Expression of steroid-converting enzymes in osteoblasts derived from rat vertebrae.

Studies in vitro and in vivo have shown that glucocorticoids and sex steroids play an important role in bone physiology and pathophysiology. In this study we investigated glucocorticoid and sex steroid conversion in osteoblasts derived from lumbar vertebrae of adult male and female rats. Progesterone was converted to inactive 20alpha-OH-progesterone and the conversion at day 5 was 16-fold greater than that at day 13 in both sexes (male/ female, 2.7/1.7 and 0.16/0.10 nM/10(5)cells/24 h, respectively). The conversion of inactive androstenedione to active androgen testosterone in males and females was 1.2- and 2.4-fold greater at day 5 than at day 13, respectively (male/female, 0.40/0.70 and 0.34/0.30 nM/ 10 cells/24 h, respectively). These results suggest that osteoblasts possess 20alpha-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD and that their activities are dependent on the stage of cell differentiation. At day 5, dehydroepiandrosterone was converted to androstenedione (male/female, 0.25/0.098 nM/10(5)cells/24 h), to 7alpha-OH-dehydroepiandrosterone (male/female, 0.49/0.39 nM/10(5)cells/24 h) and to 5-androstene-3beta,17beta-diol (male/female, 0.18/0.37 nM/10(5)cells/24 h), indicating the presence of 3beta-HSD, 7alpha-hydroxylase and 17beta-HSD, respectively. Both 3beta-HSD and 7alpha-hydroxylase activities declined with cell differentiation. Hormonally inactive cortisone was converted to active cortisol (male/female, 0.34/0.29 microM/10(6)cells/6 h) while conversion of cortisol to cortisone was not detectable, suggesting the presence of oxoreductase activity of 11beta-HSD-1. These results show, for the first time, the presence of 7alpha-hydroxylase and 20alpha-HSD in osteoblasts, and provide further evidence that osteoblasts metabolize a variety of steroid hormones and can thus regulate tissue responsiveness to them.

Quadriceps muscle function and fatigue in women with Addison's disease.

In nine patients with Addison's disease (mean +/- SE: 51 +/- 2 years) receiving conventional steroid treatment, and nine age-matched healthy controls (56 +/- 2 years), we investigated maximum voluntary quadriceps force (MVC) and contractile properties evoked with stimulation and central activation both at rest and during a submaximal intermittent fatigue task. The MVC was similar (-3%), but twitch tension (-27%) and central activation were significantly less (-7%), and tetanic half-relaxation time was approximately 40% slower in the patients. Twitch amplitudes were potentiated by 6% in the patients, but unchanged in the control group. The patients self-terminated a submaximal intermittent fatigue protocol (0.6 duty cycle) at approximately 5 +/- 1 min, whereas the controls stopped when they lost 50% of MVC force ( approximately 10 +/- 1 min). Force loss was similar between groups over the first 5 min of the fatigue task. In the patient group, maximal and submaximal relative integrated electromyogram (IEMG) increased significantly in the first minute of fatigue and remained elevated, whereas the controls exhibited a gradual increase in submaximal IEMG with little change in maximal IEMG. These results indicate that conventionally treated Addison's patients have similar MVC strength, but altered contractile properties and decreased endurance compared with controls.

A microdeletion within DAX-1 in X-linked adrenal hypoplasia congenita and hypogonadotrophic hypogonadism.

