Dihydrotestosterone Administration Does Not Increase Intraprostatic Androgen Concentrations or Alter Prostate Androgen Action in Healthy Men: A Randomized-Controlled Trial
Context:
Concern exists that androgen treatment might adversely impact prostate health in older men. Dihydrotestosterone (DHT), derived from local conversion of testosterone to DHT by 5α-reductase enzymes, is the principal androgen within the prostate. Exogenous androgens raise serum DHT concentrations, but their effects on the prostate are not clear.
Objective:
To determine the impact of large increases in serum DHT concentrations on intraprostatic androgen concentrations and androgen action within the prostate.
Design:
Double-blind, randomized, placebo-controlled.
Setting:
Single academic medical center.
Participants:
31 healthy men ages 35–55.
Intervention:
Daily transdermal DHT or placebo gel.
Main Outcome Measures:
Serum and prostate tissue androgen concentrations and prostate epithelial cell gene expression after 4 wk of treatment.
Results:
Twenty-seven men completed all study procedures. Serum DHT levels increased nearly sevenfold, while testosterone levels decreased in men treated with daily transdermal DHT gel but were unchanged in the placebo-treated group (P < 0.01 between groups). In contrast, intraprostatic DHT and testosterone concentrations on d 28 were not different between groups (DHT: placebo = 2.8 ± 0.2 vs. DHT gel = 3.1 ± 0.5 ng/g; T: placebo = 0.6 ± 0.2 vs. DHT gel = 0.4 ± 0.1, mean ± se). Similarly, prostate volume, prostate-specific antigen, epithelial cell proliferation, and androgen-regulated gene expression were not different between groups.
Conclusions:
Robust supraphysiologic increases in serum DHT, associated with decreased serum T, do not significantly alter intraprostatic levels of DHT, testosterone, or prostate epithelial cell androgen–regulated gene expression in healthy men. Changes in circulating androgen concentrations are not necessarily mimicked within the prostate microenvironment, a finding with implications for understanding the impact of androgen therapies in men.
Materials and Methods
Subjects
Healthy male volunteers, 35–55 yr old, were recruited via advertisement and gave written informed consent before screening. All study procedures took place at the University of Washington Medical Center, Seattle WA, and were approved by the University of Washington Institutional Review Board. Informed consent was obtained by the investigator in all cases before any study procedures. Inclusion criteria included the following: general good health, normal serum total T level (3–11 ng/ml) and gonadotropins, serum PSA <2.0 ng/ml, International Prostate Symptom Score (IPSS) <10, and a normal digital rectal exam and transrectal prostate ultrasound. Exclusion criteria included the following: regular medication use, a personal or first-degree relative with a history of prostate cancer, or a history of chronic use of androgens, anabolic steroids, or drugs that interfere with androgen metabolism.
Study design and randomization
After screening, subjects were assigned to treatment group by the investigational drug pharmacist using a random number sequence with a block size of four. Subjects applied 10 g of either transdermal placebo gel or 0.7% DHT gel (BHR Pharma, LLC, Herndon, VA) daily for 28 d. IPSS questionnaires (19) and blood for serum hormone measurements and chemistries were collected on d 0, 14, 28, and 56 (recovery) and a transrectal prostate ultrasound was performed at baseline. On d 28, subjects underwent an ultrasound-guided prostate biopsy using local anesthetic (1% lidocaine). Using an 18-gauge needle, ten prostate core biopsies were procured, snap frozen in liquid nitrogen, and stored at −70 C or formalin-fixed. Four weeks after cessation of drug treatment (d 56), a third transrectal prostate ultrasound was performed to assess prostate volume.
Serum hormone assays and safety laboratory tests
Serum androgens were measured by liquid chromatography-tandem mass spectrometry on a Waters Aquity UPLC coupled with a Micromass Premiere-XE tandem quadrupole mass spectrometer (Waters Corp., Milford, MA) as described previously (20). Deuterated internal standards for each androgen were included in each sample. Androgens were extracted in hexane:ethyl acetate and derivatized in 0.025 m hydroxylamine hydrochloride and methanol. The lower limit of quantification for each androgen using this assay was 0.01 ng/ml. The intraassay coefficients of variation were: T = 4.9%, DHT 4.4%, dehydroepiandrosterone (DHEA) = 7.6%, androstenedione = 3.5%. Serum LH, FSH, and SHBG were quantified by immunofluorometric assay and estradiol by RIA (20). Samples for all subjects were measured in one assay. Safety laboratories including PSA, serum chemistries, and complete blood counts were measured in the clinical laboratory at the University of Washington Medical Center, Seattle, WA.
Tissue hormone assays
Prostate tissue androgen concentrations were measured using liquid chromatography-mass tandem spectrometry as described previously (21). For each subject, two separate prostate core biopsies were thawed, homogenized using a Precellys homogenizer and individually extracted. Individual core concentrations for each androgen were averaged for each subject. Cores weighed an average of 4.5 ± 1.3 mg. In four subjects, one in the placebo and three in the DHT group, deuterated DHT internal standard recovery was <5% in both cores and DHT measurements were below the limit of detection and not included in the analyses (although inclusion of these samples did not change the conclusions).
Laser capture microdissection and microarray hybridization
Frozen prostate biopsy cores were used for laser capture microdissection, RNA isolation and amplification, and microarray hybridization as previously described, using 44K whole human genome expression oligo-microarray slides (Agilent Technologies, Inc., Santa Clara, CA) (22). Fluorescence array images were collected (Agilent DNA oligo-array scanner G2565BA, Agilent Technologies, Inc.), and Agilent Feature Extraction software used to grid, extract, and normalize data. Spots of poor quality or average intensity levels (<300) were removed from further analysis.
We evaluated expression changes using two sample t tests [Statistical Analysis of Microarray (SAM) (http://www-stat.stanford.edu/_tibs/SAM/)]. False discovery rate less than 5% was considered significant (23). Unsupervised hierarchical average linkage clustering was performed using Cluster 3.0 software (http://bonsai.ims.u-tokyo.ac.jp/∼mdehoon/software/cluster/software.htm) plotted using TreeView version 1.6 (http://rana.lbl.gov/EisenSoftware.htm), using a custom list of 90 androgen-regulated genes as we have previously described (22). Microarray data can be accessed via the Gene Expression Omnibus database (accession# {"type":"entrez-geo","attrs":{"text":"GSE20317","term_id":"20317","extlink":"1"}}GSE20317).
