a wide range of pharmacological effects, such as anti‐inflammatory, anxiolytic, anticancer and antioxidant properties.5, 6, 7

Chrysin promotes testosterone‐boosting effects, either by inhibiting aromatase catalytic activity8, 9, 10 or by stimulating testicular steroidogenesis via increased Star gene expression.11 Due to its hormonal properties, chrysin has been employed as a dietary supplement to increase lean body mass12 and for the treatment of reproductive dysfunctions.13, 14

The effects of chrysin on reproduction are better understood in males. In rodents and birds, chrysin showed beneficial effects on spermatogenesis.15, 16 In male rats, chrysin showed a protective effect against testosterone‐induced benign prostate hyperplasia.17 However, the effects of chrysin on the prostate gland remain poorly understood.

The prostate is an accessory gland of the reproductive system, whose function is to produce an alkaline secretion that promotes the nutrition and survival of spermatozoa.18 Several studies have reported the presence of a prostate in women and female rodents.19, 20, 21, 22 In gerbils, the female prostate presents structural homology with the ventral male prostate, making this a valuable model for comparative prostate studies.23

The prostate is hormone‐dependent and requires both androgens and oestrogens for growth, development and maintenance during adulthood.24 In this context, we hypothesized that exposure to hormonally active phytochemicals may alter male and female prostate morphophysiology. Thus, the purpose of this study was to evaluate the effects of different periods of chrysin exposure (3, 7 and 21 days) on the ventral male prostate and the female prostate of adult gerbils. In addition, in order to evaluate the androgenic potency of chrysin, animals treated with testosterone cypionate were employed as positive controls for the experiments.

2. MATERIAL AND METHODS

2.1. Chemical procedures

Chrysin synthesis was performed in the Laboratory of Green and Medicinal Chemistry (IBILCE/UNESP) according to the method previously described in Ribeiro et al25 Briefly, the chalcone intermediate was obtained using Claisen‐Schmidt condensation between trihydroxyacetophenone and benzaldehyde.26 The conversion from chalcone to flavone was obtained by intramolecular nucleophilic substitution.27 After complete conversion was confirmed by TLC analysis, the reaction mixture was extracted with ethyl acetate by liquid‐liquid partitioning. The crude product was purified over silica gel, yielding chrysin (23%). Chrysin was obtained as a pale yellow solid. 1H NMR (600 MHz, DMSO‐d 6): 12.82 (brs, 1H), 8.06 (d, 2H), 7.59 (m, 3H), 6.96 (s), 6.54 (d), and 6.23 (d). 13C NMR (125 MHz, DMSO‐d6): 182.3, 164.9, 163.7, 161.9, 157.9, 132.5, 131.3, 129.6, 126.8, 106.6, 105.0, 99.5 and 94.6.

2.2. Ethical approval

Animal handling and experiments were approved by the Ethics Committee on the Use of Animals of the Universidade Federal de Goias (protocol no. 111/17—CEUA/UFG), following the Guide for Care and Use of Laboratory Animals.

2.3. Animals

All animals employed in this study were maintained in a temperature‐controlled room (23°C) on a 12‐hour light/dark cycle. Gerbils were maintained on filtered water and fed standard rodent food ad libitum (Labina‐Purina®; composition: 23% protein, 4% fat, 5% fibre and 12% minerals).

2.4. Experimental design

One hundred twelve adult gerbils (Meriones unguiculatus, Gerbillinae: Muridae; 3 months old) were employed in this study. Male and female gerbils were divided into 14 groups as shown in Figure 1 (seven female groups and seven male groups, n = 8 animals/group): Animals in the control group (C) received daily oral doses of the dilution vehicle (mineral oil/Nujol‐Mantecorp, 100 μL/animal) for 21 days. Animals in the chrysin group (Chr) received daily oral doses of chrysin (50 mg/kg)15 for 3, 7 and 21 days. To evaluate the possible androgenic effects of chrysin, a group that received testosterone cypionate (T) was employed as a positive control for the experiments. T‐group animals received subcutaneous injections of testosterone cypionate (1 mg/kg/week; Deposteron/EMS)25 for 3, 7 and 21 days. All gerbils were weighed and euthanized by cervical dislocation. The prostatic complexes (corresponding urethral segment and ventral, dorsolateral and dorsal prostate lobes in males; vaginal segment, corresponding urethral segment and prostatic tissue in females) were dissected and weighed.

