Androgen-metabolizing Enzymes Research Articles

The association between missense polymorphisms in SRD5A2 and HSD3B1 and treatment failure with abiraterone for castration-resistant prostate cancer.

Missense polymorphism in HSD3B1, encoding 3β-hydroxysteroid dehydrogenase-1, was associated with outcome after abiraterone treatment. Other androgen-metabolizing enzymes may be involved in therapeutic effect in abiraterone. In this study, we investigated the significance of polymorphisms in genes involved in androgen and abiraterone metabolisms in prostate cancer patients treated with abiraterone. A total of 99 Japanese male castration-resistant prostate cancer patients treated with abiraterone between 2014 and 2018 were included. Genomic DNA was obtained from whole blood samples, and genotyping on SRD5A2 (rs523349), CYP17A1 (rs743572), CYP17A1 (rs2486758), and AKR1C3 (rs12529) was performed by PCR-based technique. Among the 99 patients, 32 (32.3%), 49 (49.5%), and 18 patients (18.2%) carried GG, GC, and CC alleles in SRD5A2, respectively. CC allele was associated with lower risk of treatment failure (hazard ratio, 0.43; 95% confidence interval, 0.20-0.87; P = 0.017) on multivariate analyses, compared with GG/GC alleles. In the combination model using HSD3B1 and SRD5A2 polymorphisms, compared with the combination of AA in HSD3B1 and GG/GC in SRD5A2, other combinations were associated with lower risk of treatment failure (hazard ratio, 0.34; 95% confidence interval, 0.17-0.62; P = 0.0003) on multivariate analyses. This study showed that SRD5A2 genetic variation was associated with the risk of treatment failure in abiraterone. Combinational use of genetic variation in HSD3B1 with SRD5A2 genetic variation augmented the ability of prognostic stratification.

Abstract B087: Role of GATA-4-Androgen-AR axis in prostate cancer from African American men

Abstract Background: African American (AA) men are 1.7 times more likely to develop and 2.4 times more likely to die of prostate cancer (PrCa) than their European American (EA) counterparts, however the underlying biology causing this disparity is unknown. Here, we show how epigenetic dysregulation of calcium signaling genes affect GATA-4, androgen receptor (AR), androgen metabolism and associate with clinical outcomes in AA men with PrCa. Methods: Using Illumina arrays we identified DNA methylation differences between 32 EA and AA PrCa patients. We used RNA-sequencing to discover transcriptomic changes potentially mediated by DNA methylation. We measured GATA-4 and AR protein expression in a tissue microarray (TMA) of 95 EA and 92 AA PrCa patients. We measured testosterone (T) and dihydrotestosterone (DHT) in serum of the same AA and EA PrCa patients. In vitro, we treated MDA PCa 2a (2a) and LaPC4 cells derived from AA and EA prostate tumors, respectively with a calcium chelator (BAPTA-AM) and T. We measured GATA-4 and AR protein expression in control and treated cells. Results: Our DNA methylation analysis showed that AA but not EA prostate tumors enriched for hypermethylated sites in calcium sensing genes was associated with worse disease-free time (21.6 vs 46.7 months, p<0.05). DNA hypermethylation of calcium signaling genes, which correlated with decreased transcript levels, can potentially raise intracellular calcium. Calcium levels regulate stability of proteins including AR and GATA-4. Therefore, we tested potential calcium mediated changes in GATA-4 and AR expression in vitro and in patient samples. In vitro, depleting calcium in the presence of T increased GATA-4 protein expression by 1.5 times and AR expression only in AA prostate tumor derived 2a cells. DNA hypermethylation of calcium sensing genes was associated with lower GATA-4 and AR expression in PrCa and adjacent non-tumor tissues of AA men. The role of GATA-4 in PrCa is unknown, but it is a known transcriptional regulator of androgen metabolizing enzymes (AME). AMEs are important for androgen metabolism that result in T and DHT production through the primary and alternative pathways. AME transcripts in the primary and alternative pathways were one of the top 5 transcriptionally dysregulated pathways in AA compared to EA prostate tumors. We found that T but not DHT is significantly lower (p<0.05) in serum samples from AA (3.64ng/ml) compared to EA (3.99ng/ml) PrCa patients. T and DHT bioavailability in prostate tumors affect AR expression and function. In our studies, AA adjacent non-tumor tissues had higher percent AR positive nuclei (p<0.05) compared to EA adjacent non-tumor tissues. Conclusion: Epigenetic alteration of calcium signaling can affect GATA-4 mediated changes in androgen metabolism and AR signaling that are well-established therapeutic targets in PrCa. This molecular alteration, unique to AA prostate tumors, can be used to rationalize specific androgen deprivation and AR-targeted therapies in these patients. Citation Format: Swathi Ramakrishnan, Xuan Peng, Eduardo Cortes Gomez, Kristopher Attwood, Li Yan, John Wilton, Gissou Azabdaftari, James Mohler, Jianmin Wang, Anna Woloszynska. Role of GATA-4-Androgen-AR axis in prostate cancer from African American men [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B087.