Ki-67 staining and quantification
Immunohistochemistry on formalin-fixed paraffin-embedded tissues sections was performed as previously described (24) using a Vectastin Elite Kit (Vector Laboratories, Burlingame, CA). The percentage of Ki-67–positive cells per 100 epithelial cells was assessed by two individuals blinded to treatment group and averaged (24).
Statistical analyses
Because hormone results were nonnormally distributed, nonparametric statistics were performed using STATA version 10.0. Specifically, Wilcoxon rank-sum (Mann–Whitney) tests were used to compare between groups, and Wilcoxon signed-rank tests were used for within group comparisons with a Bonferonni correction for multiple comparisons. An α of 0.05 was significant.
Subjects
Healthy male volunteers, 35–55 yr old, were recruited via advertisement and gave written informed consent before screening. All study procedures took place at the University of Washington Medical Center, Seattle WA, and were approved by the University of Washington Institutional Review Board. Informed consent was obtained by the investigator in all cases before any study procedures. Inclusion criteria included the following: general good health, normal serum total T level (3–11 ng/ml) and gonadotropins, serum PSA <2.0 ng/ml, International Prostate Symptom Score (IPSS) <10, and a normal digital rectal exam and transrectal prostate ultrasound. Exclusion criteria included the following: regular medication use, a personal or first-degree relative with a history of prostate cancer, or a history of chronic use of androgens, anabolic steroids, or drugs that interfere with androgen metabolism.
Study design and randomization
After screening, subjects were assigned to treatment group by the investigational drug pharmacist using a random number sequence with a block size of four. Subjects applied 10 g of either transdermal placebo gel or 0.7% DHT gel (BHR Pharma, LLC, Herndon, VA) daily for 28 d. IPSS questionnaires (19) and blood for serum hormone measurements and chemistries were collected on d 0, 14, 28, and 56 (recovery) and a transrectal prostate ultrasound was performed at baseline. On d 28, subjects underwent an ultrasound-guided prostate biopsy using local anesthetic (1% lidocaine). Using an 18-gauge needle, ten prostate core biopsies were procured, snap frozen in liquid nitrogen, and stored at −70 C or formalin-fixed. Four weeks after cessation of drug treatment (d 56), a third transrectal prostate ultrasound was performed to assess prostate volume.
Serum hormone assays and safety laboratory tests
Serum androgens were measured by liquid chromatography-tandem mass spectrometry on a Waters Aquity UPLC coupled with a Micromass Premiere-XE tandem quadrupole mass spectrometer (Waters Corp., Milford, MA) as described previously (20). Deuterated internal standards for each androgen were included in each sample. Androgens were extracted in hexane:ethyl acetate and derivatized in 0.025 m hydroxylamine hydrochloride and methanol. The lower limit of quantification for each androgen using this assay was 0.01 ng/ml. The intraassay coefficients of variation were: T = 4.9%, DHT 4.4%, dehydroepiandrosterone (DHEA) = 7.6%, androstenedione = 3.5%. Serum LH, FSH, and SHBG were quantified by immunofluorometric assay and estradiol by RIA (20). Samples for all subjects were measured in one assay. Safety laboratories including PSA, serum chemistries, and complete blood counts were measured in the clinical laboratory at the University of Washington Medical Center, Seattle, WA.
Tissue hormone assays
Prostate tissue androgen concentrations were measured using liquid chromatography-mass tandem spectrometry as described previously (21). For each subject, two separate prostate core biopsies were thawed, homogenized using a Precellys homogenizer and individually extracted. Individual core concentrations for each androgen were averaged for each subject. Cores weighed an average of 4.5 ± 1.3 mg. In four subjects, one in the placebo and three in the DHT group, deuterated DHT internal standard recovery was <5% in both cores and DHT measurements were below the limit of detection and not included in the analyses (although inclusion of these samples did not change the conclusions).
Laser capture microdissection and microarray hybridization
Frozen prostate biopsy cores were used for laser capture microdissection, RNA isolation and amplification, and microarray hybridization as previously described, using 44K whole human genome expression oligo-microarray slides (Agilent Technologies, Inc., Santa Clara, CA) (22). Fluorescence array images were collected (Agilent DNA oligo-array scanner G2565BA, Agilent Technologies, Inc.), and Agilent Feature Extraction software used to grid, extract, and normalize data. Spots of poor quality or average intensity levels (<300) were removed from further analysis.
We evaluated expression changes using two sample t tests [Statistical Analysis of Microarray (SAM) (http://www-stat.stanford.edu/_tibs/SAM/)]. False discovery rate less than 5% was considered significant (23). Unsupervised hierarchical average linkage clustering was performed using Cluster 3.0 software (http://bonsai.ims.u-tokyo.ac.jp/∼mdehoon/software/cluster/software.htm) plotted using TreeView version 1.6 (http://rana.lbl.gov/EisenSoftware.htm), using a custom list of 90 androgen-regulated genes as we have previously described (22). Microarray data can be accessed via the Gene Expression Omnibus database (accession# {"type":"entrez-geo","attrs":{"text":"GSE20317","term_id":"20317","extlink":"1"}}GSE20317).
Ki-67 staining and quantification
Immunohistochemistry on formalin-fixed paraffin-embedded tissues sections was performed as previously described (24) using a Vectastin Elite Kit (Vector Laboratories, Burlingame, CA). The percentage of Ki-67–positive cells per 100 epithelial cells was assessed by two individuals blinded to treatment group and averaged (24).
Statistical analyses
Because hormone results were nonnormally distributed, nonparametric statistics were performed using STATA version 10.0. Specifically, Wilcoxon rank-sum (Mann–Whitney) tests were used to compare between groups, and Wilcoxon signed-rank tests were used for within group comparisons with a Bonferonni correction for multiple comparisons. An α of 0.05 was significant.
Results
Study population
Of 43 screened subjects, 32 met criteria for randomization and enrollment, and 28 completed study procedures. Of four subjects who failed to complete the study, one withdrew before randomization due to relocation; one in the placebo group withdrew for personal reasons; and two in the DHT group discontinued treatment due to adverse events (see below). After completion of the study an additional subject in the DHT group was determined to have been undergoing treatment for concomitant HIV infection that he had not reported at enrollment. As chronic illness and medication use were exclusion criteria, this participant was excluded from the presented analysis (n = 27 analyzed samples), although inclusion did not affect the conclusions of the study for any parameter. There were no significant baseline differences between subjects in the two treatment groups (Table 1).