Figure 1.

Figure 1

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Schematic representation of the experimental protocol employed in this study. Asterisks (*) represent the days of euthanasia and dissection of the glands

2.5. Hormonal serum dosage

Blood samples were obtained from gerbils by cardiac puncture immediately after euthanasia (n = 5 animals/group). Serum was obtained by centrifugation (3000 rpm) and stored at −20°C for subsequent hormone analysis. Circulating serum testosterone and estradiol levels were determined by competitive enzyme immunoassay (Monobind Inc., AccuBind). All the samples were analysed in triplicate in the same assay. Sensitivity was 0.0576 ng/ml for testosterone and 8.82 pg/ml for estradiol.

2.6. Light microscopy

The prostatic complexes were fixed by immersion in methacarn (60% methanol, 30% chloroform and 10% acetic acid; n = 5 animals/group) for 4 hours at 4°C or in 4% paraformaldehyde (buffered in 0.1 M phosphate, pH 7.2; n = 3 animals/group) for 24 hours. Then, the tissues were dehydrated through a crescent ethanol series, clarified in xylol, embedded in paraplast (Histosec, Merck) and sectioned to 5 μm on a Leica microtome (Leica RM2155; Nussloch). Sections were stained by haematoxylin‐eosin (HE). Specimens were analysed and digitized using a Zeiss Axioscope A1 light microscope (Zeiss).

2.7. Stereology

Stereological analyses were carried out using Weibel's multipurpose graticulate with 130 points and 10 test lines.28 The relative frequency of each component of the prostatic tissue (epithelium, lumen, non‐muscle stroma and muscle stroma) was determined. We randomly obtained 30 microscopic fields from each experimental group (six fields per animal; n = 5). The relative frequency was determined by counting the coincident points in the test grid and dividing them by the total number of points. Stereological analysis was performed using Image‐Pro Plus software v6.1 for Windows (Media Cybernetics Inc).

2.8. Histopathological analysis

Histopathological analysis was performed using an Olympus light microscope (BX43; Olympus). Ventral male prostate and female prostate sections stained by HE (30 histological sections/group; n = 8 animals/group) were analysed to determine the frequency (%) of prostatic disorders. For this, the total number of prostatic alveoli per section was determined, and then, the alveoli that presented foci of hyperplasia, neoplasia, and luminal or perialveolar inflammation were discriminated. The percentage of altered alveoli was obtained in relation to the total alveoli number in each histological section.

2.9. Immunohistochemistry

Ventral male prostate and female prostate sections were subjected to immunohistochemistry analysis (n = 3 animals/group). Antibodies against androgen receptor (AR; rabbit polyclonal IgG, N‐20, sc‐816; Santa Cruz Biotechnology), oestrogen receptor alpha (ERα; rabbit polyclonal IgG, MC‐20, sc‐542; Santa Cruz Biotechnology) and proliferating cell nuclear antigen (PCNA; mouse monoclonal IgG2a, SC 56, Santa Cruz Biotechnology) were employed for immunostaining at a dilution of 1:100 overnight at 4°C. On the next day, Novocastra Post Primary and Polymer were used as secondary antibodies. The sections were stained with DAB Chromogen and DAB Substrate Buffer (in a proportion of 1:20) and, finally, counterstained with haematoxylin. The histological sections were analysed using a Zeiss Axioscope A1 light microscope (Zeiss).

2.10. AR, ERα and PCNA quantification

For AR, ERα and PCNA quantification, 30 microscopic fields (n = 3 animals/group; magnification of 400×) were examined for each experimental group. In each field, the total number of positive epithelial and stromal cells was obtained as a relative frequency (%) in relation to the total number of cells. All these analyses were performed using the image analysis system previously described.

2.11. Statistical analyses

The hypothesis tests employed to determine statistical significance were the Kruskal‐Wallis test followed by Dunn's test (post hoc) for non‐parametric distributions. Parametric data were compared by applying one‐way ANOVA followed by Tukey's test (post hoc). The data were analysed using Statistica 7.0 (StatSoft, Inc). Values are presented as mean ± standard error of the mean (SEM).

