5α-reduction Of Testosterone Research Articles

Structural basis for the catalysis and inhibition of human steroid 5α‐reductase 2

Human steroid 5α-reductases (SRD5As) are membrane-embedded NADPH-dependent steroid and lipid reductases with diverse physiological and pathological roles. Among them, SRD5A2 is a critical enzyme in steroid metabolism that catalyzes testosterone to dihydrotestosterone. Mutations on its gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride as SRD5A2 inhibitors are widely used anti-androgen drugs for benign prostate hyperplasia and androgenic alopecia, which have recently been indicated in the treatment of COVID-19. The molecular mechanisms underlying enzyme catalysis and inhibition had remained elusive for SRD5A2 and other eukaryotic integral membrane steroid reductases due to a lack of structural information. We recently solved a crystal structure of human SRD5A2 at 2.8 Å with finasteride using the lipid mesophase (or LCP) crystallization method. The structure reveals a unique structural topology of 7-transmembrane helices and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride, which binds in a largely enclosed binding cavity inside the membrane. To our knowledge, similar NADPH adducts have not been reported previously. Structural analysis together with computational and mutagenesis studies reveals molecular mechanisms for the 5α-reduction of testosterone and the semi-irreversible finasteride inhibition involving residues E57 and Y91 of SRD5A2. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region of SRD5A2 including three cytosolic loops regulating the NADPH/NADP+ exchange and their entry into the transmembrane region of SRD5A2. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. In summary, our results offer critical and unprecedented structural insights into the function of integral membrane steroid reductases and will facilitate drug development.

Transcriptional regulation of myotrophic actions by testosterone and trenbolone on androgen-responsive muscle

Androgens regulate body composition and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for aromatase) induces myotrophic effects in skeletal muscle without causing prostate enlargement, which is in contrast to the known prostate enlarging effects of testosterone. These previous results suggest that the 5α-reduction of testosterone is not required for myotrophic action. We now report differential gene expression in response to testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus (LABC) muscle complex of the adult rat after 6weeks of orchiectomy (ORX), using real time PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1] and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as shown by increases in LABC mass and in protein expression of embryonic myosin by western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these findings identify specific mechanisms by which testosterone and trenbolone may regulate skeletal muscle maintenance and growth.

Testosterone metabolites mediate its effects on myocardial damage induced by ischemia/reperfusion in male Wistar rats

The role of testosterone in cardiovascular (CV) homeostasis is in controversy, and the exact effects of testosterone on the cardiovascular system remain poorly understood. Testosterone is metabolized by aromatase into 17β-estradiol and by 5α-reductase into dihydrotestosterone (DHT). Thus, identification of these metabolites in the heart may help to explain the controversy regarding the cardiovascular effects of testosterone. We analyzed the expression patterns of these testosterone-metabolizing enzymes and assessed the effect of its enzymatic activity inhibition on ischemia (40min)/reperfusion (4h, I/R) via the left anterior descendent coronary artery in intact and gonadectomized male rats. Myocardial damage was measured as percentage of infarcted area vs. area at risk. Aromatase and 5α-reductase protein expression was found in the left ventricle of intact and orchidectomized rats. Exogenous testosterone had no effect on I/R induced myocardial damage in intact male rats, meanwhile exogenous testosterone protects against I/R injury in orchidectomized rats. However, enzymatic inhibition of aromatase increased myocardial damage in the presence of testosterone, while enzymatic inhibition of 5α-reductase significantly decreased the level of myocardial damage. Our results also showed that sub-chronic inhibition of 5α-reductase resulted in myocardial protection in both groups. Furthermore, in orchidectomized and intact male rats IV treatment with DHT induces a significant increase in the myocardial damage induced by I/R. Thus, the effect of testosterone on cardiovascular pathophysiology could be related, at least in part to changes in the balance of testosterone 5α-reduction and aromatization.