Missense Polymorphism in <i>SRD5A2</i> in Combination with <i>HSD3B1</i> Variation Is Associated with Outcome of Castration-Resistant Prostate Cancer Patients Treated with Abiraterone

Background: Missense polymorphism in HSD3B1, encoding 3β-hydroxysteroid dehydrogenase-1, was associated with outcome after abiraterone treatment. Other androgen-metabolizing enzymes may be involved in therapeutic effect in abiraterone. In this study, we investigated the significance of polymorphisms in genes involved in androgen and abiraterone metabolisms in prostate cancer patients treated with abiraterone. Methods: A total of 99 Japanese male castration-resistant prostate cancer patients treated with abiraterone between 2014 and 2018 were included. Genomic DNA was obtained from whole blood samples, and genotyping on SRD5A2 (rs523349), CYP17A1 (rs743572), CYP17A1 (rs2486758) and AKR1C3 (rs12529) was performed by PCR-based technique. Findings: Among the 99 patients, 32 (32.3%), 49 (49.5%), and 18 patients (18.2%) carried GG, GC, and CC alleles in SRD5A2, respectively. CC allele was associated with lower risk of treatment failure (hazard ratio, 0.43; 95% confidence interval, 0.20–0.87; P = 0.017) on multivariate analyses, compared with GG/GC alleles. In the combination model using HSD3B1 and SRD5A2 polymorphisms, compared with the combination of AA in HSD3B1 and GG/GC in SRD5A2, other combinations were associated with lower risk of treatment failure (hazard ratio, 0.34; 95% confidence interval, 0.17–0.62; P = 0.0003) on multivariate analyses. This study showed that SRD5A2 genetic variation was associated with the risk of treatment failure in abiraterone. Combinational use of genetic variation in HSD3B1 with SRD5A2 genetic variation augmented the ability of prognostic stratification. Funding Statement: This work was supported by a JSPS KAKENHI grant (17K11145) to M.S. Declaration of Interests: MS, SA, SN, OO, TH and ME received honoraria from Janssen Pharmaceutical. The other authors declare no conflict of interest. Ethics Approval Statement: This study was performed in accordance with the principles described in the Declaration of Helsinki and the Ethical Guidelines for Epidemiological Research enacted by the Japanese Government and approved by each institutional review board. Written informed consent was obtained from all patients.

SAT-LB062 Adrenal Sexual Dimorphism Is Abolished by Tissue-Targeted Deletion of the Androgen Receptor

Introduction: Sex differences in mouse and human tissues can affect cancer risks, cardiovascular, kidney and liver diseases. Mouse adrenals have several sexually dimorphic traits, but their role in physiology remain poorly understood. Differences include larger adrenals in females and earlier regression of the X-zone (the mouse fetal adrenal) in males. The X-zone regresses at puberty in males and at pregnancy in females. There are also clear sex-differences in expression of adrenal mRNA and proteins. These include higher female levels of Dax-1 (Nr0b1), a nuclear receptor critically involved in adrenal development/steroidogenesis, whereas male adrenals exhibit higher levels of the androgen-metabolizing enzyme, 5α-reductase (Srd5a2). Determining the factors that drive adrenal sexual dimorphism may provide insight into the potential impact of these differences on adrenal physiology. One potential regulator of adrenal sexual dimorphism is exposure to sex steroids, particularly androgens and estrogens. Herein, we test the hypothesis that androgens, through the adrenal androgen receptor (AR), are responsible for sexually dimorphic traits of mouse adrenal glands. This hypothesis was tested through specific disruption of adrenal cortex Ar expression. Methods: Adrenal targeted deletion of Ar was accomplished in male mice by crossing heterozygous aldosterone synthase-Cre mice to floxed Ar mice (mAdARKO). Mice were sacrificed at 25 weeks of age and comparisons were made to wild type male (mWT) and female (fWT) litter mates. Adrenals were weighed, processed for histology (H&E), immunofluorescence (IF) for AR, and whole adrenal mRNA levels analyzed using quantitative RTPCR. Results: mAdARKO mice decreased adrenal Ar mRNA by 90% compared to mWT mice (p < 0.01) and AR protein was undetectable in the adrenal cortex using IF. mAdARKO adrenals were larger than adrenals of mWT mice (1.6-fold increase in weight, p < 0.01) but comparable to that of fWT animals. Nr0b1 transcripts in mAdARKO mice increased by 8-fold compared to mWT (p < 0.01) and were not significantly different from fWT adrenals. Adrenal Sdr5a2 expression was significantly downregulated in mAdARKO (0.16-fold compared to mWT; p < 0.01) and analogous to the levels seen in fWT mice. Histological analysis revealed the retention of an X-zone in post-pubertal mAdARKO which was not present in age-matched mWT animals. Moreover, mRNA for the X-zone marker, 20α-HSD (Akr1c18), increased by 140,000-fold (p < 0.01) in mAdARKO adrenals compared to mWT, and was 20 times that seen in fWT mice. Conclusion: The current study suggests that deleting adrenal Ar is sufficient to cause male adrenal phenotypic sex-reversal including retention of the X-zone. Finally, adrenal sexual dimorphism highlights the necessity to include both sexes in mouse adrenal studies and the mAdARKO model provides a novel model to determine the impact of sex on adrenal biology. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Quantitative characterization of UDP-glucuronosyltransferase 2B17 in human liver and intestine and its role in testosterone first-pass metabolism

