Metabolite Dihydrotestosterone Research Articles

Topical finasteride in the treatment of androgenic alopecia. Preliminary evaluations after a 16-month therapy course

The enzyme 5α-reductase (5AR), which catalyzes reduction of testosterone to the more potent metabolite dihydrotestosterone, has been assumed to play a key role in a variety of skin disorders, including acne, seborrhea, hirsutism, and androgenic alopecia (AA). Also, evidences have been provided supporting the pathogenetic relevance of higher rates of testosterone reduction at lesional level. The azasteroid finasteride, a 5AR inhibitor, is widely employed in the treatment of benign prostatic hyperplasia; by contrast, its potential role in other androgen-related conditions have been, so far, only poorly evaluated.We present herein the results of a single-blind, placebo-controlled, 16-month trial carried out in 52 patients with AA using a 0.005% finasteride solution. The clinical outcome, in terms of both hair regrowth and balding areas reduction, seems to be encouraging, in the absence of either any evidence of percutaneous absorption of finasteride, or local/systemic untoward effects.We also briefly review the possible pharmacodynamic and pharmacokinetic bases of the use of topically delivered finasteride in AA.

Androgen independence of primary epithelial cultures of the prostate is associated with a down‐regulation of androgen receptor gene expression

BACKGROUND Epithelial cells cultured from prostatic acini do not demonstrate significant (P > 0.05) growth response to the testosterone metabolite dihydrotestosterone (DHT) at concentrations of 0.001–10.0 nM. In addition, the nonsteroidal antiandrogen hydroxyflutamide (HO-F) does not influence primary epithelial cell proliferation in this concentration range. METHODS Northern blotting carried out with an androgen reception (AR)-specific cDNA probe indicated that the extent of AR gene expression in six unpassaged primary prostatic epithelial cell cultures was insufficient to elicit a detectable signal upon autoradiography. However, RT/PCR analysis of total RNA using two sets of intron-spanning androgen receptor (AR) primers demonstrates the presence of full-length receptor transcripts in two BPH-derived epithelial cell cultures (BPH1 and BPH2) as well as a carcinoma-derived culture (CaP1). RESULTS AR-positive LNCaP cells transfected with the AR reporter plasmid pMMTV/SPAP exhibit significant increases (P < 0.05) in SPAP production upon treatment with DHT. pMMTV/SPAP-transfected primary epithelial cells exhibit no such response when pulsed with either androgen or anti-androgen. CONCLUSIONS These results indicate that the lack of significant AR gene expression underlies the androgen independence of primary prostatic epithelial cell cultures. © 1996 Wiley-Liss, Inc.

Androgen independence of primary epithelial cultures of the prostate is associated with a down-regulation of androgen receptor gene expression.

Epithelial cells cultured from prostatic acini do not demonstrate significant (P > 0.05) growth response to the testosterone metabolite dihydrotestosterone (DHT) at concentrations of 0.001-10.0 nM. In addition, the nonsteroidal antiandrogen hydroxyflutamide (HO-F) does not influence primary epithelial cell proliferation in this concentration range. Northern blotting carried out with an androgen reception (AR)-specific cDNA probe indicated that the extent of AR gene expression in six unpassaged primary prostatic epithelial cell cultures was insufficient to elicit a detectable signal upon autoradiography. However, RT/PCR analysis of total RNA using two sets of intron-spanning androgen receptor (AR) primers demonstrates the presence of full-length receptor transcripts in two BPH-derived epithelial cell cultures (BPH1 and BPH2) as well as a carcinoma-derived culture (CaP1). AR-positive LNCaP cells transfected with the AR reporter plasmid pMMTV/SPAP exhibit significant increases (P < 0.05) in SPAP production upon treatment with DHT. pMMTV/SPAP-transfected primary epithelial cells exhibit no such response when pulsed with either androgen or anti-androgen. These results indicate that the lack of significant AR gene expression underlies the androgen independence of primary prostatic epithelial cell cultures.