3. RESULTS

3.1. Plasma testosterone levels

Chr‐treated males showed a significant reduction of serum testosterone levels in the 3D group, but these levels gradually recovered in the 7D and 21D groups (Figure 2A). Chr‐treated females showed no significant changes in serum testosterone levels (Figure 2C). T‐treated males and T‐treated females showed a significant increase in plasma testosterone levels only in the 7D and 21D groups (Figure 2B,D).

Figure 2.

Figure 2

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A–D, Plasma testosterone levels (T, ng/mL) in all experimental groups. C: control; Chr: chrysin; T: testosterone cypionate for 3 (3D), 7 (7D) and 21 (21D) days of treatment (n = 5 animals/group). Values are mean ± SEM. Asterisks represent statistically significant differences between experimental groups (P ≤ 0.05)

3.2. Plasma 17β‐estradiol levels

Chr‐treated males showed no significant changes in 17β‐estradiol levels (Figure 3A). T‐treated males had a significant increase in estradiol levels only in the 7D group (Figure 3B). Chr‐treated and T‐treated females showed higher 17β‐estradiol levels only in the 21D group (Figure 3C‐D).

Figure 3.

Figure 3

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A–D, 17β‐Estradiol plasma levels (E2, pg/mL) in all experimental groups. C: control; Chr: chrysin; T: testosterone cypionate for 3 (3D), 7 (7D) and 21 (21D) days of treatment (n = 5 animals/group). Values are mean ± SEM. Asterisks represent statistically significant differences between experimental groups (P ≤ 0.05)

3.3. Body and prostate weight

Chr‐treated males exhibited body weight reduction in the 3D and 21D groups. In T‐treated males, a reduction in body weight was observed in the 3D group (Table 1). Chr and T treatments did not affect the prostatic complex weight of male gerbils (Table 1). Chr‐treated and T‐treated females did not show changes in body weight (Table 2). Only 7D Chr‐treated females showed a significant increase in prostatic complex weight (Table 2).

Table 1.

(1) Body and prostate complex weight in all male groups (n = 8/group). (2) Stereological data obtained for the ventral prostate in all experimental groups (n = 30 fields in three animals/group)

  Treatment Groups
C 3D 7D 21D
(1) Biometry (g)
 Body weight Chr 73.2 ± 2.2a 65.2 ± 1.2b 71.6 ± 1.7a 66.4 ± 1.5b
T 62.8 ± 2.2b 64.8 ± 1.7a 69.6 ± 3.7a
 Prostate complex weight Chr 0.7 ± 0.05 0.7 ± 0.05 0.6 ± 0.09 0.6 ± 0.02
T 0.7 ± 0.06 0.6 ± 0.07 0.9 ± 0.06
(2) Stereology (%)
 Epithelium Chr 17.4 ± 1.2a 33.6 ± 1.1b 35.5 ± 1.3b 23.5 ± 1.2c
T 31.1 ± 1.4b 28.3 ± 1.1b. c 25.8 ± 1.7c
 Lumen Chr 52.9 ± 1.9a 28.3 ± 1.9b 26.7 ± 1.5b 43.6 ± 2.7c
T 35.6 ± 2.8b 37.1 ± 2.2b 46.1 ± 3.3a
 Muscle stroma Chr 9.8 ± 0.6a 12.4 ± 0.5b 15.3 ± 0.7c 10.1 ± 0.6a
T 11.4 ± 0.7 12.3 ± 0.8 10.3 ± 0.8
 Non‐muscle stroma Chr 19.9 ± 1.7 25.7 ± 2.1 22.6 ± 1.9 22.8 ± 2.3
T 21.9 ± 2.4 22.2 ± 1.9 17.9 ± 2.2
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Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between experimental groups (P ≤ 0.05).

Table 2.