BACKGROUND: X-linked adrenal hypoplasia congenita (AHC) is a developmental disorder characterized by primary adrenal gland failure, which produces extreme and potentially fatal endocrine deficiencies. Hypogonadotrophic hypogonadism (HHG) also may be associated with AHC. AHC has been shown to result from a variety of mutations in the DAX-1 gene, which encodes a member of the nuclear hormone receptor superfamily. METHODS: The proband, one of the world's oldest living patients with AHC and HHG, was diagnosed in 1955. He was on corticosteroid replacement therapy since that time and androgen replacement therapy since puberty. We sequenced his DAX-1 gene. RESULTS: We found a 4 bp ACTC deletion between nucleotides 1464 and 1467 in the second exon of the normal DAX-1 sequence. This mutation caused a shift in the reading frame and predicted a premature stop codon at amino acid position 416. The mutation abolished a recognition site for DdeI, allowing for confirmation by restriction analysis. CONCLUSIONS: The position of the mutation confirms the functional importance of the COOH-terminal 10% of the DAX-1 sequence. The clinical history also reinforces the importance of early diagnosis in AHC, which can be associated with longevity and no obvious morbidity after more than 40 years of hormone replacement therapy.

Overexpression of the growth-hormone-releasing hormone gene in acromegaly-associated pituitary tumors. An event associated with neoplastic progression and aggressive behavior.

The clinical behavior of growth hormone (GH)-producing pituitary tumors is known to vary greatly; however, the events underlying this variability remain poorly understood. Herein we demonstrate that tumor overexpression of the GH-releasing hormone (GHRH) gene is one prognostically informative event associated with the clinical aggressiveness of somatotroph pituitary tumors. Accumulation of GHRH mRNA transcripts was demonstrated in 91 of a consecutive series of 100 somatotroph tumors by in situ hybridization; these findings were corroborated by Northern analysis and reverse transcriptase polymerase chain reaction, and protein translation was confirmed by Western blotting. By comparison, transcript accumulation was absent or negligibly low in 30 normal pituitary glands. GHRH transcripts were found to preferentially accumulate among clinically aggressive tumors. Specifically, GHRH mRNA signal intensity was 1) linearly correlated with Ki-67 tumor growth fractions (r = 0.71; P < 0.001), 2) linearly correlated with preoperative serum GH levels (r = 0.56; p = 0.01), 3) higher among invasive tumors (P < 0.001), and 4) highest in those tumors in which post-operative remission was not achieved (P < 0.001). Using multivariate logistic regression, a model of postoperative remission likelihood was derived wherein remission was defined by the single criterion of suppressibility of GH levels to less than 2 ng/ml during an oral glucose tolerance test. In this outcome model, GHRH mRNA signal intensity proved to be the most important explanatory variable overall, eclipsing any and all conventional clinicopathological predictors as the single most significant predictor of postoperative remission; increases in GHRH mRNA signal were associated with marked declines in remission likelihood. The generalizability of this outcome model was further validated by the model's significant performance in predicting postoperative remission in a random sample of 30 somatotroph tumors treated at another institution. These data indicate that overexpression of GHRH gene is an event associated with the neoplastic progression and clinical aggressiveness of somatotroph adenomas. More generally, these data merge essential elements of the hypothalamic and pituitary hypotheses of pituitary tumorigenesis, providing for a more unified concept of neoplastic progression in the pituitary.

11 beta-Hydroxysteroid dehydrogenase 1 activity and gene expression in human adipose stromal cells: effect on aromatase activity.

The biological activity of glucocorticoids in target tissues can be influenced by locally produced 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the enzyme responsible for the interconversion of cortisol and its inactive metabolite cortisone. In human adipose stromal cells, glucocorticoids are potent stimulators of the conversion of androgens to estrogens (aromatase activity). The present study was designed to determine whether 11 beta-HSD activity was present in human adipose stromal cells, and if changes in the activity of this enzyme could influence aromatase activity. 11 beta-HSD activity was determined by a radiometric conversion assay in breast adipose tissue from six patients. It was found that both dehydrogenase (cortisol to cortisone) and reductase (cortisone to cortisol) activities were present in all six subjects, and the reductase activity was always predominant. Carbenoxolone (CBX), a potent inhibitor of 11 beta-HSD, added to the culture medium at 50 and 200 microM, resulted in 39 +/- 4% and 85 +/- 1% inhibition, respectively, of both reductase and dehydrogenase activity of 11 beta-HSD. To determine whether alterations in 11 beta-HSD could influence aromatase activity, the effect of CBX (200 microM) on cortisol- and cortisone-induced changes in the conversion of androstenedione to estrone was examined. CBX prevented the stimulatory effect of cortisone and minimally potentiated the stimulatory effect of cortisol on aromatase activity, reflecting an inhibition of the local activation of cortisone and the local metabolism of cortisol, respectively. In order to determine whether the product of the 11 beta-HSD 1 gene was responsible for the observed 11 beta-HSD activity, total RNA extracts from these cells were subjected to Northern blot analysis using human 11 beta-HSD 1 cDNA as the probe. A single 1.8 11 beta-HSD 1 transcript was detected, and its abundance was reduced by CBX. No 11 beta-HSD 2 mRNA was detected. The present results demonstrate that the 11 beta-HSD 1 gene is expressed and functional in human breast adipose stromal cells and that changes in 11 beta-HSD 1 activity result in alterations in aromatase activity.

