Androgen-metabolizing Enzymes Research Articles

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, 5alpha-reductase and 5beta-reductase in free-living longspur males. Aromatase and 5alpha-reductase convert T into the active steroids 17beta-estradiol (E(2)) and 5alpha-dihydrotestosterone (5alpha-DHT), respectively. 5beta-Reductase deactivates T via conversion to 5beta-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). 5beta-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 5beta-reductase do not explain the effects of plasma T on aggressive behavior.

Regulation of aromatase, 5 alpha- and 5 beta-reductase in primary cell cultures of developing zebra finch telencephalon.

Sex steroids act on the developing and adult telencephalon of songbirds to organize and activate the neural circuits required for the learning and production of song. Presumably, the availability of active androgens and estrogens to steroid-sensitive neural circuits controlling song is modulated by the local expression of androgen-metabolizing enzymes. Two enzymes, 5 alpha- and 5 beta-reductase, are expressed widely in the songbird telencephalon, as they are in the telencephalons of other avian species. These enzymes convert circulating testosterone (T) into the active and inactive metabolites, 5 alpha- and 5 beta-dihydrotestosterone (DHT), respectively. A third enzyme, aromatase, converts T into estradiol (E2) and is expressed at unusually high levels in several regions of the songbird telencephalon. In many tissues, including the brain, the regulation of expression of one or more of these enzymes can be a critical feature of their ability to control the production of active sex steroids. We have used primary cell cultures to examine factors that might regulate the expression of these enzymes in developing zebra finch telencephalon. Cultures were treated for 0-72 h with sex steroids (T, E2, 5 alpha-DHT, and 5 beta-DHT) or with dibutyryl cAMP. Afterward, activities of aromatase, 5 alpha- and 5 beta-reductase were determined or total RNA was extracted for Northern analysis. Treatments with cAMP increased both aromatase activity and aromatase mRNA levels by 220%. E2 significantly reduced aromatase activity by an average 65%, whereas 5 alpha- and 5 beta-DHT had no effect on aromatase activity. Compared to untreated controls, E2 treatment decreased aromatase mRNA levels by 56%. None of these treatments consistently affected either 5 alpha- and 5 beta-reductase activities. These results suggest that telencephalic E2 may regulate its own synthesis by repression of aromatase expression, whereas factors that upregulate cAMP in the telencephalon can increase the local concentrations of E2.

Kinetic analysis of androstenedione 5α-reductase in epithelium and stroma of human prostate

In the human prostate, various androgen-metabolizing enzymes are present. Among these enzymes, testosterone 5α-reductase seems to be dominant. However, androstenedione is also a potential substrate of the prostatic 5α-reductase. To address the question of to what extent the reduction of androstenedione to androstanedione occurs, the present study describes in detail the kinetic characteristics ( K m and V max) and possible age-dependent alterations of this enzymatic step in epithelium and stroma of the human prostate. In normal prostate (NPR), the mean K m (nM) and V max (pmol/mg protein · h) were about twofold higher in stroma ( K m, 211; V max, 130) than in epithelium ( K m, 120; V max, 56), whereas in the benign prostatic hyperplasia (BPH), the mean K m (nM; mean ± SEM) and V max (pmol/mg protein · h; mean ± SEM) were about sixfold higher in stroma ( K m, 688 ± 121; V max, 415 ± 73) than in epithelium ( K m, 120 ± 10; V max, 73 ± 8). In BPH, those differences between epithelium and stroma were highly significant ( p < 0.001). However, the efficiency ratios ( V max K m ) of neither BPH nor NPR showed any significant differences between epithelium (NPR, 0.47; BPH, 0.62 ± 0.06) and stroma (NPR, 0.70; BPH, 0.63 ± 0.05). With respect to age-related changes, only stroma showed a significant increase of K m ( p < 0.01) and V max ( p < 0.05) with age. In summary, in both epithelium and stroma of the human prostate, a 5α-reductase converts in measurable amounts androstenedione to androstanedione. The kinetic data were, in part, different between epithelium and stroma; the reason for this difference remains unclear. In comparison to other metabolic conversions, such as testosterone to dihydrotestosterone and androstenedione to testosterone, it is unlikely that, in the human prostate, the adrenal androgen androstenedione contributes significantly to the formation of testosterone and, further, of dihydrotestosterone.