Protein abundance and activity of UGT2B17, a highly variable drug- and androgen-metabolizing enzyme, were quantified in microsomes, S9 fractions, and primary cells isolated from human liver and intestine by validated LC-MS/MS methods. UGT2B17 protein abundance showed >160-fold variation (mean ± SD, 1.7 ± 2.7 pmol/mg microsomal protein) in adult human liver microsomes (n = 26) and significant correlation (r2 = 0.77, p < 0.001) with testosterone glucuronide (TG) formation. Primary role of UGT2B17 in TG formation compared to UGT2B15 was confirmed by performing activity assays in UGT2B17 gene deletion samples and with a selective UGT2B17 inhibitor, imatinib. Human intestinal microsomes isolated from small intestine (n = 6) showed on average significantly higher protein abundance (7.4 ± 6.6 pmol/mg microsomal protein, p = 0.016) compared to liver microsomes, with an increasing trend towards distal segments of the gastrointestinal (GI) tract. Commercially available pooled microsomes and S9 fractions confirmed greater abundance and activity of UGT2B17 in intestinal fractions compared to liver fractions. To further investigate the quantitative role of UGT2B17 in testosterone metabolism in whole cell system, a targeted metabolomics study was performed in hepatocytes (n = 5) and enterocytes (n = 16). TG was the second most abundant metabolite after androstenedione in both cell systems. Reasonable correlation between UGT2B17 abundance and activity were observed in enterocytes (r2 = 0.69, p = 0.003), but not in hepatocytes. These observational and mechanistic data will be useful in developing physiologically-based pharmacokinetic (PBPK) models for predicting highly-variable first-pass metabolism of testosterone and other UGT2B17 substrates.

Hepatic Abundance and Activity of Androgen- and Drug-Metabolizing Enzyme UGT2B17 Are Associated with Genotype, Age, and Sex.

The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (∼10-fold) with ∼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.

Abstract 3537: Stem-cell based selective delivery of alpha keto reductases for therapeutic targeting of residual androgens in prostate cancer

Abstract The twice as high incidence and mortality among African Americans (AA) compared to Caucasian Americans and other ethnic minorities remains elusive. The de novo synthesis of androgens by prostate tumors provides a significant survival advantage leading to outgrowth of castration resistant tumors. The “intracrine” production of androgens in the tumor microenvironment is greatly influenced by the synthesis of androgen metabolizing enzymes (AMEs) and the level of circulating hormones. Currently, targeting de novo synthesis of androgens in tumor microenvironment is singularly the toughest challenge presented to oncologists. Our present research attempts to identify and exploit the key players among the AMEs for targeting the backdoor pathway of DHT synthesis in castration resistant prostate cancer (CRPC). Comparative analysis of gene expression of AMEs in microdissected cells versus the adjacent normal tissue revealed AKR1C4 to be the most promising candidate for targeting the backdoor pathway in DHT synthesis. Existing literature supports this view since Type 1 3a-HSD enzyme has been shown to readily oxidize testosterone to Δ4-androstene-3, 17-dione in a substrate dependent reaction. Our analysis using recombinant AKR1C4 induced cytotoxicity and apoptosis in androgen dependent PC cells in vitro. Next, using a lentivirus construct, we genetically engineered tumor-tropic adipose-derived stem cells (ASCs) to express and secreted AKR1C14 (mouse homolog of AKR1C4). The results showed that ASCs are capable of expressing the enzyme, which can catabolize DHT in vitro and induce apoptosis in androgen dependent prostate cancer cells as shown by DHT Elisa, psPSA reporter assays and cytotoxicity and survival assays. Our current research plan is to examine the efficacy of tumor-tropic AKR1C14-expressing ASCs in targeting and inhibiting prostate metastatic growth in an animal model system. Citation Format: Manish Ranjan, Zakaria Abd Elmageed, Hogyoung Kim, Amrita Datta, Nobel Bhasin, Steven E. Braun, Debasis Mondal, Asim B. Abdel-Mageed. Stem-cell based selective delivery of alpha keto reductases for therapeutic targeting of residual androgens in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2015-3537