Estrone formation from dehydroepiandrosterone in cultured human breast adipose stromal cells.

The metabolism of dehydroepiandrosterone (DHA) and androstenedione (A-dione) was studied in cultured human adipose stromal cells obtained from breast tissue of six premenopausal patients undergoing reduction mammoplasty. Cells were maintained in culture in the presence of 10% fetal bovine serum. Studies were carried out during the proliferative and confluent phases of culture with radiolabelled substrates (2 microCi, 10 nM). During the early phases of replication 7 alpha-hydroxydehydroepiandrosterone (7 alpha-OHDHA) was formed from DHA. As the cells reached confluence, the major metabolite of DHA in cells from all patients was A-dione indicating the presence of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD). The conversion of DHA to A-dione was variable among patients when cells were confluent with 30-80% of substrate being metabolized to this product. Adipose stromal cells synthesized estrone (E1) from DHA once A-dione formation was established. Under basal conditions E1 was obtained in cells from three of the six patients examined with up to 36% substrate converted to this product. Dexamethasone (Dex 10(-7) M) stimulated E1 formation in cells from all subjects with up to 50% of substrate being converted. Parallel studies comparing the conversion of DHA with A-dione to E1 revealed that as the cells became confluent, E1 formation from both substrates was similar. The pattern of steroid metabolism was also examined in primary culture and in subculture. Passage 1 cells continued to form A-dione as a major metabolite of DHA, and did not revert to the pattern of metabolism found in primary cells during the early stages of replication, when 7 alpha-hydroxylation predominated. Human adipose stromal cells actively metabolize DHA, producing 7 alpha-OHDHA, A-dione and E1 as principal metabolites. Changes in the circulating levels of DHA may directly influence the formation of E1 in peripheral tissues. This source of E1 will be modulated by factors controlling 3 beta-HSD and aromatase activities.

Substitution of arginine-839 by cysteine or histidine in the androgen receptor causes different receptor phenotypes in cultured cells and coordinate degrees of clinical androgen resistance.

We aim to correlate point mutations in the androgen receptor gene with receptor phenotypes and with clinical phenotypes of androgen resistance. In two families, the external genitalia were predominantly female at birth, and sex-of-rearing has been female. Their androgen receptor mutation changed arginine-839 to histidine. In a third family, the external genitalia were predominantly male at birth, and sex-of-rearing has been male: their codon 839 has mutated to cysteine. In genital skin fibroblasts, both mutant receptors have a normal androgen-binding capacity, but they differ in selected indices of decreased affinity for 5 alpha-dihydrotestosterone or two synthetic androgens. In transiently cotransfected androgen-treated COS-1 cells, both mutant receptors transactivate a reporter gene subnormally. The His-839 mutant is less active than its partner, primarily because its androgen-binding activity is more unstable during prolonged exposure to androgen. Adoption of a nonbinding state explains a part of this instability. In four other steroid receptors, another dibasic amino acid, lysine, occupies the position of arginine-839 in the androgen receptor. Androgen receptors with histidine or cysteine at position 839 are distinctively dysfunctional and appear to cause different clinical degrees of androgen resistance.

Effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-hydroxydehydroepiandrosterone by human adipose stromal cells.

7 alpha-Hydroxydehydroepiandrosterone (7 alpha-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-OHDHA. Dexamethasone (10(-9) to 10(-7) M) stimulated 7 alpha-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10(-7) M. The dexamethasone stimulated 7 alpha-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10(-6) M. Progesterone (10(-7) M) had no effect on 7 alpha-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7 alpha-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70-80% was obtained with ketoconazole and 25-60% with metyrapone at concentrations of 10(-5) M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7 alpha-OHDHA formation with < 30% inhibition at 10(-5) M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.

Metabolism of dehydroepiandrosterone by cultured human adipose stromal cells: identification of 7 alpha-hydroxydehydroepiandrosterone as a major metabolite using high performance liquid chromatography and mass spectrometry.

Studies of the metabolism of dehydroepiandrosterone (DHA) by cultured human adipose stromal cells revealed that the most abundant metabolite detected by HPLC was a polar compound accounting for up to 45% of total radioactivity. This metabolite was isolated by chromatography on Lipidex 5000 from the culture medium of breast adipose stromal cells cultured with unlabelled DHA (5 microM) and identified by combined capillary gas chromatography and mass spectrometry as 7 alpha-hydroxydehydroepiandrosterone (7 alpha-OHDHA). In breast adipose stromal cells, the conversion of DHA to 7 alpha-OHDHA was linear from a substrate concentration of 10 nM to 1 microM. At 1 microM substrate concentration, the formation of 7 alpha-OHDHA in four patients ranged from 6.1 to 22.5 ng/10(5) cells/24 h. Incubations carried out in primary culture and up to the fifth subculture revealed continued formation of 7 alpha-OHDHA. Adipose stromal cells from abdomen, flank and perinephric fat also produced 7 alpha-OHDHA from DHA. These studies have shown that 7 alpha-OHDHA is a major metabolite of DHA in human adipose stromal cells. The variability from patient to patient and the magnitude of this conversion suggests that this pathway may play an important role in the peripheral metabolism of DHA.

Null cell adenoma of the pituitary with features of plurihormonality and plurimorphous differentiation.

The case of a 35-year-old man with pituitary macroadenoma who was complaining of reduced sexual activity is presented. Histologic examination showed a chromophobic adenoma corresponding mainly to a null cell adenoma at the ultrastructural level. Focal plurihormonality and plurimorphous differentiation of adenoma cells were demonstrated by immunohistochemical and electron-microscopic studies. It is suggested that adenomatous null cells represent pluripotent progenitor cells capable of transforming to different hormone-producing cell types. The factors accounting for differentiating to various cell populations have yet to be elucidated.

Influence of adipose tissue distribution on the biological activity of androgens.

To establish whether the conversion of androstenedione (A) to estrogens and 5 alpha-reduced metabolites in human adipose tissue was determined by the site of origin of the tissue, studies were carried out on adipose stromal cells from different body sites. Adipose tissue was obtained from the breast, omentum, abdomen, lower thigh, upper thigh, buttock, and flank from patients undergoing liposuction for cosmetic reasons or at surgery. Stromal cells were isolated after incubation of the adipose tissue with collagenase and were grown in culture using alpha-minimal essential medium (MEM) + 15% fetal calf serum. Studies of A metabolism were carried out when the cells were between days 4 and 12 in culture. After an 8-hour incubation with (3H)-A as substrate, estrone (E1), testosterone (T), 5 alpha-androstanedione (5 alpha-A-dione), androsterone (AND), and dihydrotestosterone (DHT) were isolated using thin layer and paper chromatography. The conversion per 1 x 10(6) cells of A of E1 was more than 10-fold greater in the upper thigh, buttock, and flank than in the breast, lower thigh, abdomen, or omentum (0.13-3.0 vs 0.01-0.09%). The formation of 5 alpha-reduced androgens varied from 0.86-10% and was similar in tissue from different body sites. Cortisol (10(-7) M) stimulated E1 formation 3- to 10-fold in cells from all sites, whereas 5 alpha-reductase activity was either unchanged or increased moderately (up to twofold). In cells from the abdomen, omentum, and lower thigh, the formation of 5 alpha-reduced androgens was more than 10-fold greater than the formation of E1. In cells from the upper thigh, buttock, and flank, E1 formation was comparable to 5 alpha-reduced androgen formation. These studies show marked differences in the relative conversion of A to estrogens and 5 alpha-reduced androgens in adipose stromal cells depending on their site of origin, and they suggest that the distribution of body fat may be a major factor in determining the biologic effects of secreted androgens.