Table 1.
Placebo n = 15 | DHT gel n = 12 | P value | |
---|---|---|---|
Age, yr | 44.4 ± 6.7 | 42.7 ± 1.6 | 0.48 |
Weight, kg | 93.7 ± 17.6 | 93.0 ± 16.9 | 0.92 |
BMI | 28.9 ± 4.9 | 29.2 ± 5.6 | 0.83 |
Total T, ng/ml | 4.4 ± 1.8 | 3.8 ± 1.5 | 0.37 |
Total DHT, ng/ml | 0.35 ± 0.17 | 0.26 ± 0.17 | 0.38 |
Total E, pg/ml | 18 ± 11 | 21 ± 8 | 0.69 |
PSA, ng/ml | 1.1 ± 0.6 | 0.7 ± 0.4 | 0.09 |
E, Estradiol.
Adverse events
Two subjects in the DHT group discontinued participation due to adverse events. One experienced increased urinary frequency during the first week of drug exposure and the other had aggravation of preexisting hemorrhoids during the second week. Both conditions resolved without treatment. Four subjects in the DHT-treated group experienced one to four episodes of mild headache during treatment and one of these subjects had an episode of arthralgia, all of which resolved within 24 h without treatment. There were no significant changes or differences between groups in serum liver function tests, blood counts, or chemistries (data not shown).
Serum hormones
Serum DHT significantly increased in the DHT-treated group compared with both baseline and the placebo group by d 14 and remained more than sevenfold greater than baseline levels through d 28 of treatment (Fig. 1A, P < 0.05) These concentrations of serum DHT were significantly above the normal range throughout the treatment period. Of note, the total serum T + DHT was equivalent comparing the two treatment groups at d 28 (Fig. 1, A and B).
Both serum total T and total estradiol decreased in the DHT-treated group (Fig. 1, B and C) compared with baseline on d 14 and 28 (P < 0.05). Serum T fell nearly 50% in the DHT-treated group and was significantly lower than placebo during treatment. Serum androstenedione, a precursor of testosterone, and LH decreased only in the DHT-treated group compared with baseline (Table 2), while FSH and SHBG were not significantly affected in either group.
Table 2.
Placebo | DHT gel | |||||
---|---|---|---|---|---|---|
Day 0 Baseline | Day 28 End-of-treatment | Day 56 4-wk recovery | Day 0 baseline | Day 28 End-of-treatment | Day 56 4-wk recovery | |
LH, IU/liter | 4.3 ± 1.5 | 4.1 ± 1.8 | 4.8 ± 2.0 | 6.0 ± 5.6 | 4.5 ± 4.1a | 6.6 ± 5.0 |
FSH, IU/liter | 5.1 ± 3.3 | 5.2 ± 3.0 | 5.2 ± 3.0 | 8.1 ± 12 | 8.2 ± 12 | 8.4 ± 12.1 |
SHBG, μg/ml | 3.5 ± 1.5 | 3.5 ± 1.7 | 3.4 ± 1.2 | 3.0 ± 1.5 | 3.1 ± 1.6 | 2.9 ± 1.4 |
DHEA, ng/ml | 4.7 ± 2.3 | 4.1 ± 2.0 | 4.1 ± 2.0 | 5.8 ± 3.8 | 5.5 ± 3.8 | 6.3 ± 5.6 |
Androstenedione, ng/ml | 0.8 ± 0.2 | 0.8 ± 0.3 | 0.8 ± 0.2 | 1.0 ± 0.3 | 0.8 ± 0.3a | 0.9 ± 0.3 |
PSA, ng/ml | 1.1 ± 0.6 | 1.0 ± 0.5 | 1.8 ± 0.8a | 0.7 ± 0.4 | 0.8 ± 0.5 | 1.6 ± 1.0a |
Prostate volume, cc | 21.5 ± 4.2 | 21.0 ± 3.5 | 21.2 ± 3.9 | 20.3 ± 4.6 | 21.2 ± 4.3 | 21.8 ± 4.8 |
IPSS score | 2.5 ± 2.8 | 1.7 ± 2.0a | 1.6 ± 2.0a | 2.3 ± 1.7 | 2.8 ± 2.6 | 2.6 ± 2.3 |
Weight, kg | 93.7 ± 17.6 | 93.2 ± 17.6 | 93.3 ± 18.0 | 93.0 ± 16.9 | 93.3 ± 17.1 | 94.9 ± 17.2 |
Intraprostatic androgen concentrations
Despite a sevenfold increase in serum DHT and a significant reduction in serum T concentration, intraprostatic concentrations of DHT and T were not significantly different in the DHT-treated compared with placebo-treated group after 28 d of drug exposure (Fig. 2) (P value between groups: intraprostatic DHT, P = 0.56, intraprosatic T, P = 0.37). Similarly, despite a significant change in the ratio of T:DHT in serum, the proportion of T:DHT did not differ within the prostate during DHT or placebo administration. Moreover, intraprostatic DHEA and androstenedione, precursors in testosterone biosynthesis, were no different in the two groups (intraprostatic DHEA ng/g: placebo= 27 ± 4, DHT-treated= 43 ± 10, P = 0.07 between groups, intraprostatic androstenedione ng/g: placebo= 0.27 ± 0.03, DHT-treated= 0.28 ± 0.03, P = 0.79 between groups).
Prostate volume, PSA, and IPSS
Prostate volume and IPSS scores were unaffected by DHT treatment (Table 2). PSA concentrations were unchanged compared with baseline during the treatment period in both placebo and DHT groups. However, serum PSA increased significantly at the d 56 visit (follow-up) above baseline and on-treatment time points in both groups (Table 2), likely due to PSA release after the d 28 biopsy (25). In subjects with a PSA more than twofold above baseline (n = 9), a repeat PSA was measured 1 month later. All subjects had serum PSA of < 1.5-fold of baseline concentration before conclusion of the study. No subject had a PSA of > 4.0 ng/ml at any point during the study.