Sex-specific differences in the effects of local androgen metabolism in the heart as an indicator for the risk of myocardial infarction

Aim Testosterone influences cardiovascular risk and disease in a sex-specific manner. The more potent androgen 5α-dihydrotestosterone (DHT) can be formed through conversion of testosterone by the enzyme 5α-reductase. We hypothesized that, because of the presence of DHT in coronary and myocardial tissues, a sexually dimorphic effect can be observed if differences exist in genetics or mRNA expression in androgen-metabolizing enzymes. Materials and methods mRNA levels of steroidogenic enzymes and the androgen receptor (AR) were investigated in human myocardial tissue samples. Subsequently, all participants in the baseline cohort of the Rotterdam Study (RSI) with successful genotyping and without prevalent myocardial infarction (MI, N=5199) were recruited to study the association between single nucleotide polymorphisms (SNPs) within SRD5A1, SRD5A2, and AKR1C3 and incident MI using Cox regression models. Significant results were replicated within the Atherosclerosis Risk in Communities cohort and the second cohort of the RSII. Results The expression of SRD5A1, AKR1C3, and AR was found in all myocardial samples, whereas HSD17B3 and SRD5A2 expression levels were low and undetectable, respectively. Myocardial SRD5A1 expression was higher in women than in men. Within SRD5A1, SNP rs248805G>A was significantly associated with incident MI in western European women (hazard ratio 1.49; 95% confidence interval 1.19-1.87). This SNP is tightly linked to the HinfI polymorphism in SRD5A1 (rs248793G>C), of which the minor allele has been associated with a higher DHT/T ratio. Conclusion Genetic variation in SRD5A1 is associated with an increased risk of MI in western European women, possibly because of the sex-specific potential of local androgen conversion and effect.

Androgen and androgen-metabolizing enzymes in metastasized lymph nodes of breast cancer

Androgen receptor and androgen metabolizing enzymes, 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5) and 5α-reductase1 (5α1), are frequently detected in primary tumor of breast cancer, but their status in metastatic lymph nodes has not been examined. The biological role of androgen in breast cancer and its metastatic process also remain unknown. In this study, we used immunohistochemistry to localize the expression of androgen receptor, 17βHSD5, and 5α1 in primary tumors and paired metastatic lymph nodes and correlated the findings with clinicopathologic factors of individual patients. Approximately 70% of primary tumors and paired metastatic lymph nodes expressed androgen receptor, with significant correlation between both lesions. However, 17βHSD5 and 5α1 immunoreactivity was decreased in metastatic lymph nodes. Alone or in tandem with androgen receptor, 5α1 was associated with significantly lower Ki-67 index, lower pathologic grade, and higher estrogen receptor positivity, but androgen receptor/5α1 double positivity in lymph nodes was associated with larger lymph node metastasis and higher TNM stage. In conclusion, androgen receptor immunoreactivity remained stable during the process of metastasis, whereas androgen-metabolizing enzymes decreased. Although results of our study and previous reports imply additional roles of androgen metabolism in the metastasis process, especially conversion by 5α1, there may be divergence between its effects on primary tumor and those in metastatic lymph nodes.