(1) Body and prostate complex weight in all female groups (n = 5/group). (2) Stereological data obtained for the female prostate in all experimental groups (n = 30 fields in 3 animals/group)

  Treatment Groups
C 3D 7D 21D
(1) Biometry (g)
 Body weight Chr 54.8 ± 3.0 65.2 ± 2.2 61.2 ± 1.9 58.4 ± 4.5
T 58.0 ± 1.1 56.4 ± 2.6 59.2 ± 1.7
 Prostate complex weight Chr 0.13 ± 0.01a 0.16 ± 0.02a 0.23 ± 0.03b 0.16 ± 0.03a
T 0.17 ± 0.01 0.16 ± 0.01 0.16 ± 0.01
(2) Stereology (%)
 Epithelium Chr 16.1 ± 1.0a 31.3 ± 1.1b 32.3 ± 1.3b 24.7 ± 1.03c
T 34.6 ± 1.4b 34.3 ± 1.7b 28.6 ± 1.2c
 Lumen Chr 34.6 ± 2.2a 19.0 ± 1.9b 14.4 ± 1.2b 27.8 ± 2.3a
T 18.5 ± 1.6b 21.0 ± 1.7b 24.8 ± 2.1b
 Muscle stroma Chr 13.9 ± 0.9a 18.8 ± 1.3b 17.9 ± 1.0b 13.0 ± 0.9a
T 16.0 ± 1.0a 19.2 ± 1.1b 17.4 ± 1.2a
 Non‐muscle stroma Chr 35.7 ± 1.9a 31.0 ± 2.3 35.4 ± 1.3 34.5 ± 2.0
T 30.9 ± 1.7a 25.5 ± 1.9b 29.3 ± 1.7a
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Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between experimental groups (P ≤ 0.05).

3.4. Prostate morphology, stereology and histopathology

Ventral prostates of the 3D and 7D Chr‐treated males showed an increase in epithelium and muscle stroma and a reduction in the luminal compartment (Figure 4A‐C, Table 1). In 21D Chr‐ treated males, the ventral prostate presented both hypertrophic alveoli with large lumens (Figure 4D) and hyperplastic alveoli (Figure 4E). When compared to the C group, the ventral prostates of 21D Chr‐males showed increased epithelium and reduced luminal compartment (Table 1). In male gerbils, Chr treatment caused a gradual increase in the hyperplastic alveoli frequency (C: 1.4 ± 0.4%, 3D: 14.7 ± 1.1%, 7D: 17.5 ± 1.0%, 21D: 26.8 ± 1.1%) (Table 3). T‐treated male prostates (3D, 7D, and 21D groups) showed glandular changes similar to those found in Chr‐males, such as increased epithelium frequency, reduced lumen, and increased frequency of alveolar hyperplasia (Figure 4F‐H, Tables 1 and 3). However, when compared to Chr‐treated males, T‐treated male prostates showed higher hyperplasia rates in all treatment phases (C: 1.4 ± 0.4%, 3D: 24.9 ± 1.3%, 7D: 24.1 ± 1.0%, 21D: 25.6 ± 1.4%, P ≤ 0.05), in addition to isolated foci of neoplasia (Table 3).

Figure 4.

Figure 4

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A–H, Histological sections of ventral male prostate stained by the HE method. C: control; Chr: chrysin; T: testosterone cypionate for 3 (3D), 7 (7D) and 21 (21D) days of treatment. Epithelium (Ep), lumen (L), stroma (S), hyperplasia (Hyp), prostatic intraepithelial neoplasia (PIN), inflammatory infiltrate (*). Scale bar: 50 μm; inserts (E): 20 μm [Colour figure can be viewed at wileyonlinelibrary.com]

Table 3.

Alveolar disorders (%) in the ventral male prostate of adult gerbils (n = 8 animals/group)

Alveoli histology (%) Treatment Groups
C 3D 7D 21D
Normal Chr 98.4 ± 0.5a 84.0 ± 1.0b 80.1 ± 1.6b 72.1 ± 1.0c
T 74.7 ± 1.2b, * 74.9 ± 1.1b, * 69.6 ± 2.3b
Hyperplasia Chr 1.4 ± 0.4a 14.7 ± 1.1b 17.5 ± 1.0b 26.8 ± 1.1c
T 24.9 ± 1.3b, * 24.1 ± 1.0b, * 25.6 ± 1.4b
Inflammation Chr 0.2 ± 0.1 1.0 ± 0.3 2.5 ± 1.4 1.1 ± 0.5
T 0.4 ± 0.2 0.4 ± 0.2 4.0 ± 1.4
Neoplasia Chr 0 0 0 0
T 0 0.6 ± 0.3 0.8 ± 0.3
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Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between the control and time‐related groups (3D, 7D, 21D; P ≤ 0.05).