Inhibition of 5 alpha-reductase activity impairs the testosterone-dependent restoration of spermiogenesis in adult rats.

Testosterone (T) is required for spermatogenesis, particularly in the conversion of round spermatids between stages VII and VIII of spermatogenesis. T is generally thought to be the major androgen involved in adult spermatogenesis due to the high local concentration within the testis, whereas its more potent 5alpha-reduced metabolite dihydrotestosterone (DHT) is thought to be the active androgen in peripheral tissues. The current study investigated whether 5alpha-reduction of T to DHT is involved in the restoration of spermiogenesis in vivo in a setting in which testicular T levels were markedly reduced. Adult male rats were given 3-cm T plus 0.4-cm estradiol implants for 9 weeks to suppress serum LH and testicular T levels and thereby inhibit spermatogenesis. Increasing doses of T (3-, 6-, 10-, and 24-cm implants) were then given for 4 days to restore spermatogenesis in the presence or absence of a 5alpha-reductase inhibitor (L685,273). The hourly production rates of round spermatids in stages I-III, IV-VI, VII, and VIII were assessed using stereological techniques, and the conversion of round spermatids between stages VII and VIII was then assessed as an index of androgen action on spermiogenesis. Testicular androgen levels were measured by HPLC and RIA. The 5alpha-reductase inhibitor significantly suppressed (P < 0.05) the hourly production rate of round spermatids at the 3- and 6-cm T doses, but not at the 10- and 24-cm doses. The conversion of round spermatids between stages VII and VIII was suppressed (P < 0.05) by the inhibitor only at the 3- and 6-cm doses. The 5alpha-reductase inhibitor had no effect on testicular T levels, but suppressed (P < 0.05) DHT levels at the 6-, 10-, and 24-cm doses. We conclude that the 5alpha-reduction of T is involved in the restoration of spermiogenesis at the lower administered doses of T and that these data are the first description of a role for 5alpha-reduced androgens in adult spermatogenesis.

Effect of progesterone, testosterone and their 5α-reduced metabolites on GFAP gene expression in type 1 astrocytes

Astrocytes possess steroid receptors as well as several enzymes typical of steroid target cells, such as 5α-reductase, which converts testosterone (T) and progesterone (P) into their respective 5α-reduced metabolites, and the 3α-hydroxysteroid dehydrogenase (3α-HSD). Because of this, it was deemed of interest to analyze whether the original hormones P and T, and their 5α-reduced metabolites dihydrotestosterone (DHT), 5α-androstan-3α,17β-diol (3α-diol), dihydroprogesterone (DHP) and 5α-pregnan-3α-ol-20-one (THP), might exert some effects on the expression of the most typical astrocytec marker, i.e. the glial fibrillary acidic protein WAY). Cultures of rat type 1 astrocytes were exposed to the various steroids for 2, 6, and 24 h, and the variations of GFAP mRNA were measured by Northern blot analysis. A significant elevation of GFAP mRNA levels was observed after exposure to either P or DHP; the effect of DHP appeared more promptly (at 2 h) than that of P (at 6 h). This result suggests that the effect of P might be linked to its conversion into DHP; this hypothesis has been confirmed by showing that the addition of finasteride (a specific blocker of the 5α-reductase) is able to completely abolish the effect of P. After exposure to DHP or THP, a decrease of GFAP gene expression was observed at later intervals (24 h). In the case of androgens, T and 3α-diol did not change GFAP expression at any time of exposure, while DHT produced a significant decrease of GFAP mRNA only after 24 h of exposure. Taken together, the data indicate that the 5α-reduced metabolites of P and T may modulate the expression of GFAP in type 1 rat astrocytes.