Combination effects of dietary soy and methylselenocysteine in a mouse model of prostate cancer

High dietary intake of soy or selenium (Se) is associated with decreased risk of prostate cancer. Soy constituents and various chemical forms of Se have each been shown to downregulate expression of the androgen receptor (AR) and AR-regulated genes in the prostate. We hypothesized that downregulation of AR and AR-regulated genes by the combination of these dietary components would inhibit tumorigenesis in the TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse. Male mice were exposed from conception to stock diets high or low in soy, with or without a supplement of Se-methylseleno-L-cysteine (MSC) in a 2 × 2 factorial design. Mice were sacrificed at 18 weeks. Prostate histopathology, urogenital tract (UGT) weight, hepatic activity of androgen-metabolizing enzymes, and expression of AR, AR-regulated, and AR-associated FOX family genes, in the dorsolateral prostate were examined. High soy intake decreased activity of hepatic aromatase and 5α-reductase, expression of AR, AR-regulated genes, FOXA1, UGT weight, and tumor progression, and upregulated protective FOXO3. Supplemental MSC upregulated AKR1C14, which reduces 5α-dihydrotestosterone. Soy is an effective pleiotropic dietary agent for prevention of prostate cancer. The finding of effects of soy on FOX family gene expression in animals is novel. Combination effects of supplemental MSC may depend upon the soy content of the basal diet to which it is added.

Müllerian Inhibiting Substance Recruits ALK3 to Regulate Leydig Cell Differentiation

Müllerian inhibiting substance (MIS) not only induces Müllerian duct regression during male sexual differentiation but also modulates Leydig cell steroidogenic capacity and differentiation. MIS actions are mediated through a complex of homologous receptors: a type II ligand-binding receptor [MIS type II receptor (MISRII)] and a tissue-specific type I receptor that initiates downstream signaling. The putative MIS type I receptors responsible for Müllerian duct regression are activin A type II receptor, type I [Acvr1/activin receptor-like kinase 2 (ALK2)], ALK3, and ALK6, but the one recruited by MIS in Leydig cells is unknown. To identify whether ALK3 is the specific type I receptor partner for MISRII in Leydig cells, we generated Leydig cell-specific ALK3 conditional knockout mice using a Cre-lox system and compared gene expression and steroidogenic capacity in Leydig cells of ALK3(fx/fx)Cyp17(cre+) and control mice (ALK3(fx/fx)Cyp17(cre-) or ALK3(fx/wt)Cyp17(cre-) littermates). We found reduced mRNA expression of the genes encoding P450c17, StAR, and two enzymes (17βHSD-III and 3βHSD-VI) that are expressed in differentiated adult Leydig cells and increased expression of androgen-metabolizing enzymes (3α-HSD and SRD5A2) and proliferating cell nuclear antigen (PCNA) in Leydig cells of ALK3(fx/fx)Cyp17(cre+) mice. Despite down-regulation of steroidogenic capacity in ALK3(fx/fx)Cyp17(cre+) mice, the loss of MIS signaling also stimulates Leydig cell proliferation such that plasma testosterone and androstenedione concentrations are comparable to that of control mice. Collectively, these results indicate that the phenotype in ALK3 conditional knockout mice is similar to that of the MIS-knockout mice, confirming that ALK3 is the primary type I receptor recruited by the MIS-MISRII complex during Leydig cell differentiation.

Analysis of Polymorphisms in the SRD5A2 Gene and Semen Parameters in Estonian Men

Spermatogenesis is an androgen-dependent process and polymorphisms in genes encoding androgen-metabolizing enzymes may be associated with impaired male fertility. The enzyme steroid 5α-reductase converts testosterone into dihydrotestosterone. We analyzed genotype frequencies of 5 single-nucleotide polymorphisms (SNPs 1-5) (rs632148, rs523349, rs2300701, rs2268797, and rs12470143) in the steroid 5α-reductase type 2 gene (SRD5A2) in 132 azoospermic or oligozoospermic and 211 normozoospermic men. We found no association between investigated genotypes and the occurrence of male infertility. Linear regression analysis revealed a significant correlation between certain alleles of SNP1 and SNP5 and testicular volume among control men. Normozoospermic men carrying the minor allele of all but SNP5 polymorphism exhibited a significantly higher proportion of progressively motile spermatozoa, compared with major homozygotes. However, SRD5A2 genotypes did not influence sperm concentration, serum testosterone, or follicle-stimulating hormone levels in controls. Our results suggest that polymorphisms examined in SRD5A2 exhibit no adverse effect on semen parameters in Estonian men.