Potential Prostate Cancer Drug Target: Bioactivation of Androstanediol by Conversion to Dihydrotestosterone

High-affinity binding of dihydrotestosterone (DHT) to the androgen receptor (AR) initiates androgen-dependent gene activation, required for normal male sex development in utero, and contributes to prostate cancer development and progression in men. Under normal physiologic conditions, DHT is synthesized predominantly by 5α-reduction of testosterone, the major circulating androgen produced by the testis. During androgen deprivation therapy, intratumoral androgen production is sufficient for AR activation and prostate cancer growth, even though circulating testicular androgen levels are low. Recent studies indicate that the metabolism of 5α-androstane-3α, 17β-diol by 17β-hydroxysteroid dehydrogenase 6 in benign prostate and prostate cancer cells is a major biosynthetic pathway for intratumoral synthesis of DHT, which binds AR and initiates transactivation to promote prostate cancer growth during androgen deprivation therapy. Drugs that target the so-called backdoor pathway of DHT synthesis provide an opportunity to enhance clinical response to luteinizing-hormone-releasing hormone (LHRH) agonists or antagonists, AR antagonists, and inhibitors of 5α-reductase enzymes (finasteride or dutasteride), and other steroid metabolism enzyme inhibitors (ketoconazole or the recently available abiraterone acetate).

Dihydrotestosterone synthesis bypasses testosterone to drive castration-resistant prostate cancer

In the majority of cases, advanced prostate cancer responds initially to androgen deprivation therapy by depletion of gonadal testosterone. The response is usually transient, and metastatic tumors almost invariably eventually progress as castration-resistant prostate cancer (CRPC). The development of CRPC is dependent upon the intratumoral generation of the potent androgen, dihydrotestosterone (DHT), from adrenal precursor steroids. Progression to CRPC is accompanied by increased expression of steroid-5α-reductase isoenzyme-1 (SRD5A1) over SRD5A2, which is otherwise the dominant isoenzyme expressed in the prostate. DHT synthesis in CRPC is widely assumed to require 5α-reduction of testosterone as the obligate precursor, and the increased expression of SRD5A1 is thought to reflect its role in converting testosterone to DHT. Here, we show that the dominant route of DHT synthesis in CRPC bypasses testosterone, and instead requires 5α-reduction of androstenedione by SRD5A1 to 5α-androstanedione, which is then converted to DHT. This alternative pathway is operational and dominant in both human CRPC cell lines and fresh tissue obtained from human tumor metastases. Moreover, CRPC growth in mouse xenograft models is dependent upon this pathway, as well as expression of SRD5A1. These findings reframe the fundamental metabolic pathway that drives CRPC progression, and shed light on the development of new therapeutic strategies.

Assessment of steroidogenic pathways that do not require testosterone as intermediate.

Traditional literature and textbooks generally describe that estradiol (E2) and dihydrotestosterone (DHT) are synthesized from the aromatization and 5α-reduction of testosterone (T), respectively, following a pathway in which T is an essential intermediate (Tpath). This pathway implies that the steps of aromatization and 5α-reduction follow the reaction of the androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) that catalyzes the conversion of 4-androstenedione (4-dione) into T, and that estrogenic 17β-HSDs are not required. Contrary to this belief, the cloning of many estrogen-specific 17β-HSDs and the observation of higher affinity of aromatase and 5α-reductase for 4-dione than T are strongly in favor of biosynthetic pathways in which the steps catalyzed by aromatase and 5α-reductase precede that catalyzed by 17β-HSDs. Such pathways do not require T as an intermediate, as demonstrated by experiments using [14C]-labeled DHEA and 4-dione as substrates and incubation with SZ95 sebaceous gland, DU-145 prostate cancer and JEG-3 choriocarcinoma cell lines cultured in the presence of inhibitors of 5α-reductase and aromatase. A review of early literature about patients with testicular 17β-HSD deficiency and of steroid metabolism appears to confirm the physiological functionality of the E2 and DHT biosynthetic pathway not requiring T as intermediate (noTpath).