Role of dihydrotestosterone in androgen action

Androgen action differs from that of most hormones in the testosterone, the major androgen secreted from the testes and the most abundant androgen in the circulation of men, is not the principal androgen within target cells. Indeed, abundant evidence indicates that most androgen actions are mediated by the 5alpha-reduced metabolite dihydrotestosterone that is formed in target tissues. The conversion of testosterone to dihydrotestosterone is mediated by two isoenzymes; mutations in the steroid 5alpha-reductase 2 gene cause a rare autosomal-recessive form of male pseudohermaphroditism, and inhibition of this enzyme causes regression of the prostate gland. Dihydrotestosterone binds more tightly to the androgen receptor that does testosterone, but it is not clear whether this property is the sole explanation for its essential role in androgen action. Nor is it clear whether some androgenic effects may be mediated by circulating dihydrotestosterone acting as a hormone.

Androgens induce divergent proliferative responses in human breast cancer cell lines.

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.

Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells

We demonstrate that androgens rapidly and specifically increase intracellular calcium in Sertoli cells, investigate the mechanism, and suggest the unifying hypothesis that calcium might be a common intracellular molecular effector to explain the known synergism between FSH and testosterone (T) action on Sertoli cells in support of spermatogenesis. In freshly isolated Sertoli cells, T and its 5 alpha-reduced metabolite dihydrotestosterone increased intracellular calcium from 83 +/- 4 to 147 +/- 8 and 167 +/- 29 nM, respectively, whereas estradiol had minor (117 +/- 9 nM) and progesterone no (80 +/- 6 nM) effect. The effect of T was rapid (20-40 sec) and inhibited by 1) preincubation with either a pure nonsteroidal antiandrogen (hydroxyflutamide) or a 5 alpha-reductase inhibitor (finasteride) or 2) removal of extracellular calcium (47 +/- 4 nM) or pharmacological blockade of voltage-activated (62 +/- 5 nM) or voltage-independent (55 +/- 14 nM) membrane calcium channels. These findings suggest that the T-induced rise in Sertoli cell cytosolic calcium involves sequential 5 alpha-reduction, binding to a classical androgen receptor, and activation of transmembrane influx of extracellular calcium. Immobilization of T by conjugation to a large carrier molecule (BSA) to prevent steroid entry into Sertoli cells also resulted in a rapid increase in cytosolic calcium to a similar magnitude as unconjugated T, consistent with a plasma membrane site of action. This finding together with the rapid cytosolic calcium rise caused by T argues for the possible existence of a short term, nongenomic effects in hormonal regulation of Sertoli cell function in addition to the well known, slower genomic response.

Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells.

We demonstrate that androgens rapidly and specifically increase intracellular calcium in Sertoli cells, investigate the mechanism, and suggest the unifying hypothesis that calcium might be a common intracellular molecular effector to explain the known synergism between FSH and testosterone (T) action on Sertoli cells in support of spermatogenesis. In freshly isolated Sertoli cells, T and its 5 alpha-reduced metabolite dihydrotestosterone increased intracellular calcium from 83 +/- 4 to 147 +/- 8 and 167 +/- 29 nM, respectively, whereas estradiol had minor (117 +/- 9 nM) and progesterone no (80 +/- 6 nM) effect. The effect of T was rapid (20-40 sec) and inhibited by 1) preincubation with either a pure nonsteroidal antiandrogen (hydroxyflutamide) or a 5 alpha-reductase inhibitor (finasteride) or 2) removal of extracellular calcium (47 +/- 4 nM) or pharmacological blockade of voltage-activated (62 +/- 5 nM) or voltage-independent (55 +/- 14 nM) membrane calcium channels. These findings suggest that the T-induced rise in Sertoli cell cytosolic calcium involves sequential 5 alpha-reduction, binding to a classical androgen receptor, and activation of transmembrane influx of extracellular calcium. Immobilization of T by conjugation to a large carrier molecule (BSA) to prevent steroid entry into Sertoli cells also resulted in a rapid increase in cytosolic calcium to a similar magnitude as unconjugated T, consistent with a plasma membrane site of action. This finding together with the rapid cytosolic calcium rise caused by T argues for the possible existence of a short term, nongenomic effects in hormonal regulation of Sertoli cell function in addition to the well known, slower genomic response.

