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Pregnenolone (PREG) and dehydroepiandrosterone (DHEA), and their respective sulfated forms PREGS and DHEAS, were among the first steroids to be identified in rodent brain. However, unreliable steroid isolation and solvolysis procedures resulted in errors, particularly in the case of brain steroid sulfates analyzed by radioimmunology or GC-MS of liberated free steroids. By using a solid-phase extraction recycling/elution procedure, allowing the strict separation of sulfated, free, and fatty acid esters of PREG and DHEA, PREGS and DHEAS, unlike free PREG, were not detected in rat and mouse brain and plasma. Conversely, considerable amounts of PREG and DHEA were released from unknown precursor(s) present in the lipoidal fraction, distinct from fatty acid ester conjugates. Chromatographic and mass spectrometric studies of the nature of the precursor(s) showed that autoxidation of brain cholesterol (CHOL) was responsible for the release of PREG and DHEA from the lipoidal fraction. When inappropriate protocols were used, CHOL was also the precursor of PREG and DHEA obtained from the fraction assumed to contain sulfated steroids. In contrast, free PREG was definitely confirmed as an endogenous steroid in rat brain. Our study shows that an early removal of CHOL from brain extracts coupled to well-validated extraction and fractionation procedures are prerequisites for reliable measurements of free and conjugated PREG and DHEA by GC-MS or other indirect methods.

Previous work showed that dehydroepiandrosterone (DHEA) prevents and reverses chronic hypoxic pulmonary artery hypertension in rat via targeting smooth muscle cells. In our study, DHEA was tested on human pulmonary arterial smooth muscle cells (HPASMC) to identify its mechanism of action under hypoxia in vitro. We show that DHEA decreased HIF-1alpha accumulation under both "chemical hypoxia" with treatment by the iron chelator deferroxamin and gas hypoxia (1% O2). The mRNA levels of HIF-1alpha were unchanged whether or not DHEA was applied under chemical and gas hypoxia, as compared to controls in normoxia, suggesting a post-transcriptional effect of the steroid. Protein levels of prolyl hydroxylases responsible for HIF-1alpha degradation were not modified by DHEA treatment. In addition, a synthetic derivative of DHEA, 3beta-methyl-Delta5-androsten-17-one (which cannot be metabolized), was as active as DHEA on HIF-1alpha accumulation, as well as testosterone and 17beta-estradiol (E2). In HPASMC cultures under normoxia and both types of hypoxia, DHEA gave rise to Delta5-androstene-3beta,17beta-diol (ADIOL) and DHEA-sulfate (DHEA-S). Neither testosterone, nor E2 were found. In addition, ADIOL, DHEA-S, 7alpha-hydroxy-DHEA and Delta4-androstene-3,17-dione were ineffective on HIF-1alpha accumulation. The effect of DHEA per se reducing HIF-1alpha accumulation may be relevant to reduced hypoxia effects in pulmonary arterial hypertension.

Background: Survival remains an issue in pulmonary hypertension, a chronic disorder that often affects aged human adults. In young adult mice and rats, chronic 50% hypoxia (11% FIO2 or 0.5 atm) induces pulmonary hypertension without threatening life. In this framework, oral dehydroepiandrosterone was recently shown to prevent and reverse pulmonary hypertension in rats within a few weeks. To evaluate dehydroepiandrosterone therapy more globally, in the long term and in old age, we investigated whether hypoxia decreases lifespan and whether dehydroepiandrosterone improves survival under hypoxia., Methods: 240 C57BL/6 mice were treated, from the age of 21 months until death, by normobaric hypoxia (11% FIO2) or normoxia, both with and without dehydroepiandrosterone sulfate (25 mg/kg in drinking water) (4 groups, N = 60). Survival, pulmonary artery and heart remodeling, weight and blood patterns were assessed., Results: In normoxia, control mice reached the median age of 27 months (median survival: 184 days). Hypoxia not only induced cardiopulmonary remodeling and polycythemia in old animals but also induced severe weight loss, trembling behavior and high mortality (p < 0.001, median survival: 38 days). Under hypoxia however, dehydroepiandrosterone not only significantly reduced cardiopulmonary remodeling but also remarkably extended survival (p < 0.01, median survival: 126 days). Weight loss and trembling behavior at least partially remained, and polycythemia completely, the latter possibly favorably participating in blood oxygenation. Interestingly, at the dose used, dehydroepiandrosterone sulfate was detrimental to long-term survival in normoxia (p < 0.05, median survival: 147 days)., Conclusion: Dehydroepiandrosterone globally reduced what may be called an age-related frailty induced by hypoxic pulmonary hypertension. This interestingly recalls an inverse correlation found in the prospective PAQUID epidemiological study, between dehydroepiandrosterone blood levels and mortality in aged human smokers and former smokers.

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.

