The Biomolecular Effects of Finasteride on Male Rat Brain During Administration and After Discontinuation

Abstract

Purpose

This study aimed to investigate the biomolecular effects of finasteride on rat brain tissue during and after discontinuation.

Materials and Methods

Twenty-four 14-week-old male Wistar rats were randomly assigned to three groups: Group 1 (control), Group 2 (finasteride-treated), and Group 3 (finasteride-withdrawn). Serum testosterone and dihydrotestosterone levels were measured at the end of the study period. Brain tissues were analyzed using real-time polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry, with c-Fos employed as a non-specific biomarker of neural cellular activity.

Results

In Group 2, serum dihydrotestosterone levels, 5-alpha reductase type 1 protein expression, and c-Fos phosphorylation showed a decreasing trend (p=0.067, p=0.015, p=0.123, respectively, versus Group 1), but rebounded in Group 3 (p=0.044, p=0.033, p=0.049, respectively, versus Group 2). In contrast, 5-alpha reductase type 2 protein expression was significantly reduced in Group 2 (p=0.017 vs. group 1) and remained suppressed in Group 3 (p=0.029 versus Group 1, p>0.999 versus Group 2). The mean intensity of immunohistochemistry revealed decreased 5-alpha reductase type 2 and c-Fos protein levels in Group 2 (p=0.008 and p=0.017, respectively). After withdrawal, c-Fos protein levels recovered in Group 3 (p=0.031 versus Group 2), whereas 5-alpha reductase type 2 levels remained unchanged (p=0.682 versus Group 2).

Conclusions

Suppression of c-Fos-related neural activity in rat brain tissue could be temporarily maximized by both type 1 and type 2 5-alpha reductase inhibition by finasteride administration, and this suppression could be relieved by the restoration of 5-alpha reductase type 1 expression after finasteride discontinuation.

INTRODUCTION

5-alpha reductase (5AR) inhibitors are commonly prescribed for the treatment of benign prostatic hyperplasia and androgenetic alopecia. Although numerous studies have reported the potential serious adverse effects, such as decreased libido, cognitive impairment, anxiety, depression, and suicidal ideation [1]. These effects may persist even after discontinuation of 5AR inhibitors, for example, Irwig and Kolukula [2] reported that 94% of patients with androgenic alopecia developed low libido, maintained for up to 40 months after finasteride discontinuation. Although evidences demonstrating persistent neurological adverse events remain inconclusive, the recognition of the possibility of drug-related neurological adverse events helps the use of 5AR inhibitors more be individualized [3].

To observe the direct effect of the 5AR inhibitor on tissue, 5AR expression should be measured after administration and discontinuation of the drug. Although the change in serum dihydrotestosterone reflects the non-specific 5AR activity of an individual, it is not representative of a specific organ (e.g., brain or prostate) because the hormone acts in a paracrine manner [4].

The c-Fos protein is well-known as a non-specific but strong biomarker that represents neural cellular activity and responds immediately to recent stimuli [5,6]. Furthermore, it has been suggested that c-Fos expression in several areas of brain tissue is closely associated with sex-steroid hormones [7]. Therefore, in the brain tissue, it is possible that the expression of c-Fos could be influenced by finasteride administration and discontinuation.

In this study, we aimed to measure serum dihydrotestosterone, investigate the expression of 5AR type 2 (5AR-2), the target enzyme of finasteride, and examine c-Fos activation in the brain tissue during finasteride treatment and after cessation of the drug in a rat model. The objective of this study is to provide valuable insights into the role of finasteride in brain tissue.

MATERIALS AND METHODS

1. Ethics approval and consent to participate

All the experimental protocols were approved by the Institutional Animal Care and Use Committee of St. Vincent’s Hospital, The Catholic University of Korea (approval no. IACUC-20-08; date: Feb 20, 2020). All the experiments were performed in a laboratory at St. Vincent’s Hospital under standard conditions. All experiments adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [8].