Prostate epithelial cell androgen-regulated gene expression and proliferation
To further assess the impact of transdermal DHT gel on the prostatic environment, we used microarray profiling to quantify gene expression in prostate epithelium microdissected from posttreatment biopsy cores. Unpaired two-sample t tests revealed no gene expression differences between the placebo and DHT gel-treated subjects. Specific interrogation of known androgen regulated genes, such as KLK3 (PSA), showed similar expression across both treated and untreated samples, with no consistent differences in the DHT-treated subjects (Fig. 3). Moreover, we performed quantitative RT-PCR for several well known androgen-regulated genes (androgen receptor, FKBP5, NKX3.1, PSA, ACPP) and found no differences in expression of these genes between the two groups (data not shown). To more comprehensively evaluate the effect of DHT gel on the expression of androgen regulated genes, we focused on the expression of 90 known androgen-regulated genes. We clustered the samples using a program that grouped samples based on how similarly or differently they express a set of genes. A strong effect of DHT treatment on androgen regulated gene expression compared with placebo would be expected to cause the DHT-treated and placebo-treated samples to cluster into different groups. However, we did not observe any segregation of the DHT-treated and placebo samples, suggesting DHT treatment did not systematically affect androgen sensitive genes (Fig. 4).
We went on to perform Ki-67 inmmunohistochemial staining of prostate epithelium as a marker of cell proliferation, and found no significant differences between treatment groups (Ki-67 positive cells per 100 epithelial cells, ±sd: placebo = 1.3 ± 0.5, DHT-treated = 0.7 ± 0.9; P = 0.09 between groups).
Study population
Of 43 screened subjects, 32 met criteria for randomization and enrollment, and 28 completed study procedures. Of four subjects who failed to complete the study, one withdrew before randomization due to relocation; one in the placebo group withdrew for personal reasons; and two in the DHT group discontinued treatment due to adverse events (see below). After completion of the study an additional subject in the DHT group was determined to have been undergoing treatment for concomitant HIV infection that he had not reported at enrollment. As chronic illness and medication use were exclusion criteria, this participant was excluded from the presented analysis (n = 27 analyzed samples), although inclusion did not affect the conclusions of the study for any parameter. There were no significant baseline differences between subjects in the two treatment groups (Table 1).
Table 1.
Placebo n = 15 | DHT gel n = 12 | P value | |
---|---|---|---|
Age, yr | 44.4 ± 6.7 | 42.7 ± 1.6 | 0.48 |
Weight, kg | 93.7 ± 17.6 | 93.0 ± 16.9 | 0.92 |
BMI | 28.9 ± 4.9 | 29.2 ± 5.6 | 0.83 |
Total T, ng/ml | 4.4 ± 1.8 | 3.8 ± 1.5 | 0.37 |
Total DHT, ng/ml | 0.35 ± 0.17 | 0.26 ± 0.17 | 0.38 |
Total E, pg/ml | 18 ± 11 | 21 ± 8 | 0.69 |
PSA, ng/ml | 1.1 ± 0.6 | 0.7 ± 0.4 | 0.09 |
E, Estradiol.
Adverse events
Two subjects in the DHT group discontinued participation due to adverse events. One experienced increased urinary frequency during the first week of drug exposure and the other had aggravation of preexisting hemorrhoids during the second week. Both conditions resolved without treatment. Four subjects in the DHT-treated group experienced one to four episodes of mild headache during treatment and one of these subjects had an episode of arthralgia, all of which resolved within 24 h without treatment. There were no significant changes or differences between groups in serum liver function tests, blood counts, or chemistries (data not shown).
Serum hormones
Serum DHT significantly increased in the DHT-treated group compared with both baseline and the placebo group by d 14 and remained more than sevenfold greater than baseline levels through d 28 of treatment (Fig. 1A, P < 0.05) These concentrations of serum DHT were significantly above the normal range throughout the treatment period. Of note, the total serum T + DHT was equivalent comparing the two treatment groups at d 28 (Fig. 1, A and B).
Both serum total T and total estradiol decreased in the DHT-treated group (Fig. 1, B and C) compared with baseline on d 14 and 28 (P < 0.05). Serum T fell nearly 50% in the DHT-treated group and was significantly lower than placebo during treatment. Serum androstenedione, a precursor of testosterone, and LH decreased only in the DHT-treated group compared with baseline (Table 2), while FSH and SHBG were not significantly affected in either group.
Table 2.
Placebo | DHT gel | |||||
---|---|---|---|---|---|---|
Day 0 Baseline | Day 28 End-of-treatment | Day 56 4-wk recovery | Day 0 baseline | Day 28 End-of-treatment | Day 56 4-wk recovery | |
LH, IU/liter | 4.3 ± 1.5 | 4.1 ± 1.8 | 4.8 ± 2.0 | 6.0 ± 5.6 | 4.5 ± 4.1a | 6.6 ± 5.0 |
FSH, IU/liter | 5.1 ± 3.3 | 5.2 ± 3.0 | 5.2 ± 3.0 | 8.1 ± 12 | 8.2 ± 12 | 8.4 ± 12.1 |
SHBG, μg/ml | 3.5 ± 1.5 | 3.5 ± 1.7 | 3.4 ± 1.2 | 3.0 ± 1.5 | 3.1 ± 1.6 | 2.9 ± 1.4 |
DHEA, ng/ml | 4.7 ± 2.3 | 4.1 ± 2.0 | 4.1 ± 2.0 | 5.8 ± 3.8 | 5.5 ± 3.8 | 6.3 ± 5.6 |
Androstenedione, ng/ml | 0.8 ± 0.2 | 0.8 ± 0.3 | 0.8 ± 0.2 | 1.0 ± 0.3 | 0.8 ± 0.3a | 0.9 ± 0.3 |
PSA, ng/ml | 1.1 ± 0.6 | 1.0 ± 0.5 | 1.8 ± 0.8a | 0.7 ± 0.4 | 0.8 ± 0.5 | 1.6 ± 1.0a |
Prostate volume, cc | 21.5 ± 4.2 | 21.0 ± 3.5 | 21.2 ± 3.9 | 20.3 ± 4.6 | 21.2 ± 4.3 | 21.8 ± 4.8 |
IPSS score | 2.5 ± 2.8 | 1.7 ± 2.0a | 1.6 ± 2.0a | 2.3 ± 1.7 | 2.8 ± 2.6 | 2.6 ± 2.3 |
Weight, kg | 93.7 ± 17.6 | 93.2 ± 17.6 | 93.3 ± 18.0 | 93.0 ± 16.9 | 93.3 ± 17.1 | 94.9 ± 17.2 |
Intraprostatic androgen concentrations
Despite a sevenfold increase in serum DHT and a significant reduction in serum T concentration, intraprostatic concentrations of DHT and T were not significantly different in the DHT-treated compared with placebo-treated group after 28 d of drug exposure (Fig. 2) (P value between groups: intraprostatic DHT, P = 0.56, intraprosatic T, P = 0.37). Similarly, despite a significant change in the ratio of T:DHT in serum, the proportion of T:DHT did not differ within the prostate during DHT or placebo administration. Moreover, intraprostatic DHEA and androstenedione, precursors in testosterone biosynthesis, were no different in the two groups (intraprostatic DHEA ng/g: placebo= 27 ± 4, DHT-treated= 43 ± 10, P = 0.07 between groups, intraprostatic androstenedione ng/g: placebo= 0.27 ± 0.03, DHT-treated= 0.28 ± 0.03, P = 0.79 between groups).