*Represents statistically differences between Chr and T treatments in each treatment period.

Prostates of the 3D and 7D Chr‐treated females showed increased frequency of epithelial cells and muscular stroma, and reduction of the luminal compartment (Figure 5A‐C, Table 2). Chr‐treated females of the 21D group showed both hypertrophic and hyperplastic alveoli (Figure 5D‐E). When compared to the control group, 21D Chr‐treated female prostates showed only increased epithelial frequency (Table 2). Chr‐treated females of all experimental groups showed an increased frequency of hyperplastic alveoli and a reduction in the occurrence of inflammatory foci (Table 4). T‐treated females showed increased epithelial frequency (especially in 3D and 7D groups) and reduced luminal compartment (only in 3D and 7D groups) (Figure 5F‐I, Table 2). In addition, in 7D T‐treated females there was an increase in the muscular stroma frequency and a decrease in the non‐muscular stroma frequency (Table 2). When compared to the control group or to the Chr group, T‐treated females had a higher incidence of alveolar hyperplasia, inflammation, and neoplasia (Table 4).

Figure 5.

Figure 5

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A–I, Histological sections of female prostate stained by the HE method. C: control; Chr: chrysin; T: testosterone cypionate for 3 (3D), 7 (7D) and 21 (21D) days of treatment. Epithelium (Ep), lumen (L), stroma (S), hyperplasia (Hyp), prostatic intraepithelial neoplasia (PIN), inflammatory infiltrate (*). Scale bar: 50 μm; inserts (E, G): 20 μm [Colour figure can be viewed at wileyonlinelibrary.com]

Table 4.

Alveolar disorders (%) in the female prostate of adult gerbils (n = 8 animals/group)

Alveoli histology (%) Treatment Groups
C 3D 7D 21D
Normal Chr 90.4 ± 1.5a 77.8 ± 2.1b 71.3 ± 2.7b 70.2 ± 2.8b
T 43.5 ± 2.7b, * 47.5 ± 3.0b, * 52.3 ± 2.7b, *
Hyperplasia Chr 8.2 ± 1.2a 22.2 ± 2.1b 27.8 ± 2.4b 29.2 ± 2.8b
T 48.4 ± 2.6b, * 47.7 ± 3.2b, * 42.4 ± 2.9b, *
Inflammation Chr 2.0 ± 0.8a 0b 1.4 ± 0.7a 0.2 ± 0.2a
T 4.6 ± 0.9* 3.0 ± 0.7* 2.1 ± 0.5*
Neoplasia Chr 0.4 ± 0.2a 0 0 0
T 3.5 ± 0.9b, * 1.8 ± 0.6a, * 3.3 ± 1.0b, *
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Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between the control and time‐related groups (3D, 7D, 21D; P ≤ 0.05).

*Represents statistically significant differences between Chr and T treatments in each treatment period.

3.5. AR immunostaining and frequency

Males and females of the Chr and T groups showed a significant increase in AR‐positive cells in the epithelial and stromal compartments (Figure 6). However, in both treatments, no significant differences were observed between drug administration times (3D, 7D, and 21D) (Figure 6).

Figure 6.

Figure 6

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AR immunostaining in ventral male prostate and female prostate. C: control; Chr: chrysin; T: testosterone cypionate. A‐C, Arrows indicate AR‐positive cells in the epithelium (Ep), and arrowheads indicate AR‐positive cells in the stroma (S) of the 7D male groups. Lumen (L). D‐E, Frequency (%) of AR‐positive cells in the ventral male prostate of all experimental groups. F‐H, Arrows indicate AR‐positive cells in the epithelium, and arrowheads show positive stromal cells in the 7D female groups. I‐J, Frequency (%) of AR‐positive cells in the female prostate of all experimental groups. Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between experimental groups (P ≤ 0.05) [Colour figure can be viewed at wileyonlinelibrary.com]

3.6. ERα immunostaining and frequency

In this study, we did not demonstrate immunohistochemical reactions to ERα in males. However, Chr‐treated and T‐treated females showed a significant increase in stromal immunostaining for ERα at all treatment times (Figure 7). The effects of chrysin were more pronounced at the beginning of treatment, since a peak of ER‐α was observed in 3D, followed by a significant reduction in 7D and 21D.