Dutasteride affects progesterone metabolizing enzyme activity/expression in human breast cell lines resulting in suppression of cell proliferation and detachment

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5α-reductase and lower 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5α-reduced progesterone metabolites such as 5α-dihydroprogesterone (5αP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3α-ol-20-one (3αHP) and 4-pregnen-20α-ol-3-one (20αHP) have the opposite (anti-cancer-like) effects. The 5α-reductase inhibitor dutasteride has been shown to inhibit 5α-reduction of testosterone to 5α-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5α-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10 −6 M) inhibited progesterone conversion to 5α-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5α-reductase activity and to altered levels of expression of 5α-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5α-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5αP, providing proof-of-principle that the effects were due not to progesterone but to the 5α-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5α-reductase inhibitor and that such inhibition may be beneficial in breast cancer.

5α-reduced C 21 steroids are substrates for human cytochrome P450c17

The 5α-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5α-reduced C 19 steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5α-reduced C 21 steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C 21 steroids to C 19 steroids via its 17α-hydroxylase and 17,20-lyase activities. hCYP17 17α-hydroxylates 5α-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17α-hydroxylates 5α-pregnan-3α-ol-20-one and the 5α-pregnan-3α,17α-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5α-pregnan-3α,17α-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17α-hydroxypregnenolone and cytochrome b 5 stimulates androsterone formation only 3-fold. Both 5α-pregnan-3α-ol-20-one and 5α-pregnan-3α,17α-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5α-reduced, 3α-hydroxy-C 21 steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5α-pregnan-3α,17α-diol-20-one to androsterone by CYP17 explains how, when 5α-reductases are present, the testis can produce C 19 steroids androsterone and androstanediol from 17α-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.

Androgen, estrogen and progesterone receptor expression in the human uterus during the menstrual cycle

Cyclic changes in steroid receptor expression in endometrial cells are considered a reflection of its differential functions. Besides estrogen and progestogens, androgens have also been suggested to affect the biological function of the female reproductive tract. We investigated the distribution and intensity of immuno-cytochemical estrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) staining in the various cell types of human endometrium and myometrium during the different menstrual cycle phases in 30 paraffin-embedded sections. AR expression in endometrial stromal cells decreased gradually from early proliferative till mid secretory phase. In the late secretory phase, AR expression in all cell types distinguished. Staining of epithelial cells was moderate. The disappearance of AR expression before cyclic separation of endometrial tissue may be causally related or just an epiphenomenon. Due to local competition for 5α-reduction of testosterone and the excess of progesterone in the secretory phase, the level of dihydrotestosterone (DHT) will be diminished. Hypothetically, if AR synthesis in endometrium would be DHT-dependent, it would explain the lack of AR expression in the late secretory phase.

Ontogeny of steroid metabolizing enzymes in rat oligodendrocytes

Rat oligodendroglial cells were isolated from newborn and developing brains and used immediately after, for quantification of steroid metabolizing activities. Oligodendrocytes (Ol) and their progenitor cells were incubated with [ 14C] testosterone, [ 14C] progesterone, [ 14C] pregnenolone or [ 14C] dehydroepiandrosterone (DHEA). Oligodendrocytes and their progenitor cells expressed different steroid metabolizing enzymes. The main activities were 5α reduction of testosterone and progesterone and 3β hydroxy steroid dehydrogenase–isomerase which transformed pregnenolone into progesterone and DHEA into Δ4 androstenedione. 5α reductase activity increased in male and female rats in parallel with testosterone or progesterone. Contrary to this, 3β hydroxysteroid dehydrogenase-isomerase activity was found to be high in the young rat and to decrease when testosterone and progesterone plasma concentration increased.