Prostate volume, PSA, and IPSS
Prostate volume and IPSS scores were unaffected by DHT treatment (Table 2). PSA concentrations were unchanged compared with baseline during the treatment period in both placebo and DHT groups. However, serum PSA increased significantly at the d 56 visit (follow-up) above baseline and on-treatment time points in both groups (Table 2), likely due to PSA release after the d 28 biopsy (25). In subjects with a PSA more than twofold above baseline (n = 9), a repeat PSA was measured 1 month later. All subjects had serum PSA of < 1.5-fold of baseline concentration before conclusion of the study. No subject had a PSA of > 4.0 ng/ml at any point during the study.
Prostate epithelial cell androgen-regulated gene expression and proliferation
To further assess the impact of transdermal DHT gel on the prostatic environment, we used microarray profiling to quantify gene expression in prostate epithelium microdissected from posttreatment biopsy cores. Unpaired two-sample t tests revealed no gene expression differences between the placebo and DHT gel-treated subjects. Specific interrogation of known androgen regulated genes, such as KLK3 (PSA), showed similar expression across both treated and untreated samples, with no consistent differences in the DHT-treated subjects (Fig. 3). Moreover, we performed quantitative RT-PCR for several well known androgen-regulated genes (androgen receptor, FKBP5, NKX3.1, PSA, ACPP) and found no differences in expression of these genes between the two groups (data not shown). To more comprehensively evaluate the effect of DHT gel on the expression of androgen regulated genes, we focused on the expression of 90 known androgen-regulated genes. We clustered the samples using a program that grouped samples based on how similarly or differently they express a set of genes. A strong effect of DHT treatment on androgen regulated gene expression compared with placebo would be expected to cause the DHT-treated and placebo-treated samples to cluster into different groups. However, we did not observe any segregation of the DHT-treated and placebo samples, suggesting DHT treatment did not systematically affect androgen sensitive genes (Fig. 4).
We went on to perform Ki-67 inmmunohistochemial staining of prostate epithelium as a marker of cell proliferation, and found no significant differences between treatment groups (Ki-67 positive cells per 100 epithelial cells, ±sd: placebo = 1.3 ± 0.5, DHT-treated = 0.7 ± 0.9; P = 0.09 between groups).
Discussion
This is the first placebo-controlled trial to examine the impact of substantial supraphysiologic increases in serum DHT, the most potent endogenous androgen in men, on the intraprostatic hormonal environment in healthy men. Administration of exogenous DHT gel yielded a sevenfold increase in serum DHT levels and a 50% decline in serum T, while intraprostatic androgen concentrations, including DHT, T, and both androgen precursors and metabolites of T, were no different in men using transdermal DHT gel compared with men receiving placebo gel. Moreover, in the setting of these stable intraprostatic androgen concentrations, markers of prostate androgen response in men including serum PSA, prostate volume, prostate epithelial cell gene expression, and prostate epithelial cell proliferation were no different in men treated with DHT gel compared with placebo-treated men. Our data demonstrate that interventions which significantly raise serum DHT concentrations for 1 month and concomitantly lower serum T levels do not result in marked macroscopic or microscopic effects on healthy prostate homeostasis.
The administration of exogenous T increases both serum T and DHT concentrations (26, 27). In contrast, exogenous DHT, by lowering pituitary LH production via negative feedback, increases serum DHT while concomitantly lowering serum T concentrations (15, 16, 28). The degree of increase in serum DHT that we observed in this study, and the lack of increase in PSA and prostate volume, are consistent with previous reports of these parameters using transdermal DHT gel (15,–17), an approved therapy for the treatment of hypogonadism in parts of Europe. Our data are consistent with these, as intraprostatic androgens, including DHT, T, adrenal androgen precursors, DHEA and androstenedione, and androgen-regulated gene expression, were unchanged despite very high serum DHT levels with DHT gel administration. Despite data implicating DHT as important in the pathophysiology of benign prostatic hypertrophy, long-term follow-up of men taking oral testosterone undecanoate, which also raises serum DHT well above the normal range (29), have not demonstrated an increase in serum PSA or prostate-related adverse events (30). While the pharmacokinetics of oral testosterone undecanoate and that of transdermal DHT gel are significantly different, our data demonstrate that interventions that significantly raise serum levels of the potent androgen DHT, and lower serum testosterone, do not markedly alter the prostate microenvironment.
Our findings that manipulation of serum androgens in men does not result in similar changes within the prostate have been observed in other studies involving androgen supplementation and suppression. Marks and colleagues found that modest levels of testosterone-replacement in older men with low serum T levels did not impact intraprostatic T or DHT concentrations nor androgen-regulated gene expression, although serum PSA levels were modestly increased (7). We extend these findings by demonstrating similar prostate stability in the setting of markedly supraphysiologic circulating androgen concentrations produced by a high dose of the pure, more potent, nonaromatizable (that is, not converted to estrogen) androgen, DHT. We have shown previously that medical castration in healthy men significantly decreases intraprostatic T and DHT concentrations (8). However, similar to findings in men with prostate cancer (18, 21), there is a relative preservation of intraprostatic androgen levels compared with decrements in serum T and DHT in healthy men in this setting (8). Moreover, these levels of intraprostatic androgens appear to maintain important biological processes within the gland, because a paucity of changes in androgen-regulated gene expression were observed in healthy men treated with androgen deprivation therapy (31). Together, these data support the proposal that the prostate is able to maintain intraprostatic androgen concentrations and action in the setting of significant alterations in serum levels, perhaps by regulating intraprostatic androgen transport, synthesis and metabolism (32, 33). These mechanisms of maintenance of intraprostatic androgen concentrations also may explain the lack of change with supraphysiological DHT administration in the present study.