Silent somatotroph adenomas of the human pituitary. A morphologic study of three cases including immunocytochemistry, electron microscopy, in vitro examination, and in situ hybridization.

Pituitary adenomas, removed surgically from three women with normal or slightly elevated serum growth hormone levels and no evidence of acromegaly, were studied. The tumor cells were shown by electron microscopy to correspond to sparsely granulated somatotrophs but immunocytochemistry showed that they contained no, moderate, or little growth hormone. Two tumors examined in vitro secreted small amounts of growth hormone in the tissue culture medium initially with a spontaneous rise after several days, and responded to growth hormone-releasing hormone stimulation with increased growth hormone release. In situ hybridization demonstrated growth hormone mRNA expression in adenoma cells. Clinically silent somatotroph adenomas represent a hitherto undescribed entity; electron microscopy shows that they consist of somatotrophs, and express growth hormone mRNA but do not secrete growth hormone in amounts needed to raise substantially serum growth hormone levels and cause acromegaly. Further work is required to clarify the mechanisms accounting for the lack of clinical and biochemical evidence of hormone excess associated with these tumors.

Impaired spermatogenesis is not an obligate expression of receptor-defective androgen resistance.

We are studying a man who presented at age 21 years with severe extragenital subvirilization despite high-normal to above-normal levels of plasma testosterone for at least 5 years. At puberty, his penis, scrotum, and testes matured normally, and he did not develop gynecomastia; however, his voice, muscularity, and facial, sexual, and body hair remained immature. A 2.5-ml ejaculate yielded normal results for sperm density, morphology, and motility. Because persistent undervirilization was emotionally disabling, he has received pharmacologic doses of testosterone enanthate intramuscularly for 3.5 years. The treatment has improved his virilization and masculine self-image substantially, and his semen analysis has remained well within the normal range. The androgen receptor in his genital skin fibroblasts has a distinctively mutant phenotype: it has a low affinity (increased apparent equilibrium dissociation constant, Kd) for 5 alpha-dihydrotestosterone and two synthetic androgens, mibolerone (MB) and methyltrienolone (MT), and its binding capacity (Bmax) is normal for the other two ligands, but questionably low for MT. In addition, it up-regulates its activity normally in response to prolonged incubation with androgen, and its androgen-receptor complexes are not thermolabile. Our study of this man permits two conclusions: impaired spermatogenesis is not the irreducible expression of receptor-defective androgen resistance in man; and androgen pharmacotherapy may be remedial for those in whom extragenital subvirilization is emotionally costly and subnormal spermatogenesis is not an inevitable side effect of such therapy.

A novel type of pituitary adenoma: morphological features and clinical correlations.