Figure 7.

Figure 7

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ERα immunostaining in the female prostate. C: control; Chr: chrysin; T: testosterone cypionate. A‐C, Arrows indicate ERα‐positive cells in the stroma (S) of the 3D female groups. Epithelium (Ep), Lumen (L). D‐E, Frequency (%) of ERα‐positive cells in the female prostate of all experimental groups. Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between experimental groups (P ≤ 0.05) [Colour figure can be viewed at wileyonlinelibrary.com]

3.7. PCNA immunostaining and frequency

Males (Figure 8) and females (Figure 9) of the Chr and T groups showed a significant increase in PCNA‐positive cells in the epithelium and stroma. In Chr‐treated and T‐treated males, the peak of epithelial and stromal proliferation was in the 3D group (Figure 8B,E,F,I). In Chr‐treated females, the peak of epithelial proliferation was in the 7D group, and greater stromal proliferation was observed in the 3D and 7D groups (Figure 9A‐E). On the other hand, in T‐treated females, peak epithelial proliferation occurred in the 3D group, and there was a higher index of stromal proliferation in the 3D and 7D groups (Figure 9F‐I).

Figure 8.

Figure 8

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A‐D, F‐H, PCNA immunostaining in the ventral male prostate. C: control; Chr: chrysin; T: testosterone cypionate in 3 (3D), 7 (7D) and 21 (21D) days of treatment. Arrows indicate PCNA‐positive cells in the epithelium (Ep), and arrowheads represent positive cells in the stroma (S). Lumen (L). E, I, Frequency (%) of PCNA‐positive cells in the ventral male prostate of Chr and T groups. Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between experimental groups (P ≤ 0.05) [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 9.

Figure 9

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A‐D, F‐H, PCNA immunostaining in the female prostate. C: control; Chr: chrysin; T: testosterone cypionate in 3 (3D), 7 (7D) and 21 (21D) days of treatment. Arrows indicate PCNA‐positive cells in the epithelium (Ep), and arrowheads represent positive cells in the stroma (S). Lumen (L). E, I, Frequency (%) of PCNA‐positive cells in the female prostate of Chr and T groups. Values are mean ± SEM. Superscript letters (a,b,c) represent statistically significant differences between experimental groups (P ≤ 0.05) [Colour figure can be viewed at wileyonlinelibrary.com]

4. DISCUSSION

In this study, we evaluated the ventral male prostate and the female prostate after 3, 7, and 21 days of exposure to either chrysin or testosterone (positive control). In contrast to testosterone treatment, chrysin did not increase plasma testosterone levels in male or female gerbils. However, chrysin showed similar potential to increase AR immunoreactivity and cellular proliferation in male and female prostates to that of testosterone. In addition, Chr‐treated females showed an increase in serum estradiol levels at 21 days of treatment and an increase in ERα‐positive stromal cells at all treatment times, especially at the beginning of treatment (3D group).

Previous research has shown the testosterone‐boosting properties of chrysin. Male rats that received 50 mg/kg/day of chrysin for 60 consecutive days showed a significant increase in serum testosterone levels.15 Arthritic rats had serum testosterone levels restored after 21 days of chrysin supplementation (50 mg/kg/day).29 In contrast, in our study, male and female gerbils supplemented with 50 mg/kg/day of chrysin did not show a significant increase in testosterone levels at any of the analysed time points (3, 7, and 21 days). A recent study by Altawash and coworkers demonstrated that roosters treated for 12 consecutive weeks with chrysin at 25, 50 and 75 mg/kg/day showed an increase in serum testosterone levels only at 75 mg/kg/day.30 Thus, we believe that the chrysin dose employed in our study, as well as the short exposure times, was not sufficient to promote testosterone‐boosting responses in male and female gerbils.