2. Experimental setting

Twenty-four 14-week-old male Wistar rats (approximately 300–350 g; KOATECK) were used. The age of each rat used in the present study was determined according to the results conducted by Banerjee et al [9]. Each rat was acclimated singly in each standard polypropylene cage in a temperature-controlled room (25±1 ℃) with a 12:12-h light/dark cycle (lights on at 7 am) and were allowed free access to food and water for 1 week. Mean water intake during the acclimation period was approximately 30 mL/rat/day. Eight rats were randomly assigned to each group (Group 1, control group; Group 2, finasteride group; Group 3, finasteride withdrawal group). The rats were housed individually in cages throughout the experimental period. Long-term 5AR-2 inhibition by finasteride is not determined by its dosage; it inhibits over 90% of 5AR-2 for at least 3 days after a single dose of finasteride 1 mg, whereas it does not affect 5AR type 1 (5AR-1) after 24 hours [10]. We determined to administer 1 mg of finasteride orally per day. Next, 30 mL of water was required for each rat per day, and to prevent water depletion during the experiment, 10 mg of finasteride was dissolved in 300 ml of water using 0.6 ml DMSO (dimethyl sulfoxide). The solution was freely fed for 5 days, 1 mg of finasteride per day equates to finasteride 3 mg/kg/day, considering that the weight of each rat was 300–350 g. Then, we checked the residual solution volume while changing the solution every 5 days. The same amount of DMSO without finasteride was applied to the water in Group 1. Rats in Groups 1 and 2 were sacrificed for tissue retrieval at 4 weeks, and an additional four weeks without finasteride (only with DMSO solution) was required for Group 3 with discontinuation of finasteride (Fig. 1).

Fig. 1. Study design. Finasteride was administered for 4 weeks, and rats in Group 2 were sacrificed, whereas rats in Group 3 were sacrificed after the drug withdrawal period.

3. Sacrifice, blood sampling and tissue retrieval

Rats were anesthetized with isoflurane in N2O-O2 (70%:30%) in a small plastic chamber. The gas flow rate was 500 mL/min, and the isoflurane flow rate was set to 4% during induction. Once rats lost consciousness, the gas flow was switched from the chamber to an anesthetic nose cone, and the cone was placed over the rats’ noses on the experimental table where the isoflurane flow rate used for maintenance was set to 1.5% to 2%, with a gas flow rate of 200 mL/min. Blood samples were obtained by direct puncture of the right atrium after opening the chest wall immediately before decapitation and retrieval of brain tissues. The brains were retrieved using the protocol described and suggested by Jaszczyk et al [11]. Once the brain was retrieved, one hemisphere was immediately fixed with 10% formalin for histological evaluation, whereas the other hemisphere was used to evaluate RNA and protein expression. After the specimens were retrieved, only one coordinator was aware of the sources from which the groups were retrieved until data analysis was completed.

4. Hormonal assay

Plasma was obtained by centrifuging blood samples for 10 minutes at 8,000 rpm, and the samples were stored at –20 ℃ using a standard method [12]. Enzyme-linked immunosorbent assay (ELISAs) was performed to determine the serum levels of testosterone and dihydrotestosterone (E05100r; Cusabio Biotech) using commercial kits.

5. Immunohistochemistry

Fixed tissues were embedded in paraffin, cut into 4-µm sections, and placed on glass slides. As finasteride is a 5AR-2 inhibitor, a primary antibody targeting 5AR-2 (Cat #orb100170; Biorbyt) was used to evaluate the treatment effect. c-Fos is induced by a broad range of stimuli, and is a reliable marker of neural activity [13,14]. Therefore, we used another primary antibody targeting c-Fos (cat #ab190289; Abcam). Heat-induced antigen retrieval was performed, in which tissues were boiled in a citrate buffer solution by heating in a microwave for 2 minutes. The ImmPress kit (Vector Laboratories) was used for secondary antibodies, and Tris-buffered saline was used to wash the specimen. For localization, we used a preoptic based on a previous study [7]. A 3,3-diaminobenzidine tetrahydrochloride (DAB) kit was used for visualization. Three sections from each rat were analyzed bilaterally by a pathologist who was blinded to the groups. The H-score was used to compare groups semi-quantitatively, as suggested by McCarty et al [15] (1×percentage of weak staining)+(2×percentage of moderate staining)+(3×percentage of strong staining) within the target region, ranging from 0 to 300.