Twenty patients with a novel, frequently aggressive type of pituitary adenoma, termed silent subtype 3 adenoma on the basis of fine structural criteria, are reported. The surgically removed tumors were studied by morphological techniques, and the findings were correlated with clinical and biochemical data. All tumors were macroadenomas, often with parasellar extension. The histologically diffuse tumors frequently exhibited focal immunopositivity for one or more adenohypophysial hormones, although the majority of adenoma cells were negative. The tumors had characteristic electron microscopic features, assuring specific diagnosis and delineating this tumor type as a distinct ultrastructural entity. The tumors were removed from 9 women and 11 men (median ages, 27 and 41 yr, respectively). In all women, mild to moderate hyperprolactinemia and its sequelae were present from the early phase of the disease, leading to the erroneous diagnosis of prolactinoma. Bromocriptine therapy (3 patients) reduced serum PRL levels to normal, but failed to halt tumor growth. In men, most adenomas were nonfunctioning; 4 men had mild to moderate hyperprolactinemia. Three men had elevated serum GH levels and acromegaly, suggestive of multidirectional differentiation. Although the putative cell type giving rise to silent subtype 3 adenomas is not known, the tumor should be recognized to avoid erroneous diagnosis and inappropriate treatment.

Steroid modulation of aromatase activity in human cultured breast carcinoma cells.

Cortisol and steroids with progestational or androgenic activity were studied to determine the effects of these steroids on the conversion of androstenedione (A) to estrone (E1) in human cultured breast carcinoma cells. Cortisol (10(-6) M) stimulated aromatase activity in two estrogen unresponsive cell lines (MD, DM) and in an estrogen responsive cell line (MCF7) with the maximum stimulation occurring during confluence. Cortisol inhibited the replication of MCF7 cells but not MD and DM. Dihydrotestosterone, androsterone and 5 alpha-androstanedione (10(-6) M) inhibited the conversion of A to E1 by greater than 90% under basal and cortisol stimulated conditions. Progesterone (10(-6) M) had no effect on aromatase activity while the progestational agent R5020 (10(-6) M) produced a 30% inhibition. The anabolic steroids 19-nortestosterone and 19-norandrostenedione which also have progestational activity inhibited the conversion of A to E1 in a dose dependent manner with 90% inhibition at 10(-6) M. Danazol (10(-6) M) a drug with both androgenic and progestational activity inhibited E1 formation by 30%. Under the same conditions, the known inhibitor of aromatase, 4-hydroxyandrostenedione (10(-6) M) decreased E1 formation by more than 90% and aminoglutethimide (10(-6) M) caused only 25% inhibition. These studies demonstrate that endogenous and exogenous steroids may have significant effects in modulating the local formation of estrogens from androgen precursors in cultured breast carcinoma cells. This effect on estrogen formation may be a factor in the biological response of breast tissue.

Structure-function correlations of human pituitary gonadotroph adenomas in vitro.

Two pituitary gonadotroph adenomas were studied in vitro to characterize structure-function correlations. Both tumors were from men, aged 63 and 69 years, who had elevated blood levels of follicle-stimulating hormone (FSH) and normal blood luteinizing hormone (LH) and testosterone values. The surgically resected adenomas contained diffuse immunohistochemical positivity for beta-FSH, beta-LH, and alpha-subunit of glycoprotein hormones; by electron microscopy they were composed of well-differentiated gonadotrophs. In vitro, both tumors released FSH, LH, and alpha-subunit. Morphometric studies were performed on surgically resected and cultured adenoma cells. Compared with the surgical specimens, the cultured cells had decreased cytoplasmic volume densities of endoplasmic reticulum and Golgi apparatus and slightly increased cytoplasmic volume densities of secretory granules. Incubation with gonadotropin-releasing hormone (GnRH) for 2 and 24 hours increased FSH, LH, and alpha-subunit release by both tumors; morphometry after 2 consecutive days of exposure confirmed significant increases in cytoplasmic volume densities of endoplasmic reticulum and Golgi regions and marked decreases in that of secretory granules. There was no significant change in cell size, nuclear/cytoplasmic ratio, or secretory granule diameter. The two tumors differed in their response to gonadal steroids. Estradiol, testosterone, and progesterone stimulated release of FSH, LH, and alpha-subunit by one tumor and the morphologic changes paralleled the biochemical response; addition of testosterone suppressed the secretory and morphologic response to GnRH. The other tumor showed no significant response to estradiol or testosterone and addition of these steroids did not alter the response to GnRH. The results are consistent with the interpretation that GnRH stimulates not only release but also synthesis of gonadotropins by gonadotroph adenomas of men. The data also indicate variable sensitivity of these tumors to gonadal steroids with paradoxical stimulation alone and inhibition of response to GnRH. The structural changes correlate with the hormone release response in vitro.