The Activity and Expression of Aromatase in Songbirds

Songbirds have emerged as extremely important animals for investigating sex steroid hormone effects on the central nervous system. The masculinizing effects of exogenous estrogen on the neural circuits controlling song in female zebra finches are well documented. There is evidence that estrogens are necessary for the full activation of singing behavior in several species. These kinds of studies have led us and others to investigate the mechanisms whereby estrogens are made available to the brains of songbirds during development and adulthood. In this article, I review results of some of these studies examining the estrogen synthetic enzyme aromatase and its expression and activity in brain and in other tissues of songbirds. I will discuss some results and thoughts we have about the interactions of aromatase with the two remaining androgen-metabolizing enzymes in the avian brain, 5α-reductase, the enzyme that converts T into the active androgen 5α-dihydrotestosterone (DHT); and 5β-reductase, the enzyme that converts T into the inactive 5β-DHT. Finally, I will consider some ideas raised by these studies concerning potential sources of the androgen substrate for brain aromatization as well as some possible new functions that aromatase might be playing in the songbird telencephalon.

CLINICAL UPDATES IN HAIR

It is now known that there are major differences in the systemic and cellular mechanisms that mediate hair loss in women versus men. The severity or degree of hair loss in women with androgenetic alopecia is usually much less than in men. It is usually assumed that the hormonal basis for androgenetic alopecia in women is the same as in men; that is, the same target tissue-active androgens, testosterone and dihydrotestosterone, are being produced systemically to aggravate scalp hair follicles, resulting in hair loss. This article reviews some of the latest findings in androgenetic alopecia for both men and women, along with new and old treatments and drugs, doses, and effectiveness. Research findings indicate that levels of androgen-metabolizing enzymes and receptors differ in the scalps of women versus men, which may be important in formulating more effective hair growth treatments in the future.

Testosterone metabolism in primary cultures of epithelial cells and stroma from benign prostatic hyperplasia.

We studied the metabolism of testosterone in primary cultures of prostate epithelial cells and fibroblasts obtained from patients with benign prostatic hyperplasia (BPH). The conversion of 3H-testosterone in both cell cultures was predominantly to the oxidative pathway, with the formation of 3H-androstenedione increasing with cell number and time of incubation. Although we also detected some 5 alpha-reductase activity in these cells, the activity in the stroma component (0.00688 pmol/mg protein/min) was nonetheless insignificant when compared to the 5 alpha-reductase activity in the tissue of origin (0.0616 pmol/mg protein/min) and well below the 17 beta-hydroxysteroid dehydrogenase activity of the same cells (0.0518 pmol/mg protein/min). The aromatase activity in our cells was also measured by two separate techniques, but neither the deuterium procedure nor the production of oestrone from androgen precursors yielded any positive results, suggesting that under these experimental conditions there was no aromatase activity within the cells. The shift from the reductive to the oxidative pathways in these primary cell cultures was reminiscent of the androgen-metabolizing enzyme profiles seen in poorly differentiated prostate cancer. Whether this transition is an obligatory step in the development of hormone refractiveness remains to be elucidated.

Fadrozole: A Potent and Specific Inhibitor of Aromatase in the Zebra Finch Brain

Aromatase and 5β-reductase activity are expressed at high levels in the zebra finch brain, especially in the telencephalon. Aromatization of androgens to estrogens is thought to be a critical step in the organization and activation of avian sexual behaviors. In contrast, 5β-reductase is thought to be an inactivating enzyme, one that catalyzes the conversion of androgens to biologically inactive metabolites. To address the importance of aromatase activity in this system, it is necessary to find an effective and selective aromatase inhibitor, one that has little or no effect on other androgen-metabolizing enzymes. The potency and specificity of fadrozole hydrochloride as an aromatase inhibitor was tested in zebra finch telencephalon. The compound was tested in vitro in primary dissociated cell cultures made from hatchling telencephalon and compared to a commonly used inhibitor, 1,4,6-androstatriene-3,17-dione (ATD). Untreated, these cultures express extremely high levels of aromatase and 5β-reductase activity and therefore allow sensitive measurement of the effectiveness of inhibitors. Aromatase activity was also measured in homogenates of adult telencephalon following in vivo fadrozole injection. Finally, aromatase and 5β-reductase activity were quantified in zebra finch telencephalon following similar intramuscular injections in 4- to 6-day-old birds. In all three cases, fadrozole was highly effective in reducing aromatase activity. Fadrozole increased 5β-reductase activity, presumably due to an increase in available substrate, but had no inhibitory effect on the enzyme. ATD was less effective in inhibiting aromatase, and it also inhibited 5β-reductase activity at high concentrations.