Genetic aspects of epitestosterone formation and androgen disposition: influence of polymorphisms in CYP17 and UGT2B enzymes

Testosterone is a commonly abused androgen in sports and in the gym culture of the society. Its abuse is conventionally disclosed by urinary assay of the testosterone/epitestosterone (T/E) glucuronide ratio, which should not exceed 4. A noteworthy number of athletes, however, have higher natural ratios than 4, most likely because of decreased excretion of epitestosterone glucuronide. Falsely positive doping test results are of great concern for the legal rights of the sportsman. Our objective was to study the genetic aspects of epitestosterone formation, and to elucidate the impact of genetic variation in androgen-metabolizing enzymes. Urine from different study populations was analysed for androgen glucuronides by gas chromatography-mass spectrometry. All men were genotyped for the uridine diphospho-glucuronosyltransferase (UGT) 2B17 deletion polymorphism and single nucleotide polymorphisms in the cytochrome P-450c17alpha (CYP17), UGT2B15 and UGT2B7 genes. Expression of UGT2B15 mRNA in human liver samples was analysed using real-time PCR. A T>C (A1>A2) promoter polymorphism in the CYP17 gene was associated with the urinary glucuronide levels of epitestosterone and its putative precursor androstene-3beta, 17alpha-diol, resulting in 64% higher T/E ratios in A1/A1 homozygotes. Individuals devoid of UGT2B17 had significantly higher UGT2B15 mRNA levels in liver than individuals carrying two functional UGT2B17 alleles. The CYP17 promoter polymorphism may partly explain high natural (>4) T/E ratios. Our data indicate that 5-androstene-3beta, 17alpha-diol is an important precursor of epitestosterone and that CYP17 is involved in its production. In addition, we found that lack of the UGT2B17 enzyme may be compensated for by increase in UGT2B15 transcription.

Topography and function of androgen-metabolizing enzymes in the central nervous system

The present review describes concisely the topography and function of the three androgen-metabolizing enzymes, namely aromatase, 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase, in the central nervous system (CNS). Aromatase, estrogen synthetase, is the key enzyme for converting androgens to estrogens. Aromatase is indispensable for the sexual differentiation of the brain and the enzyme activity and expression of aromatase are high during the critical period of neural development, which extends from the late embryonal to the early neonatal period in rodents. Aromatase is expressed in neurons within specific hypothalamic and limbic regions. The locations of aromatase-immunoreactive neurons are divided into three groups according to the period of enzyme expression. Steroid 5alpha-reductase converts a number of steroids with a C3 ketone group and a C4-C5 double bond (delta4; androgens, progestins and glucocorticoids) to their 5alpha-reduced metabolites. Two isoforms of 5alpha-reductase are found and type 1 is predominant in neural tissues. The enzyme activity of 5alpha-reductase is found widely in the CNS and is high in white matter regions. The enzyme expression of 5alpha-reductase peaks during the late embryonic period. 3alpha-Hydroxysteroid dehydrogenase is the oxidoreductase that interconverts 3-ketosteroids to 3alpha-hydroxysteroids. Four isozymes have been found in humans and only one type has been found in rats. The enzyme converts 5alpha-reduced steroids (e.g. 5alpha-dihydroprogesterone) to tetrahydrosteroids (e.g. 3alpha,5alpha-tetrahydroprogesterone). The latter steroid is a potent stimulator of the GABA(A) receptor. The activity of 3alpha-hydroxysteroid dehydrogenase is high during the first 1-2 postnatal weeks, decreases with development and this enzyme is highly expressed in astrocytes.

Lycopene reduced gene expression of steroid targets and inflammatory markers in normal rat prostate

Epidemiological evidence links consumption of lycopene, the red carotenoid of tomato, to reduced prostate cancer risk. We investigated the effect of lycopene in normal prostate tissue to gain insight into the mechanisms, by which lycopene can contribute to primary prostate cancer prevention. We supplemented young rats with 200 ppm lycopene for up to 8 wk, measured the uptake into individual prostate lobes, and analyzed lycopene-induced gene regulations in dorsal and lateral lobes after 8 wk of supplementation. Lycopene accumulated in all four prostate lobes over time, with all-trans lycopene being the predominant isoform. The lateral lobe showed a significantly higher total lycopene content than the other prostate lobes. Transcriptomics analysis revealed that lycopene treatment mildly but significantly reduced gene expression of androgen-metabolizing enzymes and androgen targets. Moreover, local expression of IGF-I was decreased in the lateral lobe. Lycopene also consistently reduced transcript levels of proinflammatory cytokines, immunoglobulins, and immunoglobulin receptors in the lateral lobe. This indicates that lycopene reduced inflammatory signals in the lateral prostate lobe. In summary, we show for the first time that lycopene reduced local prostatic androgen signaling, IGF-I expression, and basal inflammatory signals in normal prostate tissue. All of these mechanisms can contribute to the epidemiologically observed prostate cancer risk reduction by lycopene.