Changes in intraprostatic DHT levels may be important in the pathogenesis of prostate cancer. Recent trials demonstrate that 5α-reductase inhibitors reduce the risk of prostate cancer in older men (10, 11). 5α-reductase inhibitors drastically reduce both serum and prostate DHT levels, and while they have little affect on serum T concentrations, they cause a concomitant 10- to 100-fold increase in intraprostatic T levels (12, 13). Because it appears that lowering intraprostatic DHT may contribute to lowering the risk for prostate cancer, one might infer that exogenous androgens might increase the risk of prostate cancer in men. However, a meta-analyses of small trials of androgen replacement did not find a cumulative increase in prostate cancer risk (34), and higher levels of endogenous androgens are not associated with an increased risk of developing prostate cancer (4). While no trial to date has been powered to assess the true risk, if any, of androgen-based therapies on prostate cancer risk, our data support the notion that robust changes in serum androgen levels may not be associated with marked disturbances within the prostate and perhaps may not alter prostate disease risk. Longer-term studies adequately powered to address this question directly are clearly needed.
As T is the precursor of estradiol via aromatization, serum estradiol concentrations were decreased in parallel with serum T after transdermal DHT administration. Unfortunately, we were not able to measure intraprostatic estradiol concentrations in this study due to a lack of sensitivity in our tissue assay and the low concentrations of estradiol within the prostate. This will be of interest in future studies as the complex role of estradiol and other estrogen ligands in prostate health continues to be elucidated (35). In addition, estradiol appears to be critical for long-term bone health in men (36). Studies addressing the impact of transdermal DHT on bone mineral density and fracture risk will be important in assessing clinical benefits of exogenous DHT.
This study is limited by small numbers of participants and a relatively short duration of drug exposure; thus we cannot exclude the possibility that small, and perhaps clinically significant, changes in intraprostatic hormones, gene expression, or epithelial cell proliferation occur with transdermal DHT gel. We performed our analyses in healthy men only. This allowed us to study the normal physiologic response to changes in serum androgens within the prostate, but we cannot be certain that our findings can be extrapolated to hypogonadal men who might have a less responsive hypothalamic-pituitary-gonadal axis or who may have underlying prostate pathology.
In summary, in this study of human prostate physiology we have demonstrated that substantial increases in serum DHT concentrations mediated by high-dosage exogenous DHT, which concurrently lower serum T concentrations, do not raise intraprostatic concentrations of T, DHT, DHEA, or androstenedione in healthy men. In addition, supraphysiologic levels of serum DHT for 1 month were not associated with alterations in prostate epithelial cell proliferation or androgen-regulated gene expression. Longer-term studies that directly examine the prostate effects of androgen replacement strategies should be undertaken but, lacking those data, we should not infer that changes in serum androgen concentrations conferred by exogenous androgens are reflected within the prostate.
Abstract
Context:
Concern exists that androgen treatment might adversely impact prostate health in older men. Dihydrotestosterone (DHT), derived from local conversion of testosterone to DHT by 5α-reductase enzymes, is the principal androgen within the prostate. Exogenous androgens raise serum DHT concentrations, but their effects on the prostate are not clear.
Objective:
To determine the impact of large increases in serum DHT concentrations on intraprostatic androgen concentrations and androgen action within the prostate.
Design:
Double-blind, randomized, placebo-controlled.
Setting:
Single academic medical center.
Participants:
31 healthy men ages 35–55.
Intervention:
Daily transdermal DHT or placebo gel.
Main Outcome Measures:
Serum and prostate tissue androgen concentrations and prostate epithelial cell gene expression after 4 wk of treatment.
Results:
Twenty-seven men completed all study procedures. Serum DHT levels increased nearly sevenfold, while testosterone levels decreased in men treated with daily transdermal DHT gel but were unchanged in the placebo-treated group (P < 0.01 between groups). In contrast, intraprostatic DHT and testosterone concentrations on d 28 were not different between groups (DHT: placebo = 2.8 ± 0.2 vs. DHT gel = 3.1 ± 0.5 ng/g; T: placebo = 0.6 ± 0.2 vs. DHT gel = 0.4 ± 0.1, mean ± se). Similarly, prostate volume, prostate-specific antigen, epithelial cell proliferation, and androgen-regulated gene expression were not different between groups.
Conclusions:
Robust supraphysiologic increases in serum DHT, associated with decreased serum T, do not significantly alter intraprostatic levels of DHT, testosterone, or prostate epithelial cell androgen–regulated gene expression in healthy men. Changes in circulating androgen concentrations are not necessarily mimicked within the prostate microenvironment, a finding with implications for understanding the impact of androgen therapies in men.
In hypogonadal men, androgen replacement increases muscle mass and bone mineral density and appears to improve strength, physical function, and libido (1). With the recognition that testosterone (T) levels decline with age (2), there is increasing interest in treating elderly men who have low T levels with T replacement. However, there is some concern that exogenous androgens may increase the risk of prostate cancer and/or may increase prostate growth and the frequency of related complications in men with benign prostatic hyperplasia (BPH) (3). Conversely, high endogenous serum androgen levels are not associated with increased risk of prostate cancer (4). Despite the paucity of data regarding absolute risk of androgen therapy on prostate health, prescriptions for exogenous androgens are clearly on the rise (5). An improved understanding of the hormonal and molecular consequences of androgen manipulation within the human prostate might help guide future trials designed to assess risks and benefits of these hormonal therapies.