In vitro potency and selectivity of the non-steroidal androgen aromatase inhibitor CGS 16949A compared to steroidal inhibitors in the brain

A sensitive in vitro 3H 2O microassay for aromatase activity was used to evaluate the potency and selectivity of three aromatase inhibitors in mammalian (gerbil) and avian (ring dove) hypothalamus. The steroidal inhibitors, 1,4,6-androstatrien-3,17-dione (ATD) and 4-hydroxy-androstenedione (4-OH-A) were compared with a new non-steroidal imidazole inhibitor, CGS 16949A [4-(5,6,7,8-tetrahydroimidazo-[1,5-a]-pyridin-5-yl)benzonitrile HCl]. Adult male dove hypothalamic aromatase is highly active [ V max = 5.3 pmol testosterone (T) converted/h/mg pprotein], has high substrate binding affinity ( K m = 4.0 nM), and direct involvement in control of sexual behaviour. With [1β- 3H]T or [1β- 3H]A as substrate, male dove preoptic aromatase activity was inhibited more effectively and selectively by CGS 16949A. Thus, K i s and IC 50s for aromatization were ∼50 times lower for the non-steroidal inhibitor, and inhibition of the other major androgen-metabolizing enzymes (5α/β-reductase) occured at concentrations at least one order of magnitude greater than for ATD and 4-OH-A. Neonatal male gerbil hypothalamic aromatase activity ( V max = 1.3 pmol T converted/h/mg protein) was lower than in the dove. Aromatase inhibition by CGS 16949A is more potent in the neonatal gerbil than in the dove ( K i s of 0.03 and 0.06 nM, respectively, with A as substrate). We conclude that the imidazole is an effective aromatase inhibitor in both the adult and developing brain.

Hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes: Potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5α-reductase inhibitors

5α-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5α-reductase. The inactivation of 5α-dihydrotestosterone involves reversible reduction to 5α-androstane-3β,17β-diol by 3β-hydroxysteroid oxidoreductase followed by 6α-, 7α-, or 7β-hydroxylation. 5α-Androstane-3β,17β-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity ( K m ~ 0.5 μM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5α-androstane-3β,17β-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5α-reductase (4-azasteroid analogs; 10 μ m) or inhibitors of 3β-hydroxy-steroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 μ m) had no appreciable effect on the 6α-, 7α-, or 7β-hydroxylation of 5α-androstane-3β,17β-diol (10 μ m) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1–10 μ m) all markedly inhibited 5α-androstane-3β,17β-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1–10 μ m) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole > clotrimazole > ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a K i of 0.03 μ m. The imidazole-type antimycotic drugs inhibited all three pathways of 5α-androstane-3β,17β-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5α-androstane-3β,17β-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.

Ontogeny of testosterone-inducible brain aromatose activity

The effects of testosterone (T) treatment on androgen-metabolizing enzymes and especially on aromatase were examined in the developing hypothalamus of quail using an in vitro microassay. Testosterone propionate (TP) injected into the pectoral muscles of one-day-old chicks had no effect on formation of 5α-dihydrotestosterone (5α-DHT), 5β-dihydrotestosterone (5β-DHT) and 5β-androstane-3α,17β-diol (5β,3α-diol) in the hypothalamus. By contrast, aromatase activity was significantly enhanced in the anterior (PA) and in the posterior (HT) parts of the hypothalamus of these chicks 18–23 h after a single injection of TP. This increase was site specific as it did not occur in control non-target areas, neostriatum intermediate and hyperstriatum ventrale (VN). The increase in hypothalamic aromatase activity was accompanied by T-dependent changes in behavior and somatic development. Although aromatase activity is present as early as day 10 of embryonic life in PA and HT, it becomes inducible by circulating T only after this stage of development. There is also an important increase in basal aromatase activity during embryonic development. At day 10, when the brain is particularly sensitive to the differentiating effects of gonadal steroids, formation of 5α-DHT and 5β-DHT in the hypothalamus is higher in females than in males. We conclude that although all important pathways of T are already present on day 10 of embryonic life, the brain metabolism of androgens continues maturing during the whole embryonic life.

A comparison of aromatase, 5 alpha-, and 5 beta- reductase activities in the brain and pituitary of male and female quail (C. c. japonica).