Although dehydroepiandrosterone (DHEA) is widely used in the elderly to prevent some adverse effects of ageing, possible deleterious side effects have not been fully assessed. We evaluated the direct actions of DHEA and DHEA sulphate on angiogenesis, a critical event in pathologies that are common in the elderly (cancer, atherosclerosis, inflammation... etc.). At physiological concentrations found in human plasma following DHEA therapy (1-50 nM), DHEA had no action on angiogenesis in vitro. In contrast, higher concentrations of DHEA (10-100 microM), which can be found in tissues after local administration or storage, inhibited in vitro endothelial cell proliferation (blockage in G2/M), migration and capillary tube formation and in vivo angiogenesis in the Matrigel plug assay. This inhibition might be due to a decreased glucose-6-phosphate dehydrogenase activity and to a modification of the tubulin network involved in cell proliferation and migration. The sulphate ester form of DHEA had no effect on angiogenesis.

Pulmonary artery (PA) hypertension was studied in a chronic hypoxic-pulmonary hypertension model (7-21 days) in the rat. Increase in PA pressure (measured by catheterism), cardiac right ventricle hypertrophy (determined by echocardiography), and PA remodeling (evaluated by histology) were almost entirely prevented after oral dehydroepiandrosterone (DHEA) administration (30 mg/kg every alternate day). Furthermore, in hypertensive rats, oral administration, or intravascular injection (into the jugular vein) of DHEA rapidly decreased PA hypertension. In PA smooth muscle cells, DHEA reduced the level of intracellular calcium (measured by microspectrofluorimetry). The effect of DHEA appears to involve a large conductance Ca2+-activated potassium channel (BKCa)-dependent stimulatory mechanism, at both function and expression levels (isometric contraction and Western blot), via a redox-dependent pathway. Voltage-gated potassium (Kv) channels also may be involved because the antagonist 4-amino-pyridine blocked part of the DHEA effect. The possible pathophysiological and therapeutic significance of the results is discussed.

The description of dehydroepiandrosterone (DHEA) as a neuroactive neurosteroid has raised the important question of whether the steroid itself and/or its metabolite(s) are active in the brain. Classical transformations of DHEA in brain and peripheral tissues include its conversion to testosterone and estradiol. In the human brain, the metabolism of DHEA to other metabolites is still poorly understood, particularly in aging people and Alzheimer's patients. The present study describes the in vitro transformation of DHEA into 7alpha-hydroxy-DHEA and Delta5-androstene-3beta,17beta-diol, for the first time in the aging brain of patients with Alzheimer's disease in comparison with non-demented controls. Formal identification of DHEA metabolites is provided by gas chromatography-mass spectrometry, thus indicating the presence of NADPH-dependent 7alpha-hydroxylase and 17beta-hydroxysteroid oxidoreductase activities. Under our experimental conditions, the synthesis of 7alpha-hydroxy-DHEA and Delta5-androstene-3beta,17beta-diol occurs in the frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimer's patients and non-demented controls. In both groups of patients, the pattern of DHEA metabolism is similar, but significant higher synthesis of 7alpha-hydroxy-DHEA in the frontal cortex and Delta5-androstene-3beta,17beta-diol in the cerebellum and striatum were observed compared with those in other brain regions. In addition, a trend toward a significant negative correlation is found between the density of cortical amyloid deposits and the amount of 7alpha-hydroxy-DHEA formed in the frontal cortex and that of Delta5-androstene-3beta,17beta-diol in the hippocampus. Therefore, the biosynthesis of 7alpha-hydroxy-DHEA and/or Delta5-androstene-3beta,17beta-diol is likely to regulate DHEA cerebral concentrations and may contribute to the control of DHEA activity in the aging brain including in Alzheimer's disease.

Background: The age-related decline of dehydroepiandrosterone and its sulfate ester levels is thought to be related to the development of age-associated usual modifications, such as neuromuscular function impairments. It is often claimed that individuals can enhance their muscular capacity by boosting dehydroepiandrosterone levels through oral supplementation. However, to our knowledge, there have been no controlled studies on a significant number of individuals demonstrating positive effects on the neuromuscular system. This study determines if 1-year administration of a replacement dose of dehydroepiandrosterone, 50 mg/d, orally administered, could have a beneficial influence on several determinants of the muscular function altered during aging., Methods: This work was completed within the frame of the DHEAge Study, which was conducted in France from March 1, 1998, to October 31, 1999. It was performed on 280 healthy ambulatory and independent men and women aged 60 to 80 years. The study design was a double-blind placebo-controlled trial. Dehydroepiandrosterone sulfate serum concentration, handgrip strength, isometric and isokinetic knee muscle strength, and thigh (fat and muscle) cross-sectional area were analyzed before and just after 12 months of placebo or dehydroepiandrosterone treatment., Results: The results give evidence that dehydroepiandrosterone administration restores dehydroepiandrosterone sulfate serum concentrations to the normal range for young adults (aged 20-50 years). However, no positive effect inherent to dehydroepiandrosterone treatment was observed either on muscle strength or in muscle and fat cross-sectional areas., Conclusions: The compensation of the deficit of dehydroepiandrosterone during aging using a 50-mg/d dose does not induce beneficial effects on muscle state in healthy subjects. The conditions in which dehydroepiandrosterone could contribute to preserve or improve muscle strength and morphological features still need to be determined.