Aromatase activity in the breast and other peripheral tissues and its therapeutic regulation.

Studies using [3H]androstenedione (A) demonstrated that this substrate can be aromatized to estrone (E1) in homogenates of breast carcinoma tissue and breast adipose tissue, in breast carcinoma and breast adipose stromal cells in culture, and in cultured adipose stromal cells from sites remote from the tumor. Using cultured breast carcinoma cells, it was shown that estrogen formation was stimulated by cortisol (10(-6) M) and inhibited by endogenous 5 alpha-reduced androgens: 5 alpha-androstene-dione greater than androsterone greater than dihydrotestosterone greater than epiandrosterone greater than 3 alpha- and 3 beta- androstanediol. It was also shown that 19-nortestosterone and 19-norandrostenedione (10(-6) M) inhibited E1 formation by 80%. Progesterone (10(-6) M) had no effect on aromatase activity, while the progestational agent R5020 (10(-6) M) caused a 70% inhibition. These studies emphasize that a variety of compounds can influence aromatase activity and that drugs which are used as aromatase inhibitors in patients with breast carcinoma may have multiple sites of action.

The relationship between aromatase activity and body fat distribution.

The metabolism of androstenedione (A) to estrone (E1) and 5 alpha-reduced androgens was studied in stromal cells derived from human adipose tissue from different body sites. The tissue was obtained from non-obese patients undergoing cosmetic liposuction or at the time of surgery for reduction mammoplasty. The conversion of A to E1 per 1x 10(6) cells was between 6- and 30-fold greater in the upper thigh, buttock, and flank than in the abdomen. These differences were present in primary culture and persisted to at least the third subculture. Estrogen formation in breast adipose tissue was similar to that found in cells from abdominal fat. The formation of 5 alpha-reduced metabolites (5 alpha-androstenedione, androsterone, and dihydrotestosterone) varied from patient to patient but was similar in cells from different body sites. These studies show that the regional distribution of fat may influence the metabolism of androgens in adipose tissue, with upper body fat tending to form a lower ratio of estrogens to 5 alpha-reduced androgens than lower body fat.

Ligand-specific thermal misbehavior of synthetic androgen-receptor complexes in genital skin fibroblasts of subjects with familial ligand-sensitive androgen resistance.

We have used 5 alpha-dihydrotestosterone (DHT) and two synthetic, non-metabolizable androgens, methyltrienolone (MT) and mibolerone (MB), to study intact genital skin fibroblasts from four subjects with familial incomplete androgen resistance. In each, the free androgen receptor has normal binding capacity at 37 degrees C and normal half-lives at 37-43 degrees C. In three the mutant receptor misbehaves in a pattern that is ligand-specific and temperature-dependent. At 37 degrees C the equilibrium (Kd) and non-equilibrium (k) dissociation constants, and the ability to augment binding activity during prolonged exposure to androgen, are impaired with DHT, but not with MT; with MB, only the k is abnormal. Mutant MT-receptor complexes dissociate normally even at 42 degrees C; yet, in cells post-incubated at 42 degrees C with cycloheximide and a saturating concentration of ligand, their pool size decays in the rank order, MT greater than MB greater than normal. This measure of lability is nonlinear as a semilogarithmic function of time; it varies directly with temperature and the concentration of cycloheximide, but inversely with that of ligand. Thus, MT and MB evoke distinct forms of thermal dysfunction from the androgen receptor in ligand-sensitive androgen resistance. This observation will help to elucidate the combinatorial properties of normal androgen-receptor complexes that enable them to regulate gene transcription differentially in various androgen target tissues.