6. RNA extraction (real-time PCR)

Total RNA was extracted using an RNeasy Mini Kit (Cat#74004; Qiagen). RNA was reverse transcribed using a cDNA synthesis kit (PrimeScript RT Reagent Kit; Takara Bio) according to the manufacturer’s instructions. Quantitative polymerase chain reaction was performed using LC SYBR Green (Cat# 03 003 230 001; Roche Diagnostics) on a LightCycler 2.0 Real-Time PCR System (Roche Diagnostics). Reactions for each sample were run in triplicate and cycle thresholds were normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Comparative quantitation was performed using the LightCycler 167 software (version 4.1; Roche). Real-time polymerase chain reaction (RT-PCR) was performed under the conditions described in Supplement Table 1. We used GAPDH as the housekeeping gene (National Center for Biotechnology Information accession number NG028301).

7. Western blotting

The brain tissue was frozen and homogenized in protein extraction buffer (Intron) containing protease inhibitors (Sigma). The homogenates were centrifuged at 12,000 rpm for 20 minutes. The total protein concentration in the supernatant was measured using the Bradford assay (Bio-Rad). Equal amounts of protein (10 µg) were electrophoretically separated on 10% SDS-PAGE gels and transferred onto PVDF membranes (Bio-Rad). The membrane was blocked with 5% skim milk (Bio-Rad) containing 0.1% Tween 20 for 1 hour and incubated overnight with antibodies against 5AR-2 (Cat#LS-C780518; LifeSpan BioSciences), 5AR-1 (Cat#APR21337N; Leading Biology), c-Fos (Cat#ab190289; Abcam), phosphor-c-Fos (Ser374) (Cat#PA5-104728; Invtrogen), and β-actin (Cat#4967S; Cell signaling). The membrane was then incubated with secondary antibody at room temperature for 1 hour. Protein expression was visualized by enhanced chemiluminescence (ECL) (Bio-Rad). ECL was measured using LAS 4000 mini (GE Healthcare), and the results were quantified using densitometry.

8. Statistical analysis

Statistical analysis was conducted using SPSS (IBM SPSS Statistics for Windows, Version 26.0, Released 2019; IBM Corp.). The Kruskal–Wallis test was used to compare the three groups. For all between-group comparisons, Bonferroni correction was used as a post hoc test, and the relevant p-value was ‘adjusted’ automatically by SPSS software. The number of experimental animals required (a total of 24, with eight for 8 each group) was determined based on the ‘resource equation’ approach owing to the exploratory nature of the current study [16]. Data from statistical analyses are presented as the means±standard errors. Values of p<0.05 were considered statistically significant.

RESULTS

1. Hormonal assay

For 4 weeks, the mean±standard variation of estimated consumption of DMSO (mL) was 1.58±0.17, 1.69±0.11 and 1.63±0.05 in Groups 1, 2, and 3, respectively (Kruskal–Wallis test, p=0.111). No deaths occurred in any group during the experiment. Testosterone (ng/dL) levels did not differ among the groups (p=0.162, Kruskal–Wallis test). The mean dihydrotestosterone level (ng/dL) tended to be lower in Group 2 than in Group 1, and this level was ameliorated after discontinuation of the drug (Table 1).

Table 1. Testosterone and dihydrotestosterone levels.