Chemoprevention for prostate cancer

With prostate cancer representing the most common male cancer and the second most common cause of cancer-related death in men in the United States, increasing attention is being directed at providing early detection, as well as improvement in therapy. The third option to decrease cancer-related deaths, decreasing the incidence, has only recently gained attention. If tumor promoters can be removed from the patient environment and/or agents administered that inhibit cancer progression, we may indeed be able to decrease the incidence of this most common neoplasm. The prostate is a suitable site for chemoprevention strategies. High-risk populations, including those men with a strong family history of prostate cancer, black men, and/or men with prostatic intraepithelial neoplasia, a putative premalignant change identified on prostate biopsy or simple prostatectomy, represent useful target populations. If a chemopreventive strategy could be developed that was free of toxicity and also simple, inexpensive, and readily administered, indeed all men could be targeted. Several potential agents are available for chemoprevention in the prostate. The retinoids moderate differentiation and proliferation in several prostate cell lines. Severe toxicity, however, may preclude their widespread application. Difluoromethylornithine has also been investigated. A chemopreventive trial directed against lung cancer showed that alpha-tocopherol is associated with a decreasing incidence of prostate cancer. The greatest interest in the chemopreventive strategies for prostate cancer, however, has focused on decreasing the male androgens. Although most agents, such as luteinizing hormone-releasing hormone agonists and antiandrogens, have severe toxicity, the 5-alpha reductase inhibitors, because they do not, are thought to be excellent agents for a chemopreventive trial. The dependence of prostate epithelial differentiation and maintenance of transformed cells on circulating androgens is widely acknowledged. This is the impetus for the hypothesis that reduction of circulating androgens or blockage of testosterone to the more active metabolite dihydrotestosterone by agents such as finasteride or epristeride will reduce the incidence of prostate cancer. The National Cancer Institute Intergroup Prostate Cancer Prevention Trial, now underway, will randomize 18,000 men to receive 5 mg finasteride or a placebo daily for 7 years in a chemopreventive strategy. Entry requirements include a normal result of digital rectal examination and a serum prostate-specific antigen result less than 3.0 ng/ml. Abnormalities on digital rectal examination results or prostate-specific antigen level greater than 4.0 ng/ml on annual evaluation indicate the need for biopsies in the men receiving the placebo. An equal number of men will undergo biopsy in the active arm of the study. At the end of the 7-year study, all men will have biopsies. Although the primary endpoint is a reduction in prostate cancer incidence, additional benefits include long-term follow-up of the patients receiving finasteride, with potential salutary benefits with regard to symptoms of benign prostatic hyperplasia. This trial, which already has accrued more than 75% of the required participants, should resolve the issue of whether reduction in effective androgen level will prevent the development of prostate cancer. Cancer 1995 ;75 :1783-9.

Ultrastructural effects of estradiol and 5 alpha-androstane-3 alpha, 17 beta-diol on neurons within the ventromedial nucleus of the hypothalamus.