IN VITRO AND IN VIVO EFFECTS OF VITAMIN D (CALCITRIOL) ADMINISTRATION ON THE NORMAL NEONATAL AND PREPUBERTAL PROSTATE

Although many studies have investigated the role of calcitriol in the growth regulation of normal and cancerous prostates, little is known about its role in early prostatic development. The interactions between calcitriol and androgens, and their actions on the normal prostate have similarly been proposed but not evaluated. Previous studies in our laboratory have revealed that in utero administration of 1,25-dihydroxycholecalciferol or calcitriol can influence prostate growth and differentiation throughout the life of the animal. We further examined the influence of calcitriol on the normal prostate in vitro and in vivo by focusing on early stages of prostatic development. The effects of calcitriol on the growth of the normal human neonatal prostatic epithelial cell line 267B-1 was determined in the presence and absence of dihydrotestosterone (DHT). We also examined the effect of calcitriol on the growth of maturing rat prostates in vivo. Before puberty 4 groups of rats 27 to 38 days old were treated with vehicle (controls) or calcitriol. When the rats reached adulthood at age 100 to 110 days a control group and a calcitriol group were sacrificed. The other 2 groups were given exogenous DHT for 5 days. For the animals to become adapted to DHT they were kept alive for 1 additional week and sacrificed at about age 120 days. In vitro studies demonstrated that 267B-1 cells possess vitamin D receptors and their growth was inhibited by calcitriol with an IC50 (concentration resulting in 50% cytotoxicity) of 30 microM. Proliferation of these neonatal prostate cells was also inhibited by calcitriol in the presence of DHT in vitro. Our studies indicate that, although calcitriol was administered at the apparently important prepubertal period, there was no difference in prostatic weights between the control and calcitriol treated rats. Exogenous administration of DHT decreased prostatic weight of control rats but in rats treated with 1,25-dihydroxycholecalciferol DHT did not have any significant effect on prostatic weight. No statistically significant differences were observed in seminal vesicle weights among the different groups of animals. Analysis of the nuclear matrix protein composition of the prostatic tissue showed differences in composition between the DHT, and calcitriol and DHT treated rat prostates. These studies indicate that calcitriol administered just before puberty does not significantly influence prostatic growth in the presence of endogenous or exogenous administered DHT, and has an inhibitory effect on neonatal prostate epithelial cell growth in vitro in the presence and absence of DHT. Treatment with calcitriol and DHT also results in differences in nuclear matrix protein composition. Prepubertal administration of calcitriol may inhibit the exogenous DHT action in decreasing epithelial growth and stimulating stromal proliferation in the rat prostate.

Effects of the anti-androgen finasteride on 5 alpha-reduction of androgens in the presence of progesterone in human gingival fibroblasts: modulatory actions of the alkaline phosphatase inhibitor levamisole.

Oestrogens and androgens stimulate collagen matrix synthesis, while progesterone is a competitive inhibitor for the 5 alpha-reduction of testosterone to 5 alpha-dihydrotestosterone (DHT). The anti-androgen finasteride is a specific inhibitor of the 5 alpha-reductase type 2 isoenzyme, associated with anabolic functions. The aim of this investigation is to study the effects of progesterone and finasteride on 5 alpha-reduction of androgen substrates by human gingival fibroblasts. Monolayer cultures of human gingival fibroblasts (HGF) of the 4th 9th passage were established in Eagle's minimum essential medium (MEM). Duplicate incubations were performed with 14C-testosterone/14C-4-androstenedione as substrates and progesterone (P) or finasteride (F), at concentrations of 0.5, 1, 3 and 5 microg/ml, alone and in combination, for 24 h. Similarly, the effects of the alkaline phosphatase inhibitor levamisole (L, 30 microg/ml) and P were studied. Steroid metabolites were analysed and quantified, using a radioisotope scanner. Progesterone inhibited DHT synthesis in HGF from 14C-testosterone by 24-62% (n = 8; p < 0.01). Finasteride caused 59 82% inhibition (n=8;p<0.01). The combination of P+F showed a similar degree of inhibition (68-78%) of DHT synthesis to that of F alone (n = 8; p<0.01). There was 35-56% stimulation of 17beta-HSD (hydroxysteroid dehydrogenase) activity by P, F and P + F (n = 8; p < 0.01). When 14C-4-androstenedione was used as substrate there was 47% inhibition of 5 alpha-reductase activity at higher concentrations of P and 63 and 44% stimulation at 0.5 and 1 microg/ml (n = 8;p < 0.01). F and P + F caused 40-67% inhibition of this activity. P, F and P + F caused 2-2.7-fold stimulation of 17beta-HSD activity in response to all concentrations studied. L inhibited DHT synthesis from both substrates by 36-38%, with further inhibition of 55-70% (n = 4; p < 0.01), with P; this is suggestive of ligand-independent alkaline phosphatase activity mediated by 5 alpha-reductase. Inhibition of 5 alpha-reductase activity by finasteride in gingival fibroblasts is suggestive of target tissue anabolic functions in gingivae and competitive inhibition by progesterone, is suggestive of regulation of hormone mediated tissue responses during repair.