Approximately 5% of circulating T is converted to the more potent androgen dihydrotestosterone (DHT) by the enzymes, type I and II 5α-reductase. Type II 5α-reductase is abundantly expressed in the human prostate, where it maintains high intraprostatic DHT concentrations, thereby amplifying androgen action (6). Intraprostatic DHT concentrations are roughly 10-fold those found in serum and 10-fold greater than intraprostatic T concentrations (7, 8). The conversion of T to DHT within the prostate is critical for prostate maturation, as 5α-reductase deficiency impairs prostate differentiation and development (9). 5α-reductase inhibitors reduce prostate size and improve lower urinary tract symptoms in older men with BPH and can decrease the risk of developing prostate cancer, but the mechanisms responsible for these reductions are not known (10, 11). It is likely these effects are mediated by a reduction in intraprostatic androgen concentrations because 5α-reductase inhibitors lower both serum and intraprostatic concentrations of DHT considerably (12, 13).
Exogenous androgens are used by men for the correction of hypogonadism, as performance-enhancing drugs, and experimentally, in hormonal male contraceptive regimens. T treatment raises both serum T and DHT levels (14). Interestingly, administering DHT to hypogonadal men for six months had no effect on prostate size or prostate-specific antigen (PSA), despite raising serum DHT fivefold (15, 16). A possible explanation for these findings is that exogenous DHT might paradoxically lower intraprostatic DHT by decreasing serum T, the substrate for intraprostatic 5α-reductase. In support of this hypothesis, DHT administration lowers both serum LH and T concentrations via negative feedback at the pituitary and hypothalamus (15,–17). However, intraprostatic androgen concentrations have not been measured in studies of DHT administration. Moreover, recent studies have demonstrated that alterations in serum androgen concentrations do not result in parallel changes in the intraprostatic hormonal milieu (7, 8, 18).
We sought to further characterize the relationship between serum and intraprostatic DHT levels to better understand the impact of high circulating concentrations of exogenous androgens within the human prostate. We hypothesized that supraphysiologic increases in serum DHT would not increase intraprostatic DHT and would have little impact on androgen-regulated processes within the prostate. To test this hypothesis, we conducted a double-blind, placebo-controlled trial of high-dosage transdermal DHT gel administration in healthy men to determine the effect of increases in serum DHT on intraprostatic hormone concentrations and androgen action.
E, Estradiol.
Acknowledgments
We thank Ilsa Coleman for assistance with data analysis and William J. Bremner and Bradley Anawalt for critical reading of this manuscript. We are grateful for Marilyn Busher, RN and Kathryn Torrez Duncan, Research Study Coordinator, for their assistance conducting the study, and our research study volunteers, without whom this work would not be possible.
This work was supported by the National Institute of Aging (K23-AG027238), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54-HD-42454), the Pacific Northwest Prostate Cancer SPORE (P50-CA097186), an Endocrine Society Clinical Research Award, an investigator-initiated grant from BHR Pharma LLC, and the Prostate Cancer Foundation. S.T.P. received grant support from BHR Pharma LLC.
Clinical Trial Registry Number: {"type":"clinical-trial","attrs":{"text":"NCT00490022","term_id":"NCT00490022"}}NCT00490022.
Disclosure Summary: The authors have nothing to declare.
Footnotes
Abbreviations:
- BHT
- Benign prostatic hyperplasia
- DHEA
- dehydroepiandrosterone
- DHT
- dihydrotestosterone
- IPSS
- International Prostate Symptom Score
- PSA
- prostate-specific antigen
- T
- testosterone.
References
- 1. Beg S, Al-Khoury L, Cunningham GR. 2008. Testosterone replacement in men. Curr Opin Endocrinol Diabetes Obes15:364–370 [[PubMed]
- 2. Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR. 2001. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab86:724–731 [[PubMed]
- 3. Behre HM, Bohmeyer J, Nieschlag E. 1994. Prostate volume in testosterone-treated and untreated hypogonadal men in comparison to age-matched normal controls. Clin Endocrinol (Oxf)40:341–349 [[PubMed]
- 4. Roddam AW, Allen NE, Appleby P, Key TJ. 2008. Endogenous sex hormones and prostate cancer: a collaborative analysis of 18 prospective studies. J Natl Cancer Inst100:170–183
- 5. Liverman C, Blazer DG, editors. eds 2003. Testosterone and Aging: clinical research directions. Institute of Medicine of the National Academies, Washington, DC, 2003 [PubMed]
- 6. Bartsch W, Klein H, Schiemann U, Bauer HW, Voigt KD. 1990. Enzymes of androgen formation and degradation in the human prostate. Ann NY Acad Sci595:53–66 [[PubMed]
- 7. Marks LS, Mazer NA, Mostaghel E, Hess DL, Dorey FJ, Epstein JI, Veltri RW, Makarov DV, Partin AW, Bostwick DG, Macairan ML, Nelson PS. 2006. Effect of testosterone replacement therapy on prostate tissue in men with late-onset hypogonadism: a randomized controlled trial. JAMA296:2351–2361 [[PubMed]
- 8. Page ST, Lin DW, Mostaghel EA, Hess DL, True LD, Amory JK, Nelson PS, Matsumoto AM, Bremner WJ. 2006. Persistent intraprostatic androgen concentrations after medical castration in healthy men. J Clin Endocrinol Metab91:3850–3856 [[PubMed]
- 9. Wilson JD. 2001. The role of 5alpha-reduction in steroid hormone physiology. Reprod Fertil Dev13:673–678 [[PubMed]
- 10. Thompson IM, Goodman PJ, Tangen CM, Lucia MS, Miller GJ, Ford LG, Lieber MM, Cespedes RD, Atkins JN, Lippman SM, Carlin SM, Ryan A, Szczepanek CM, Crowley JJ, Coltman CA., Jr 2003 The influence of finasteride on the development of prostate cancer. N Engl J Med349:215–224 [[PubMed][Google Scholar]