Effects of insulin-like growth factor I on steroidogenic enzyme expression levels in mouse leydig cells.

The role of IGF-I in Leydig cell maturation was studied by evaluation of: 1) steady state levels for nine mRNA species expressed specifically in Leydig cells of 35- and 50-d-old IGF-I-null mice and wild-type controls; 2) protein levels for 17 alpha-hydroxylase/C17-20 lyase, cholesterol side-chain cleavage, and type I 5 alpha-reductase (5 alpha R-1) in Leydig cells by immunocytochemistry; and 3) serum testosterone (T) and testicular interstitial fluid IGF-I levels. Expression levels of all mRNA species associated with T biosynthesis were lower in the absence of IGF-I stimulation. In contrast, androgen-metabolizing enzyme mRNA species had either normal (3 alpha-hydroxysteroid dehydrogenase) or higher expression (5 alpha R-1) levels in IGF-I-null mice (P < 0.05) relative to wild-type controls. None of the mRNA species studied changed developmentally in the mutant, whereas there were increases or decreases between d 35 and 50 in normal controls. Parallel trends were observed for average Leydig cell 5 alpha R-1 immunostaining intensity. T levels in mutants were initially higher during d 14-21, equivalent to normal on d 28, and then failed to increase pubertally, remaining at 30% of control levels (P < 0.01) in 90-d-old adult animals. In normal wild-type mice, interstitial fluid and plasma IGF-I levels were highest (P < 0.05) on d 24, indicating that the action of this growth factor on the testis peaks during pubertal development. These results show that in the absence of IGF-I, there is a failure of adult Leydig cells to mature, and that the reduced capacity for T production is caused by disproportionate expression of T biosynthetic and metabolizing enzymes.

Neurobehavioral effects of dietary soy phytoestrogens

Phytoestrogens, plant-derived nonsteroidal estrogens found in high abundance in most soy food products, have been studied for their potential beneficial effects against hormone-dependent cancers and age-related diseases. However, little is known about the influence of phytoestrogens on the brain or behavior. This brief review describes mainly our own studies in rodents that have examined the influence of dietary soy isoflavones on certain aspects of brain structure, learning, memory and anxiety along with the brain androgen-metabolizing enzyme, aromatase. These studies used a commercially available diet rich in phytoestrogens (Phyto-rich) vs. a custom diet relatively free of phytoestrogens (Phyto-free). The phytoestrogen content of each diet was determined by high-performance liquid chromatography analysis, circulating plasma phytoestrogen levels were quantified by gas chromatography mass spectroscopy and concentrations of phytoestrogens in specific brain regions were measured by time-resolved fluoroimmunoassay (TR-FIA). Our studies showed that brain aromatase levels were not significantly altered by phytoestrogen diet treatments in perinatal, maternal or adult rats. However, volumes of the sexually dimorphic nucleus of the preoptic area (SDN-POA) were significantly affected by the Phyto-free diet treatment in male rats during adulthood, where SDN-POA volumes were smaller compared to Phyto-rich male values. Additionally, the Phyto-rich diet fed to adult male and female rats produced anxiolytic effects as assessed in the elevated plus maze vs. Phyto-free fed animals. Finally, when learning and memory parameters were examined in a radial arm maze testing visual–spatial memory (VSM), the diet treatments significantly changed the typical sexually dimorphic pattern of VSM. Specifically, adult Phyto-rich fed females outperformed Phyto-free fed females, while in males on the same diets, the opposite pattern of maze performance was observed. When female vs. male performance was compared, Phyto-rich females executed the VSM task in a manner similar to that of Phyto-free fed males, while Phyto-free fed female's VSM was comparable to Phyto-rich males. These results indicate that consumption of dietary phytoestrogens resulting in very high plasma isoflavone levels (in many cases over a relatively short interval of consumption in adulthood) can significantly alter sexually dimorphic brain regions, anxiety, learning and memory. The findings of these studies identify the biological actions of phytoestrogens, specifically isoflavones and their metabolites, found in animal soy-containing diets on brain and behavior and implicate the importance of phytoestrogens given the recognized significance of estrogens in brain and neural disorders, such as Alzheimer's disease, especially in women.