Identification and partial characterization of two steroid 5 alpha-reductase isozymes in the canine prostate.

The dog has been extensively used as an in vivo model to test the pharmacokinetics and effects on pathological prostatic growth of 5 alpha-reductase inhibitors. However, no information is available on the existence or characteristics of canine 5 alpha-reductase isozymes. The 5 alpha-reduction of testosterone is analyzed in dog prostatic homogenates. Three human-specific inhibitors are tested for their activity against dog 5 alpha-reductase. Two pH optima of 5 alpha-reductase activity in dog prostatic homogenates are described, comparable to the pH optima of rat and human 5 alpha-reductase isozymes. Kinetic analysis of 5 alpha-reductase enzymatic activity at pH 7.0 revealed isozymes with a low apparent affinity constant (Km = 2.67 nM) and a high apparent affinity constant (Km = 1.23 microM). These apparent affinity constants compare favorably to the human and rat isozymes types II and I, respectively. The human type II inhibitor finasteride selectively inhibited the low Km isozyme, whereas the human type I inhibitor MK386 preferentially inhibited the high Km isozyme. The human type I inhibitor LY306089 was nonspecific for the dog isozymes. We postulate that the high and low Km isozymes described here represent the dog type I and type II 5 alpha-reductase isozymes, respectively.

Kinetic analysis of rat steroid 5α-reductase activity in prostate and epididymis homogenates at neutral pH: Evidence for type I activity in epididymis

Immunocytochemical studies and mRNA measurements have shown that the rat epididymis—like the rat prostate—expresses both rat steroid 5α-reductase isozymes, i.e. type I and II. So far, enzyme activity measurements in rat epididymis homogenates, however, do not support the presence of type I 5α-reductase activity. Incubating homogenates of both tissues with a wide range of substrate concentrations, we were able to detect activity of both isozymes in rat prostate and epididymis tissues at neutral pH. In rat prostate the amount of type I activity, as measured by the V max at pH 7.0, exceeds that of type II 5α-reductase 50-fold. The efficiency ratio, V max K m , of the type I isozyme accounts for 25% of the total in vivo potential activity. A possible anabolic role for the type I isozyme in rat prostate was thus surmised. In rat epididymis the V max of type I and type II 5α-reductase at pH 7.0 were similar. Comparison of the efficiency ratio V max K m of either isozyme in the rat epididymis, however, suggested that the type II isozyme would play the major role in the 5α-reduction of testosterone at physiological concentrations and at neutral pH. The specific localization of the isozymes should be considered to allow for correct quantification of their in vivo contribution to dihydrotestosterone formation.