In numerous vertebrate species including Japanese quail (Coturnix coturnix japonica), actions of testosterone (T) on neuroendocrine target tissues are mediated in part by conversion to estrogenic and androgenic metabolites. In order to assess which pathways were favored in each identified androgen target area in quail brain and whether there were discernible sex differences, we developed an assay for simultaneously quantifying aromatase, 5 alpha-, and 5 beta-reductase. In addition, we made the first definitive identification of aromatase in quail pituitary and compared all three enzyme activities in the pituitary of males and females. Enzymes were measured in tissue homogenates by the conversion of [3H]androstenedione to [3H]estrone, [3H]5 alpha-androstanedione, and 5 beta-androstanedione. Aromatase activity was restricted to limbic tissues (anterior hypothalamus greater than posterior hypothalamus greater than septum greater than archistriatum containing nucleus taenia) while hyperstriatum, cerebellum, and midbrain containing nucleus intercollicularis were aromatase-negative. Quail pituitary aromatized androgen at rates equivalent to anterior hypothalamus/pre-optic area (aHPOA). 5 alpha- and 5 beta-reductase were present in all tissues tested. Aromatase was significantly higher in aHPOA and pituitary of males, whereas 5 alpha-reductase was significantly higher in female pituitary. These data suggest that a complex of androgen-metabolizing enzymes controls the neuroanatomic (spatial) distribution of active hormone in neuroendocrine tissues and that quantitative differences between males and females may account for sex differences in behavior.

Effects of dicyclohexane derivatives on androgen metabolism in the rat prostate

Dicyclohexane derivatives, which inhibit the binding of testosterone and dihydrotestosterone (DHT) to the androgen-binding protein (ABP) of rat epididymis without interfering with their binding to the androgen receptor, show a similar selectivity in their effects on androgen metabolism. Their ability to inhibit the aromatization of testosterone has been reported previously. This paper demonstrates that they are potent inhibitors of 3ga(β)-hydroxysteroid:NAD(P) + oxidoreductase activity (3-HSD) in the particulate fraction from rat prostate gland; the values of K i for their inhibition of this enzyme are similar to that of the K m for DHT as substrate. The dicyclohexane derivatives are markedly less effective against the cytosolic NADPH-dependent 3-HSD, and they do not appear to inhibit testosterone 5α-reductase activity. These characteristics are likely to complicate the proposed use of the dicyclohexane derivatives as probes for the role of ABP in vivo. However, they may be of interest in the study of structure-activity relationships in androgen-metabolizing enzymes, particularly in the examination of the different forms of 3-HSD

Effects of androgens on the activity of steroid-metabolizing enzymes in the murine submaxillary gland.

Effects of androgens on the activity of androgen-metabolizing enzymes have been investigated in the murine submaxillary gland. 5 alpha-reductase activity is androgen-dependent, and 17 beta-hydroxysteroid dehydrogenase activity is androgen-resistant. The level and androgen-responsiveness of 5 alpha-reductase in the murine submaxillary gland are not imprinted during the neonatal period by testicular androgens. Circulating androgen may have a cumulative effect on androgen response of 5 alpha-reductase.

Subcellular distribution of 3 alpha-hydroxysteroid dehydrogenase and antiestrogen action on androgen-metabolizing enzymes in rat pituitary gland.

Gonadectomy of male rats led to a threefold increase of 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH) activity in pituitary homogenates that could be completely reversed by chronic administration of estradiol or 5 alpha-dihydrotestosterone (DHT). 3 alpha-HSDH was found to be distributed mainly between the 10,000 g and 100,000 g sediments from whole homogenates. The microsomal enzyme activity showed a substantial specificity for NADH whereas the cytosolic enzyme (100,000 g supernatant) demonstrated a slight preference for NADPH. The changes in Vmax found in homogenates following gonadectomy and gonadal steroid administration reflected changes in NADH-linked activity of the microsomal, but not the cytosolic enzyme. Estradiol-induced suppression of NADH-linked 3 alpha-HSDH activity in pituitary homogenates from gonadectomized rats of either sex was accompanied by a similar suppression of NADPH-linked 5 alpha-reductase activity and a marked decrease of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release. In the ovariectomized rat chronic administration of nonsteroidal antiestrogens had strong estrogenic effects on 3 alpha-HSDH activity and LH release, but not on 5 alpha-reductase activity and FSH release. In the gonadectomized male rat, which was much less sensitive to intrinsic estrogenicity of the antiestrogens tested, nafoxidine completely blocked estradiol-induced suppression of 5 alpha-reductase activity and FSH release and partially antagonized suppression of LH release. The trans-isomeric, substituted triphenylethylenes, tamoxifen, and enclomiphene, as well as nitromifene (mixture of trans and cis isomers) were able partially to counteract estradiol-induced suppression of 5 alpha-reductase, but not 3 alpha-HSDH activity. It is concluded that estradiol action on pituitary 5 alpha-reductase, but not 3 alpha-HSDH activity, involves an estrogen receptor mechanism.

97. Induction of androgen-metabolizing enzymes by testosterone in female rat liver

97. Induction of androgen-metabolizing enzymes by testosterone in female rat liver