5α‐reductase isozymes and aromatase are differentially expressed and active in the androgen‐independent human prostate cancer cell lines DU145 and PC3

BACKGROUND The presence and possible role of androgen-metabolizing enzymes in androgen-independent prostate carcinoma (CaP) are still unclear. The aim of the present study was: 1) to evaluate the pattern of androgen metabolism (relative production of 5α-reduced vs. 17-keto androgens); and 2) to analyze whether one or both the two known 5α-reductase isoforms (5α-R1 and 5α-R2) and the aromatase (Aro) are expressed and active in this pathology. METHODS Two different cell lines (DU145 and PC3) were used as a model of androgen-independent human CaP. In these cells, the expression of the two 5α-Rs and of Aro were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot, using specific sets of oligoprimers and of [32P]-labeled oligoprobes; the enzymatic activities of 5α-R and of Aro were evaluated by radioenzymatic methods. The pH optimum for the activity of the two 5α-Rs was assessed in cell homogenates at different pH (from 3.5–8), using substrate concentrations similar either to 5α-R1 or to 5α-R2 Kms. RESULTS The two CaP cell lines DU145 and PC3, although unresponsive to androgens, possess the enzymatic machinery involved in the metabolism of this class of hormonal steroids: 5α-Rs, which allow their transformation into 5α-reduced steroids (5α-dihydrotestosterone, DHT, and 5α-androstandione, 5α-A), and 17β-hydroxysteroid-oxidoreductase (17β-HSD), which interconverts testosterone (T) and androstenedione (ADIONE); however, the two cell lines show differences in the rate of formation of these metabolites. Furthermore, two cell lines expressed the type 1 isoform of 5α-R, but only DU145 cells also possess 5α-R2. Aro is expressed and active in DU145 as well as in PC3 cells. CONCLUSIONS The present findings suggest that T might still be indirectly active in androgen-unresponsive CaP through its local conversion into estrogens by the action of Aro; the biological role played by the two 5α-Rs in androgen-independent CaP deserves further investigation. Prostate 41:224–232, 1999. © 1999 Wiley-Liss, Inc.

5alpha-reductase isozymes and aromatase are differentially expressed and active in the androgen-independent human prostate cancer cell lines DU145 and PC3.

The presence and possible role of androgen-metabolizing enzymes in androgen-independent prostate carcinoma (CaP) are still unclear. The aim of the present study was: 1) to evaluate the pattern of androgen metabolism (relative production of 5alpha-reduced vs. 17-keto androgens); and 2) to analyze whether one or both the two known 5alpha-reductase isoforms (5alpha-R1 and 5alpha-R2) and the aromatase (Aro) are expressed and active in this pathology. Two different cell lines (DU145 and PC3) were used as a model of androgen-independent human CaP. In these cells, the expression of the two 5alpha-Rs and of Aro were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot, using specific sets of oligoprimers and of [(32)P]-labeled oligoprobes; the enzymatic activities of 5alpha-R and of Aro were evaluated by radioenzymatic methods. The pH optimum for the activity of the two 5alpha-Rs was assessed in cell homogenates at different pH (from 3.5-8), using substrate concentrations similar either to 5alpha-R1 or to 5alpha-R2 Kms. The two CaP cell lines DU145 and PC3, although unresponsive to androgens, possess the enzymatic machinery involved in the metabolism of this class of hormonal steroids: 5alpha-Rs, which allow their transformation into 5alpha-reduced steroids (5alpha-dihydrotestosterone, DHT, and 5alpha-androstandione, 5alpha-A), and 17beta-hydroxysteroid-oxidoreductase (17beta-HSD), which interconverts testosterone (T) and androstenedione (ADIONE); however, the two cell lines show differences in the rate of formation of these metabolites. Furthermore, two cell lines expressed the type 1 isoform of 5alpha-R, but only DU145 cells also possess 5alpha-R2. Aro is expressed and active in DU145 as well as in PC3 cells. The present findings suggest that T might still be indirectly active in androgen-unresponsive CaP through its local conversion into estrogens by the action of Aro; the biological role played by the two 5alpha-Rs in androgen-independent CaP deserves further investigation.

Androgen‐metabolizing enzymes show region‐specific changes across the breeding season in the brain of a wild songbird

The Lapland longspur (Calcarius lapponicus) is an arctic-breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate-zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen-metabolizing enzymes in the longspur brain explain hormone-behavior patterns in this species. We measured the activities of aromatase, 5α-reductase and 5β-reductase in free-living longspur males. Aromatase and 5α-reductase convert T into the active steroids 17β-estradiol (E2) and 5α-dihydrotestosterone (5α-DHT), respectively. 5β-Reductase deactivates T via conversion to 5β-DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5β-Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region-specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5β-reductase do not explain the effects of plasma T on aggressive behavior. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 176–188, 1999