	Group 1 a	Group 2b	Group 3c	p-value
Dihydrotestosterone (ng/dL)	8.82 ± 2.98	4.71 ± 1.74	9.38 ± 2.97	0.024
Testosterone (ng/dL)	321.96 ± 41.27	288.11 ± 13.50	293.06 ± 18.96	0.162

Values are presented as mean±standard deviation. Statistics was calculated by Kruskal-Wallis test. Post-hoc analysis was performed using Mann-Whitney U test with Bonferroni correction. For dihydrotestosterone, adjusted p-values between group 1 and 2, group 2 and 3, and group 3 and 1 were 0.067, 0.044, and >0.999, respectively.

^aGroup 1 (control), ^bGroup 2 (finasteride-treated), and ^cGroup 3 (finasteride withdrawal).

2. Western blotting

5AR-2 expression differed among the 3 groups (p=0.009). 5AR-2 expression sharply decreased in Group 2 (p=0.017 versus Group 1) and was persistently low in Group 3 (p=0.029 versus Group 1), which means a 1 month discontinuation of the drug may not be sufficient to recover 5AR-2 function (Fig. 2A). The expression of 5AR-1 was diminished in Group 2 (p=0.015 versus Group 1), but rebounded in Group 3 (Fig. 2B, p=0.033 versus Group 2). c-Fos protein activation also differed among the three groups (p<0.001). c-Fos protein phosphorylation tended to decrease, but not significantly, in Group 2 compared with Group 1 (p=0.123). Nevertheless, the activation of c-Fos increased significantly in Group 3 compared with Group 2 (p=0.049). The mean value of c-Fos protein activation with western blot in Group 3 was no longer different from that in Group 1 (Fig. 2C, p>0.999).

Fig. 2. Western blotting for 5-alpha reductase (5AR) type 1, 5AR type 2, and c-Fos. Statistical analyses were performed using the Kruskal–Wallis test. Post-hoc analysis was performed using Mann–Whitney U test with Bonferroni correction. (A) Comparison of 5AR type 1 expression among groups 1, 2, and 3 (p=0.008). Adjusted p-values using the Bonferroni correction between Groups 1 and 2, Groups 2 and 3, and Groups 3 and 1 were 0.015, 0.033, and >0.999, respectively. (B) Comparison of 5AR type 2 expression among groups 1, 2, and 3 (p=0.009). Adjusted p-values using the Bonferroni correction between Groups 1 and 2, Groups 2 and 3, and Groups 3 and 1 were 0.017, >0.999, and 0.029, respectively. (C) Comparison of c-Fos phosphorylation among groups 1, 2, and 3 (p=0.037). Adjusted p-values using the Bonferroni correction between Groups 1 and 2, Groups 2 and 3, and Groups 3 and 1 were 0.123, 0.049, and >0.999, respectively. ^*p<0.05.

3. Real-time PCR

The mean RT-PCR values for each group did not differ significantly (p=0.777 for 5AR-2 and p=0.157 for c-Fos). In contrast to 5AR-2 mRNA expression, c-Fos mRNA expression tended to increase in Group 3, although the difference was not statistically significant (Fig. 3).

Fig. 3. Real-time PCR for 5-alpha reductase (5AR) type 2 and c-Fos mRNA expression. Comparisons among groups 1, 2, and 3 were performed using the Kruskal–Wallis test. For (A) and (B), the p-values were 0.777 and 0.157, respectively.

4. Immunohistochemistry

Fig. 4 shows the immunohistochemistry results for 5AR-2, where the mean intensity was significantly lower in Group 2 than in Group 1 (p=0.008). However, the mean H-score of Group 3 was neither superior to that of Group 2 (p=0.682) nor inferior to that of Group 1 (p=0.219). The mean intensity of c-Fos immunohistochemistry was significantly lower in Group 2 than in Group 1 (p=0.017) and Group 3 (p=0.031). The intensity in Group 3 was similar to that in Group 1 (p>0.999) (Fig. 4, Supplement Table 2).