The metabolite of dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-Diol), is a potent inhibitor of estrogen-induced gonadotropin and prolactin secretion and lordosis behavior in the female rat. This study examined whether 3 alpha-Diol can counteract the ultrastructural changes which are known to occur in the ventromedial nucleus (VMN) of the hypothalamus following estrogen treatment. Ovariectomized rats were treated with estradiol (E2; n = 7), 3 alpha-Diol (n = 5), E2 and 3 alpha-Diol (n = 6), or received control (n = 6) treatments. E2 was administered in subcutaneous capsules for two discontinuous 2-hour periods separated by 5 h, a 'pulsed' treatment regimen known to mimic the timing of endogenous E2 action and to influence neuronal ultrastructure in the VMN. Animals given 3 alpha-Diol received subcutaneous injections (6 mg/kg) 3 h prior to each implantation of E2 or empty capsules. Control animals received vehicle 3 h prior to implantation of blank capsules. Animals were perfused 24 h after initial hormone treatment and neurons from the ventrolateral portion of the VMN were examined using electron microscopy. Separately, both E2 and 3 alpha-Diol treatment increased somal and nuclear size, altered somal and nuclear shape, and increased the numbers of lysosomes present in the cytoplasm above control levels. E2 treatment resulted in increased stacking of the rough endoplasmic reticulum while 3 alpha-Diol treatment resulted in an unusual plexiform rough endoplasmic reticulum distribution. In contrast, combined treatment with E2 and 3 alpha-Diol resulted in cells which were similar to ovariectomized control cells on these measures. All steroid treatments decreased the amount of heterochromatin present within the nucleus compared to that seen in controls. Thus, 3 alpha-Diol influences the ultrastructural characteristics of neurons within the VMN in a manner somewhat though not altogether similar to E2. However, 3 alpha-Diol given in combination with E2 counteracts or prevents the actions of E2 within these same neurons.

Androgen metabolism and inhibition of interleukin‐1 synthesis in primary cultured human synovial macrophages

The presence of androgen receptors on synovial macrophages in human normal and rheumatoid synovial tissues has been described previously. It is now reported that primary cultured human macrophages obtained from normal and rheumatoid synovia express functional androgen receptors. We have investigated the capacity of cultured macrophages to metabolize androgens and have found that these cells were capable of metabolizing testosterone to the bioactive metabolite dihydrotestosterone. Therefore, macrophages contain the key enzymes of steroidogenesis, in particular the 5α-treductase. Furthermore, interleukin-1β production by primary cultured rheumatoid macrophages was analysed, following exposure to physiological concentrations of testosterone (10−8 M). A significant decrease of IL-1β levels in conditioned media after 24 h (p < 0.05) was observed. It is concluded that androgens may act directly on human macrophages and may interfere with some of their functions via receptor-dependent mechanisms.

Changes in serum concentrations of conjugated and unconjugated steroids in 40- to 80-year-old men.

It is well recognized that aging in men is accompanied by a decline in the serum levels of some adrenal and testicular steroids, but little or no attention has focused on the multiple steroid metabolites that are formed by steroid-converting enzymes in target tissues. In the present study, we have examined in detail the serum concentrations of a large series of adrenal and testicular steroids and their most significant metabolites produced in intracrine peripheral tissues. The serum concentrations of 26 conjugated and unconjugated C21-, C19-, and C18-steroids were measured in 2423 men aged 40-80 yr. The serum concentrations of the major circulating adrenal C19-steroids, namely dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S), androst-5-ene-3 beta, 17 beta-diol and its sulfate, and androstenedione, decreased by about 60% between the ages of 40-80 yr. The small decrease in the serum concentrations of progesterone and pregnenolone in the presence of increased levels of cortisol and markedly decreased levels of DHEA, androst-5-ene-3 beta, 17 beta-diol, and their polar metabolites suggests that adrenal 17,20-lyase is particularly affected by aging. In addition to a marked decline in the serum concentrations of adrenal C19-steroids, a smaller, but significant, decrease occurred in serum testosterone. However, serum dihydrotestosterone levels remained constant, but the glucuronidated derivatives of dihydrotestosterone metabolites (androstane-3 alpha, 17 beta-diol glucuronide, androstane-3 beta, 17 beta-diol glucuronide, and androsterone glucuronide) were reduced by 45-50%, suggesting that 5 alpha-reductase activity in peripheral tissues may show a compensatory increase during aging. Analysis of the fatty acid esters of DHEA (DHEA-FA) also revealed that these nonpolar steroids markedly decrease between 40-80 yr of age, although such a decrease in DHEA-FA levels was smaller than that in DHEA and DHEA-S, suggesting that the formation of DHEA-FA may be specifically increased during aging. In summary, the present study suggests that in contrast to the marked decline in activity of steroidogenic enzymes in the adrenals and the small decrease in the testis, the activity of the steroid-converting enzymes present in peripheral tissues does not decrease during aging. In fact, the marked decrease in DHEA formation by the adrenals leads to a decrease of about 50% in total androgens in men between the ages of 40-80 yr. Such a decrease probably affects many physiological processes during aging.