Although plasma testosterone levels were not significantly increased, chrysin promoted considerable morphological changes in the male and female prostates. These morphological alterations were detected after three days of treatment and were maintained until the end of the experiment (21 days). Chr‐treated male and female prostates showed intense epithelial changes, glandular hyperplasia, increased AR immunoreactivity and higher cell proliferation. These morphological changes were also observed, at least in part, in T‐treated males and females, which showed an obvious increase in serum testosterone levels. These results suggest that chrysin may operate locally, causing increased intraprostatic testosterone bioavailability or activating AR itself. Chrysin has been described as inhibiting aromatase, an enzyme that catalyses the conversion of androgens to oestrogens.7, 31, 32 Since prostate cells express aromatase,33, 34 it is presumed that chrysin blocked androgen conversion into oestrogens, increasing the intraprostatic bioavailability of androgens in male and female gerbils.

In this study, chrysin and testosterone presented a similar potential to increase AR immunoreactivity in the male and female prostates. The effects of chrysin on AR regulation are poorly understood. In vitro assays have shown that other flavonoid types, such as apigenin, fisetin, and quercetin, have the potential to inhibit the AR signalling axis.35, 36 On the other hand, a recent study published by our research group showed that prepubertal chrysin exposure increased AR immunoreactivity in the prostates of male and female adult gerbils.25 These contradictory results indicate possibly different mechanisms of action for chrysin using in vitro and in vivo models. The AR is a transcription factor that controls the expression of specific genes. When androgens bind to the AR, it is translocated to the nucleus. Then, the AR binds to androgen response elements in the promoter regions of target genes, eliciting responses such as growth and survival.37, 38 AR activation and upregulation have also been reported in prostate proliferative disorders, such as benign prostatic hyperplasia and cancer.39, 40 In this study, AR upregulation in Chr‐treated males and females may be directly related to the increase in cell proliferation and to the emergence of benign prostatic hyperplasia.

Chr‐treated females, but not Chr‐treated males, had increased 17β‐estradiol serum levels at 21 days of treatment. This result was unexpected, since aromatase inhibitors tend to suppress serum estradiol levels.41, 42 Thus, the lower tissue estradiol bioavailability may have acted as positive feedback on the hypothalamic‐hypophyseal‐gonadal axis, culminating in ovarian stimulation to restore oestrogen levels. In addition, Chr‐treated females showed a peak of ERα reactivity at 3D, followed by a significant reduction at 7D and 21D. These dissimilarities between males and females indicate that chrysin may present different mechanisms of action in the two sexes and demonstrate the oestrogenic activity of this flavone in female gerbils. In vitro assays demonstrated that chrysin and other phytoestrogens were able to bind to ERα, triggering oestrogenic agonist responses.43, 44, 45 Since health and normal prostate morphology are maintained by the precise balance between androgens and oestrogens,24 dietary chrysin supplementation should be considered with caution, especially in females.

Although the results of this study demonstrated that chrysin and testosterone promoted similar effects on the prostates of both male and female gerbils, our histopathological examination indicated that testosterone was more deleterious than chrysin. T‐treated males and T‐treated females showed a prevalence of prostatic hyperplasia, whereas only T‐treated females had a higher incidence of inflammatory and neoplastic foci. This evidence suggests that chrysin may be better than synthetic androgens in the treatment of reproductive dysfunctions, such as infertility and low libido.

Even in treatment as short as three days, chrysin caused AR upregulation and increased cell proliferation rates in the ventral male and female prostates of adult gerbils. These precocious changes resulted in epithelial development and hyperplastic growth of the gland. In addition, chrysin treatment increased ERα immunoreactivity in the female prostate and raised serum estradiol levels in 21D females, indicating that chrysin exerted differential oestrogenic effects in females. Although chrysin and testosterone evoked, at least in part, similar morphological changes in the male and female gerbil prostates, chrysin supplementation caused less damage to prostate health, since it resulted in a lower incidence of hyperplasia and absence of neoplastic foci.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

ACKNOWLEDGEMENTS

This paper was supported by a grant from the Brazilian agency CNPq (Brazilian National Research and Development Council, Procs. Nrs. 475148/2012‐6, 471129/2013‐5 and 306251/2016‐7, FAPEG (Goiás Research Foundation, Nr. 08/2018) and FAPESP (São Paulo Research Foundation, Procs. Nr. 2014/18330‐0). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)— Finance code 001.

Campos MS, Silva JPA, Lima DS, et al. Short‐term exposure to chrysin promotes proliferative responses in the ventral male prostate and female prostate of adult gerbils. Int J Exp Path. 2019;100:192‐201. 10.1111/iep.12317

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