- 11. Andriole GL, Bostwick DG, Brawley OW, Gomella LG, Marberger M, Montorsi F, Pettaway CA, Tammela TL, Teloken C, Tindall DJ, Somerville MC, Wilson TH, Fowler IL, Rittmaster RS. Effect of dutasteride on the risk of prostate cancer. N Engl J Med362:1192–1202 [[PubMed]
- 12. Norman RW, Coakes KE, Wright AS, Rittmaster RS. 1993. Androgen metabolism in men receiving finasteride before prostatectomy. J Urol150:1736–1739 [[PubMed]
- 13. Rittmaster R, Hahn RG, Ray P, Shannon JB, Wurzel R. 2008. Effect of dutasteride on intraprostatic androgen levels in men with benign prostatic hyperplasia or prostate cancer. Urology72:808–812 [[PubMed]
- 14. Qoubaitary A, Swerdloff RS, Wang C. 2005. Advances in male hormone substitution therapy. Expert Opin Pharmacother6:1493–1506 [[PubMed]
- 15. Kunelius P, Lukkarinen O, Hannuksela ML, Itkonen O, Tapanainen JS. 2002. The effects of transdermal dihydrotestosterone in the aging male: a prospective, randomized, double blind study. J Clin Endocrinol Metab87:1467–1472 [[PubMed]
- 16. Ly LP, Jimenez M, Zhuang TN, Celermajer DS, Conway AJ, Handelsman DJ. 2001. A double-blind, placebo-controlled, randomized clinical trial of transdermal dihydrotestosterone gel on muscular strength, mobility, and quality of life in older men with partial androgen deficiency. J Clin Endocrinol Metab86:4078–4088 [[PubMed]
- 17. Swerdloff RS, Wang C. 1998. Dihydrotestosterone: a rationale for its use as a non-aromatizable androgen replacement therapeutic agent. Baillieres Clin Endocrinol Metab12:501–506 [[PubMed]
- 18. Mohler JL, Gregory CW, Ford OH, 3rd, Kim D, Weaver CM, Petrusz P, Wilson EM, French FS. 2004. The androgen axis in recurrent prostate cancer. Clin Cancer Res10:440–448 [[PubMed]
- 19. Barry MJ, Fowler FJ, Jr, O'Leary MP, Bruskewitz RC, Holtgrewe HL, Mebust WK, Cockett AT. 1992. The American Urological Association symptom index for benign prostatic hyperplasia. The Measurement Committee of the American Urological Association. J Urol148:1549–1557 [[PubMed]
- 20. Roth MY, Page ST, Lin K, Anawalt BD, Matsumoto AM, Snyder CN, Marck BT, Bremner WJ, Amory JK. 2010. Dose-dependent increase in intratesticular testosterone by very low dose human chorionic gonadotropin in normal men with experimental gonadotropin deficiency. 2010 J Clin Endocrinol Metab95:3806–3813
- 21. Montgomery RB, Mostaghel EA, Vessella R, Hess DL, Kalhorn TF, Higano CS, True LD, Nelson PS. 2008. Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth. Cancer Res68:4447–4454
- 22. Mostaghel EA, Geng L, Holcomb I, Coleman IM, Lucas J, True LD, Nelson PS. Variability in the androgen response of prostate epithelium to 5alpha-reductase inhibition: implications for prostate cancer chemoprevention. Cancer Res70:1286–1295
- 23. Tusher VG, Tibshirani R, Chu G. 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA98:5116–5121
- 24. Wu JD, Lin DW, Page ST, Lundgren AD, True LD, Plymate SR. 2009. Oxidative DNA damage in the prostate may predispose men to a higher risk of prostate cancer. Transl Oncol2:39–45
- 25. Yuan JJ, Coplen DE, Petros JA, Figenshau RS, Ratliff TL, Smith DS, Catalona WJ. 1992. Effects of rectal examination, prostatic massage, ultrasonography and needle biopsy on serum prostate specific antigen levels. J Urol147:810–814 [[PubMed]
- 26. Amory JK, Watts NB, Easley KA, Sutton PR, Anawalt BD, Matsumoto AM, Bremner WJ, Tenover JL. 2004. Exogenous testosterone or testosterone with finasteride increases bone mineral density in older men with low serum testosterone. J Clin Endocrinol Metab89:503–510 [[PubMed]
- 27. Steidle C, Schwartz S, Jacoby K, Sebree T, Smith T, Bachand R. 2003. AA2500 testosterone gel normalizes androgen levels in aging males with improvements in body composition and sexual function. J Clin Endocrinol Metab88:2673–2681 [[PubMed]
- 28. Wang C, Dobs A, Matsumoto A, Spark R, Redmond G, Weber T, Iranmanesh A, Schwartz S, Christenson P, Hull L, Swerdloff R. 2004. Effects of long term transdermal administration of dihydrotestosterone (DHT) gel in older men. 12th International Congress of Endocrinology, Lisbon, Portugal, p OR119 [PubMed]
- 29. Schnabel PG, Bagchus W, Lass H, Thomsen T, Geurts TB. 2007. The effect of food composition on serum testosterone levels after oral administration of Andriol Testocaps. Clin Endocrinol (Oxf)66: 579–585
- 30. Legros JJ, Meuleman EJ, Elbers JM, Geurts TB, Kaspers MJ, Bouloux PM. 2009. Oral testosterone replacement in symptomatic late-onset hypogonadism: effects on rating scales and general safety in a randomized, placebo-controlled study. Eur J Endocrinol160:821–831 [[PubMed]
- 31. Mostaghel EA, Page ST, Lin DW, Fazli L, Coleman IM, True LD, Knudsen B, Hess DL, Nelson CC, Matsumoto AM, Bremner WJ, Gleave ME, Nelson PS. 2007. Intraprostatic androgens and androgen-regulated gene expression persist after testosterone suppression: therapeutic implications for castration-resistant prostate cancer. Cancer Res67:5033–5041 [[PubMed]
- 32. Marks LS, Mostaghel EA, Nelson PS. 2008. Prostate tissue androgens: history and current clinical relevance. Urology72:247–254 [[PubMed]
- 33. Luu-The V, Belanger A, Labrie F. 2008. Androgen biosynthetic pathways in the human prostate. Best Pract Res Clin Endocrinol Metab22:207–221 [[PubMed]
- 34. Calof OM, Singh AB, Lee ML, Kenny AM, Urban RJ, Tenover JL, Bhasin S. 2005. Adverse events associated with testosterone replacement in middle-aged and older men: a meta-analysis of randomized, placebo-controlled trials. J Gerontol A Biol Sci Med Sci60:1451–1457 [[PubMed]
- 35. Ellem SJ, Risbridger GP. 2009. The dual, opposing roles of estrogen in the prostate. Ann NY Acad Sci1155:174–186 [[PubMed]
- 36. Burnett-Bowie SA, McKay EA, Lee H, Leder BZ. 2009. Effects of aromatase inhibition on bone mineral density and bone turnover in older men with low testosterone levels. J Clin Endocrinol Metab94:4785–4792