Effect of inhibiting 5 alpha-reductase activity on the ability of testosterone to inhibit luteinizing hormone release in male sheep.

The extent to which inhibitory effects of testosterone (T) on LH secretion in the ram are mediated by its metabolite dihydrotestosterone (DHT) is unknown. Our objective was to determine the effect of inhibiting 5 alpha-reductase activity on pulsatile patterns of LH release in castrated, T-treated male sheep. Nine Dorset and six Hampshire castrated male sheep were allocated equally to one of three treatment groups: 1) infusion of T (768 micrograms/kg/day), 2) infusion of the reductase inhibitor (RI) L-651,723 (0.6 mg/kg/day), and 3) T+RI infusion. Treatments were continuously infused for 3 days. Blood samples were collected via an indwelling jugular catheter at 10-min intervals for 4 h immediately prior to (Day 0) and during the final 4 h of infusion (Day 3). Changes in mean LH, LH pulse amplitude, LH interpulse interval (IPI), T, 17 beta-estradiol (E), and DHT were derived for each animal by subtracting values for Day 0 from Day 3. Data were subjected to one-way analysis of variance. The increase in T and E after infusion of T was similar (p > 0.10) in T- and T+RI-treated males and greater (p < 0.01) than in RI-treated males. The increase of DHT was greater (p < 0.01) in T-treated than either T+RI- or RI-treated males whereas the change was similar (p > 0.10) for T+RI- and RI-treated males. T decreased mean LH more (p < 0.01) than RI. T+RI suppressed mean LH more (p < 0.01) than RI but not as much (p < 0.01) as T alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of parathyroid hormone-related peptide production in vitro by the rat hypercalcemic Leydig cell tumor H-500

The transplantable rat Leydig cell tumor H-500 is known to cause hypercalcemia in vivo by the release of abundant PTH-related peptide (PTHRP) and to closely reproduce the human syndrome of malignancy-associated hypercalcemia. In the rat only a single messenger RNA species of 1.4 kilobases is expressed which encodes a peptide of 141 amino acid as the sole molecular form. We have examined in cultured rat Leydig tumor cells H-500, the capacity of multiple factors to regulate PTHRP messenger RNA expression and secretion. Both fetal bovine serum and epidermal growth factor stimulated PTHRP gene expression and secretion into conditioned culture medium. Dexamethasone and 1,25-dihydroxyvitamin D3 produced inhibition of PTHRP gene expression and secretion. Furthermore, in these testicular cells, after 12 h or more of incubation, testosterone produced a dose-dependent (10(-9)-10(-7) M) inhibition of PTHRP production. No significant difference in this inhibitory response was seen between testosterone and its 5 alpha-reduced metabolite dihydrotestosterone whereas 17 beta-estradiol, progesterone, LH, FSH, and PRL were ineffective. An androgen receptor antagonist Win 49596 blocked the androgen-mediated inhibition of PTHRP gene expression and secretion, but not that due to dexamethasone. Epidermal growth factor caused an increase, whereas androgen caused a decrease in PTHRP gene transcription. These studies demonstrated that growth factors, dexamethasone, and 1,25-dihydroxyvitamin D3 are broadly active regulatory agents of PTHRP production which cross species and tissue barriers. Testosterone may be a more selective modulator which can regulate PTHRP in tissues such as Leydig cell neoplasms which express the androgen receptor.