Fig. 4. Immunohistochemistry for 5-alpha reductase (5AR) type 2 and c-Fos. (A–C) Represent immunohistochemistry for 5AR type 2, and (E–G) show immunohistochemistry for c-Fos (A, E) Group 1; B and F: Group 2; C and G: Group 3). (D) Comparison of 5AR type 2 expression among groups 1, 2, and 3 (p=0.010). Adjusted p-values using the Bonferroni correction between Groups 1 and 2, Groups 2 and 3, and Groups 3 and 1 were 0.008, 0.682, and 0.219, respectively. (H) Comparison of c-Fos expression among groups 1, 2, and 3 (p=0.009). Adjusted p-values using the Bonferroni correction between Groups 1 and 2, Groups 2 and 3, and Groups 3 and 1 were 0.017, 0.031, and >0.999, respectively. Arrows: optic chiasm. Dotted Arrows: Anterior commissure. Circles: Medial preoptic area. Scale bar: 200 µm. Asterisks (*) in (D) and (H) mean adjusted p-value <0.05.

DISCUSSION

The main finding of the present study was that the expression of 5AR-2 protein decreased and remained at a lower level 4 weeks after discontinuation of the drug in rat brain tissue. However, considering that serum dihydrotestosterone levels were ameliorated after discontinuation of finasteride in this study, the persistently low expression of 5AR-2 in the brain tissue remains a complex and unresolved question.

To the best of our knowledge, the main source of circulating dihydrotestosterone is derived from the skin and liver via conversion of testosterone to dihydrotestosterone by 5AR-1 [17]. Although the half-life of finasteride is approximately 4.7 to 7.1 hours [18], a previous study reported irreversible suppression of 5AR-2 by finasteride, irrespective of dosage, whereas 5AR-1 inhibition by finasteride was shown to be dose-dependent and valid for approximately 24 hours [10]. Therefore, during finasteride administration, inhibition of both 5AR-1 and 5AR-2 may be maximized, leading to a reduction in serum dihydrotestosterone levels. When finasteride is discontinued, serum dihydrotestosterone could be ameliorated alongside a decrease in 5AR-1 suppression, which was supported by our data.

The mRNA expression could reciprocally increase if there was a feedback mechanism. We had expected that 5AR-2 mRNA, as well as 5AR-2 protein, should respond shortly after cessation of the drug. However, 5AR-2 mRNA levels did not change during finasteride administration or after drug cessation. An animal study of rat brain tissue supported this phenomenon, where 5AR-2 mRNA expression was positively correlated with serum testosterone levels rather than with dihydrotestosterone levels [19]. In the present study, serum testosterone levels did not differ between the groups. Consequently, 5AR-2 mRNA expression did not show reciprocal elevation during administration or after discontinuation of finasteride, which could explain why 5AR-2 protein levels did not significantly improve shortly after discontinuation of the drug.

Compelling evidence can support findings from human research conducted by Clark et al [20], who demonstrated that serum dihydrotestosterone levels could be restored within 4 weeks after discontinuation of finasteride (5.0 mg). In contrast, serum dihydrotestosterone levels did not improve soon after cessation of dutasteride, and the duration of suppression was dose-dependent [20]. This suggests that circulating dihydrotestosterone could be ameliorated earlier in finasteride users than in dutasteride users by alleviating the suppression of 5AR-1 protein.

According to a previous study [10], 5AR-2 inhibition by finasteride was not determined by dosage. Currently, there is little evidence to elucidate the cumulative effects (e.g., long-term use) of finasteride, especially regarding nerve system. However, a clinical study regarding persistent erectile dysfunction showed prolonged exposure to 5AR inhibitors was more important factor rather than other factors such as age, drug dose, depression, obesity, and so on [21]. An animal study conducted by Sung et al [22] reported that fibrosis-related factors could permanently change the erectile tissue using 0.5 mg dutasteride daily for 8 weeks, whereas 4 weeks of treatment led to reversible effects after discontinuation.