Kinetic analysis of steroid 5α-reductase activity at neutral pH in benign prostatic hyperplastic tissue: Evidence for type I isozyme activity in the human prostate

In human benign prostatic hyperplastic (BPH) tissue homogenates 5α-reduction of testosterone was examined at neutral pH. As Lineweaver-Burk and Eadie-Scatchard plots of estimated initial velocities against a wide range of substrate concentrations of 2nM to 3.2 μM were non-linear, the existence of two 5α-reductase isozymes in this tissue was surmised. Indeed, enzyme parameters at pH 7.0 suggested the presence of two isozymes with affinity constants of 1995 and 11.8 nM, characteristic of the well established human steroid 5α-reductase isozymes type I and II, respectively. The physiological roles of these isozyme activities remain puzzling. The specific activities, V max, of these subtypes indicated an approx. 6-fold higher maximum velocity of type I than of type II 5α-reductase in the human hyperplastic prostate at this pH. In contrast, the efficiency ratios, V max K m , demonstrated that the type II isozyme had a nearly 27 times higher potential in vivo activity than the type I isozyme, and is therefore most probably quantitatively responsible for dihydrotestosterone formation at physiological testosterone levels in this tissue at neutral pH. This is the first full paper on type I 5α-reductase activity in human BPH tissue.

5α-Reductase activity in isolated and cultured neuronal and glial cells of the rat

The distribution of the 5α-reductase, the enzyme which converts testosterone into its ‘active’ metabolite dihydrotestosterone (DHT), has been studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation and in neurons and glial cells grown in cultures. Purity of cellular preparations was examined by electron and light microscopy. Purified neurons, astrocytes and oligodendrocytes, obtained from the brain of adult male rats, are all able to form DHT from testosterone and consequently possess a 5α-reductase activity. Among the 3 cell types studied, neurons appear to be more active than oligodendrocytes and astrocytes. Moreover, between the two population of glial cells, the oligodendrocytes seem to possess a slightly higher enzymatic activity than that present in the astrocytes. Neurons appeared more active in metabolizing testosterone than glial cells also in cell culture experiments. It is presently believed that the 5α-reduction of testosterone to DHT provides one of the mechanisms through which the hormone becomes effective in the CNS. This is supported by the present findings, which indicate that neurons are the cell population in which the 5α-reductase is more concentrated. However, the presence of a considerable 5α-reductase activity in glial cells indicates that also non-neuronal cells might participate in androgen-mediated events occurring in the brain.

The influence of steroidal and nonsteroidal estrogens on the 5α-reduction of testosterone by the ventral prostate of the rat

The 5α-reduction of testosterone to dihydrotestosterone (DHT) correlates with the androgen-mediated growth of the prostate under different experimental and clinical conditions. The aim was to study the regulation of the prostatic growth and enzyme activity by steroidal and nonsteroidal estrogens. Estrogens did not activate the androgen-dependent 5α-reductase activity in cultured prostate of the rat. The direct inhibition of the enzyme activity by estrogens at the concentrations achievable in the male is not probable either. However, early estrogenization of the male rats in utero (on Day 17 of pregnancy) with diethylstilbesterol (DES) resulted in a persistent decrease of the enzyme activity and growth of the prostate indicating a critical estrogen-sensitive period in the regulation of the ultimate enzyme activity. The similar DES-like inhibitory effect on the growth of the prostate was achieved by keeping animals from fertilization throughout the pregnancy until weaning on diet containing soy, rich in environmental estrogens. Zearalenone (Zeranol) and coumestrol, two nonsteroidal estrogens found in human and animal food mimicked estradiol action in culture, but they were not estrogenic or antiestrogenic when administered to normal adults.

Comparison of the nuclear 5α-reduction of testosterone and androstenedione in human prostatic carcinoma and benign prostatic hyperplasia