Regulation of parathyroid hormone-related peptide production in vitro by the rat hypercalcemic Leydig cell tumor H-500.

The transplantable rat Leydig cell tumor H-500 is known to cause hypercalcemia in vivo by the release of abundant PTH-related peptide (PTHRP) and to closely reproduce the human syndrome of malignancy-associated hypercalcemia. In the rat only a single messenger RNA species of 1.4 kilobases is expressed which encodes a peptide of 141 amino acid as the sole molecular form. We have examined in cultured rat Leydig tumor cells H-500, the capacity of multiple factors to regulate PTHRP messenger RNA expression and secretion. Both fetal bovine serum and epidermal growth factor stimulated PTHRP gene expression and secretion into conditioned culture medium. Dexamethasone and 1,25-dihydroxyvitamin D3 produced inhibition of PTHRP gene expression and secretion. Furthermore, in these testicular cells, after 12 h or more of incubation, testosterone produced a dose-dependent (10(-9)-10(-7) M) inhibition of PTHRP production. No significant difference in this inhibitory response was seen between testosterone and its 5 alpha-reduced metabolite dihydrotestosterone whereas 17 beta-estradiol, progesterone, LH, FSH, and PRL were ineffective. An androgen receptor antagonist Win 49596 blocked the androgen-mediated inhibition of PTHRP gene expression and secretion, but not that due to dexamethasone. Epidermal growth factor caused an increase, whereas androgen caused a decrease in PTHRP gene transcription. These studies demonstrated that growth factors, dexamethasone, and 1,25-dihydroxyvitamin D3 are broadly active regulatory agents of PTHRP production which cross species and tissue barriers. Testosterone may be a more selective modulator which can regulate PTHRP in tissues such as Leydig cell neoplasms which express the androgen receptor.

Dihydrotestosterone Is a Peripheral Paracrine Hormone

Androgen action in sexual tissues, especially skin and the prostate, is expressed by dihydrotestosterone (DHT) acting at the nuclear level. Dihydrotestosterone in the circulation and target tissues is almost solely derived from the peripheral conversion of secreted testosterone (T) in men and androstenedione in women. The general pathway is testosterone----DHT in equilibrium with androstanediol (3 alpha diol). However, a number of studies suggest that blood DHT or 3 alpha diol are not reliable indicators of peripheral DHT formation. This is particularly suggested by discrepancies in the specific activity of DHT in blood and urine following infusion of labeled DHT, suggesting that total body DHT formation is not reflected by blood levels. Thus, DHT should be thought of as a paracrine hormone formed and acting primarily in target tissues. 3 alpha androstanediol glucuronide (3 alpha diol G) is a major metabolite of DHT. An important site of its formation is the skin. Levels in blood and urine are increased in hirsutism and acne, and blood levels closely parallel pubertal development. 3 alpha diol G levels are especially increased in adrenal disorders of androgenicity such as andrenogenital syndrome; it is also a good marker of response to therapy. Levels are reduced in various forms of male pseudohermaphroditism. 3 alpha androstanediol glucuronide appears to be the best marker available of DHT formation in target tissues such as skin.

Lysosomal glycosidase activities in rat testis during sexual maturation

Concentrations of testosterone and its metabolite dihydrotestosterone were determined in whole rat testis during the transition from the prepuberal to the mature status. The activities of five glycosidases (beta-N-acetylhexosaminidase, alpha-L-fucosidase, beta-D-galactosidase, alpha-D-glucosidase and alpha-D-mannosidase) were also investigated in the seminiferous tubules and interstitial tissues. The same androgens and enzyme activities were measured in testis of rats treated with 17-beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5-alpha-androstan-3-on e, a potent inhibitor of the 5-alpha-reductase-mediated conversion of testosterone into dihydrotestosterone. Lysosomal glycosidic activities in seminiferous tubules were found to vary slightly with the age of the animals, and thus seemed not to depend on the hormonal status of the tested animals. In contrast, the enzyme activities were low in the immarure interstitium but increased sharply during the onset of sexual maturity. The activity of each glycosidase reached a maximum between the 45th and the 65th day, except for beta-N-acetylhexosaminidase which did not decrease significantly following the 55th day. The five lysosomal enzyme activities decreased in the interstitial compartment of rat testis treated with DMAA, suggesting a relationship between these glycosidic enzyme activities and dihydrotestosterone.