Although substantial data support the post-finasteride syndrome, the underlying causes of this phenomenon remain poorly understood. However, when assessing neural cellular function, introducing an appropriate biomarker may be useful. More than 20 years ago, a previous study suggested that c-Fos expression in the brain tissue was closely related to testosterone-derived estradiol, mediated by the estrogen-responsive element of the c-Fos gene regulatory region [7]. Yoshikage et al [23] attempted to explore how hormonal modulation could occur along with the signals of volatile odors within the anterior olfactory bulb using c-Fos immunohistochemistry. The authors suggested that androgens, such as dihydrotestosterone, are mandatory for maintaining neuronal activity, and that estrogen disrupts the effect of dihydrotestosterone. Recent studies investigating the role of androgens in the hypothalamic-pituitary-adrenal axis used c-Fos expression, in which sex-steroid hormone modified corticotropin-releasing factor neurons [24] and dihydrotestosterone treatment were significantly related to positive c-Fos expression in neurons [25]. Therefore, c-Fos activity may serve as a sex-steroid (such as dihydrotestosterone) related neuro-biomarker in this context.

Finasteride administration reduced dihydrotestosterone levels, which correlated with a decrease in c-Fos expression. Upon discontinuation of the drug, we observed a trend where both dihydrotestosterone and c-Fos expression levels began to recover. Altogether, finasteride administration may reduce certain neuronal activities, which appear to recover shortly after discontinuation, while restoration of 5AR-2 expression may require a prolonged period.

Finally, we discussed the tissue distribution of dihydrotestosterone. It should be noted that dihydrotestosterone acts in a paracrine signaling pathway. For instance, Page et al [26] revealed that intraprostatic dihydrotestosterone levels were 10-fold higher than circulating dihydrotestosterone level. Another study comparing serum dihydrotestosterone levels with those in cerebrospinal fluid showed that dihydrotestosterone in cerebrospinal fluid could be significantly influenced by serum testosterone levels rather than serum dihydrotestosterone, which indicates that target tissue dihydrotestosterone converted by the local 5AR could be more relevant to tissue function [4]. Therefore, we should not confirm local tissue function by measuring circulating dihydrotestosterone alone; however, it could be acceptable for a general assessment using circulating sex hormones. Regarding 5AR expression in the brain tissue, the slow return of sexual desire in some individuals after finasteride use may be due to strong and long-term inhibition of 5AR-2, coupled with the temporary inhibition of 5AR-1 in the brain tissue.

The authors acknowledge the limitations of the present study. Owing to ethical issues, only a minimal number of animals were sacrificed. Therefore, the standard errors are relatively large for several items. Another limitation of this type of animal study is that the genetic responses to a drug are not always similar between animals and humans. Although we tried to show biomolecular effect of brain tissue using the expression of 5AR-1, 5AR-2 and c-Fos, in the absence of behavioral data, our study did not provide sufficient information regarding specific drug-related neural functional changes.

CONCLUSIONS

Finasteride can suppress 5AR-2 in rat brain tissue one month after discontinuation of the drug. However, serum dihydrotestosterone levels along with 5AR-1 in the brain tissue were ameliorated after drug discontinuation, which may have led to the recovery of c-Fos protein activation. Therefore, the negative impact of finasteride on c-Fos activation in the rat brain may decrease one month after cessation of the drug. Further studies using other biomarkers or other methods (e.g., brain tissue dihydrotestosterone levels) are required to clarify the detailed mechanism of 5AR in the brain tissue.

Data Sharing Statement

The data analyzed for this study have been deposited in HARVARD Dataverse and are available at https://doi.org/10.7910/DVN/UTP1M5.

Supplementary Materials

Supplementary materials can be found via https://doi.org/10.5534/wjmh.250105.

Supplement Table 1

Primer data

Supplement Table 2

Comparison of H-score in immunohistochemistry