The nuclear conversion of testosterone (T) to dihydrotestosterone (DHT) and androstenedione (Δ 4A) to androstanedione (5α-Adione) was compared in the separated stromal and epithelial fractions of hyperplastic ( n = 6) and malignant ( n = 3) prostatic tissues. Assay conditions were linear with respect to time and protein concentration and were optimal for NADPH concentration. The apparent K m values for the stromal enzymes were 0.2 and 0.02 μM for hyperplasia and carcinoma, respectively, using T as substrate. The apparent K m values, using Δ 4A as substrate, were 0.03 and 0.02 μM, respectively. Apparent V max values for the stromal formation of DHT were 16.5 ± 5.4 and 1.97 ±0.45 pmol/mg protein/30min incubation, respectively, for the hyperplastic and malignant tissues. The apparent V max values for the formation of 5α-Adione were 2.8 ± 1.3 and 6.5 ± 1.2 pmol/mg/protein/30 min incubation. The apparent K m values for the epithelial enzyme, for hyperplastic and malignant tissue were 0.04 and 0.04 μM, for T, and 0.05 and 0.03 μM for Δ 4A. The respective apparent V max values were 4.6 ±0.93 and 0.65 ±0.07 for DHT and 2.0 ±0.86 and 6.4 ± 0.45 pmol/mg protein/30 min incubation for 5α-Adione. Δ 4A was a competitive inhibitor of T 5α-reduction. These results provide further evidence that different rates of 5α-reduction at least partially explain the differences in androgen levels seen in the hyperplastic and the malignant prostate.

The influence of 4-hydroxy-4-androstene-3,17-dione on androgen metabolism and action in cultured human foreskin fibroblasts

4-hydroxy-4-androstene-3,17-dione (4-OHA) has been shown to be a potent inhibitor of aromatase activity. It is effective in the control of estrogen-dependent processes in female subjects and may potentially be useful in the treatment of estrogen-dependent processes in men. Human foreskin fibroblasts grown in cell culture provide a model to investigate the effects of 4-OHA on extraglandular aromatase activity as well as the ability of the compound to influence androgen receptor binding and the 5α-reduction of testosterone (T). Initial experiments were carried out to determine the potency of 4-OHA in genital skin fibroblasts by incubating cells with 4-OHA over a range of concentrations. When aromatase activity was determined at a substrate concentration close to the apparent K m of the enzyme, a 44% inhibition of enzyme activity occurred at a mean concentration of 5 nM 4-OHA. Enzyme kinetic studies analyzed by Eadie-Hofstee plots demonstrated competitive inhibition by 4-OHA with a mean apparent K i of 2.7 nM. When 5α-reductase activity was determined in the presence of 200 nM [ 3H]T, in the absence or presence of 4-OHA, a 50% inhibition of enzyme activity occurred at an inhibitor concentration of 3 μM. In androgen receptor binding studies, 4-OHA possessed 1% of the affinity of dihydrotestosterone (DHT) for [ 3H]DHT binding sites. In summary: 4-OHA is a potent and specific inhibitor of aromatase activity in human genital skin fibroblasts, the affinity of the enzyme for 4-OHA being greater than its affinity for the substrate, adrenostenedione. The influence of 4-OHA on 5α-reductase activity and androgen receptor binding is minimal.

Metabolism of testosterone to 17β-hydroxy-5α-androstane-3-one and 5α-androstane-3α,17β-diol in alveolar macrophages from rat lung

5α-Reduction of testosterone was observed in lung cells obtained by bronchoalveolar lavage (> 95% macrophages) from the rats. This activity was inhibited by progesterone and corticosterone. Production of 17β-hydroxy-5α-androstane-3-one and 5α-androstane-3α,17β-diol from testosterone was higher in rat pulmonary alveolar macrophages than by the 800 g supernatant fraction of whole lung homogenate from the same animals. Alveolar macrophages from rats treated with the 5α-reductase inhibitor 17 β- N, N-diethylcarbamoyl-4-aza-4-methyl-5 α-androstane-3-one (5 mg/100 g b.w., s.c.) showed decreased metabolism of testosterone to 17β-hydroxy-5α-androstane-3-one and 5α-androstane3α, 17β-diol 4 h after treatment. This metabolism was also decreased in alveolar macrophages from rats exposed to potassium dichromate by intratracheal instillation. When bovine alveolar macrophages were incubated with potassium dichromate, 5α-reduction of testosterone decreased significantly. The function of steroid 5α-reduction in alveolar macrophages is currently not known.