Androsterone Glucuronide is a Marker of Adrenal Hyperandrogenism in Hirsute Women

Androsterone glucuronide (Andros-G), a dihydrotestosterone metabolite, is present in serum at concentrations at least tenfold greater than those of androstanediol glucuronide. To investigate the significance of serum androsterone glucuronide, we developed a direct radioimmunoassay for this compound and measured its levels in normal women, women with mild or severe idiopathic hirsutism (IH), hirsute women with polycystic ovarian syndrome (PCO), and non-hirsute obese women. To determine the source of Andros-G precursors, serum levels were measured before and after selective ovarian suppression with leuprolide, combined ovarian and adrenal suppression with leuprolide and dexamethasone, and adrenal stimulation with ACTH. Androsterone glucuronide levels (nmol/l; mean +/- SD) were significantly higher (P less than 0.025) in women with mild idiopathic hirsutism (IH) (185 +/- 91), severe IH (173 +/- 97), and hirsute women with polycystic ovarian syndrome (PCO) (178 +/- 102) than in normal women (110 +/- 26). Levels in non-hirsute obese women (64 +/- 19) were lower than in normal women (P less than 0.01). Baseline levels (mean +/- SEM) in hirsute women given 20 micrograms/kg/day leuprolide for 5-9 months (171 +/- 15) were not significantly changed after leuprolide alone (153 +/- 18), and were decreased after adding dexamethasone (19 +/- 6; P less than 0.001). Andros-G levels did not increase significantly in normal women 60 min after i.v. ACTH (112 +/- 14 to 126 +/- 19), but rose in IH (170 +/- 24 to 216 +/- 26; P less than 0.001) and in PCO (179 +/- 26 to 238 +/- 31; P = 0.002). We conclude that Andros-G in women arises primarily from adrenal gland precursors and is elevated in hirsute women as a group. Its levels do not correlate with the severity of hirsutism, or the presence or absence of PCO, but reflect an increased production of adrenal androgens in both IH and PCO.

Synthesis of new steroid haptens for radioimmunoassay—VIII. Development and validation of a specific radioimmunoassay for serum 5α-androstane-3α,17β-diol 17-glucuronide

5α-Androstane-3α,17β-diol glucuronide (androstanediol-G) is a dihydrotestosterone metabolite whose serum levels are elevated in hirsute women. Current assay methods do not distinguish between the two androstanediol-G isomers, androstanediol 3-G and androstanediol 17-G. Since the production of these isomers may be influenced by different factors, we have developed a specific radioimmunoassay for androstanediol 17G. The antibody was raised against 5α-androstane-3α,17β-diol 17-G conjugated to bovine serum albumin (BSA). [9,11- 3H]5α-Androstane-3α,17β-diol 17-G was used for determination of procedural losses and as the labeled ligand in the assay. Unlabeled androstanediol 17-G was used as assay standard. Serum levels of total androstanediol-G, androstanediol 3-G and androstanediol 17-G were measured in 8 normal men. Total androstanediol-G levels were 16.5 ± 5.2 nmol/l, androstanediol 17-G levels were 12.9 ± 5 nmol/l, and androstanediol 3-G levels were 30.3 ± 1.8 nmol/l. 77 ± 13% of total androstanediol-G was androstanediol 17-G. These results confirm our previous findings that androstanediol 17-G is the predominant androstanediol-G isomer in human serum and suggests that 5α-dihydrotestosterone (DHT) is preferentially metabolized to androstanediol 17-G.