Neurosteroids are synthetisized in the central and the peripheral nervous system, in glial cells, and also in neurons, from cholesterol or steroidal precursors imported from peripheral sources. They include 3 beta-hydroxy-delta 5-compounds, such as pregnenolone (PREG) and dehydroepiandrosterone, their sulfate esters, and compounds known as reduced metabolites of steroid hormones, such as the tetrahydroderivative of progesterone 3 alpha-hydroxy-5 alpha-pregnan-20-one. These neurosteroids can act as modulators of neurotransmitter receptors, such as GABAA, NMDA, and sigma 1 receptors. Progesterone itself is also a neurosteroid, and a progesterone receptor has been detected in peripheral and central glial cells. At different sites in the brain, neurosteroid concentrations vary according to environmental and behavioral circumstances, such as stress, sex recognition, or aggressiveness. A physiological function of neurosteroids in the central nervous system is strongly suggested by the role of hippocampal PREGS with respect to memory performance, observed in aging rats. In the peripheral nervous system, a role for PROG synthesized in Schwann cells has been demonstrated in remyelination after cryolesion of the sciatic nerve in vivo and in cultures of dorsal root ganglia. A new mechanism of PREG action discovered in the brain involves specific steroid binding to microtubule associated protein and increased tubulin polymerization for assembling microtubules. It may be important to study the effects of abnormal neurosteroid concentration/metabolism in view of the possible treatment of functional and trophic disturbances of the nervous system.

Dehydroepiandrosterone (DHEA; 50 and 25 mg) and placebo tablets were orally administered daily to 24 healthy aging men and women (67.8 +/- 4.3 yr) for 8 days according to a balanced incomplete block design. Nine blood tests on both the first and eighth days allowed the measurement of DHEA, its sulfate DHEAS, and metabolites: testosterone, 5alpha-androstan-3alpha,17beta-diol glucuronide, estradiol, and estrone. Relatively low background levels of DHEA(S) were observed, and with the reestablishment of "young" levels, four important results were obtained. 1) Blood DHEA had an apparent terminal half-life of more than 20 h, the same order of magnitude as that of blood DHEAS, a result explainable by back-hydrolysis of the large amount of DHEAS formed after oral administration of DHEA, a mechanism providing long-lived unconjugated DHEA and metabolites. 2) The metabolic conversion of DHEAS to DHEA was significantly greater in women than in men. 3) No accumulation of steroids was observed. 4) No worrying transformation to androgen and estrogen was recorded; indeed, the limited increased estradiol in aged women could be predicted to be beneficial. These results suggested that daily oral administration of DHEA (25/50 mg) is safe in elderly subjects. The 50-mg dose was chosen for a 1 yr, double blind, placebo-controlled trial of daily oral administration of DHEA in 60- to 80-yr-old individuals (DHEAge).

Steroid hormones mostly act via nuclear receptors. Dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulphate(s), classically known only as hormonal precursors, are also neurosteroidal efficacious signals, modulating neurotransmitter receptor function at the membrane level. An additional unforeseen mechanism of steroid action is reported here: PREG binds to neural microtubule-associated protein MAP2 and increases both the rate and extent of tubulin polymerization studied in vitro with purified tubulin and MAP2, forming microtubules of normal electron microscopic appearance. Progesterone and PREGS also bind to MAP2, but counteract PREG activity. In cultured neurons, PREG specifically increased immunostaining by an antiMAP2 monoclonal antibody and its extension into neurites. This novel mechanism may play a role in regulating microtubule formation and dynamics, which are altered in brain ageing and diseases.

The secretion and the blood levels of the adrenal steroid dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) decrease profoundly with age, and the question is posed whether administration of the steroid to compensate for the decline counteracts defects associated with aging. The commercial availability of DHEA outside the regular pharmaceutical-medical network in the United States creates a real public health problem that may be resolved only by appropriate long-term clinical trials in elderly men and women. Two hundred and eighty healthy individuals (women and men 60-79 years old) were given DHEA, 50 mg, or placebo, orally, daily for a year in a double-blind, placebo-controlled study. No potentially harmful accumulation of DHEAS and active steroids was recorded. Besides the reestablishment of a "young" concentration of DHEAS, a small increase of testosterone and estradiol was noted, particularly in women, and may be involved in the significantly demonstrated physiological-clinical manifestations here reported. Bone turnover improved selectively in women >70 years old, as assessed by the dual-energy x-ray absorptiometry (DEXA) technique and the decrease of osteoclastic activity. A significant increase in most libido parameters was also found in these older women. Improvement of the skin status was observed, particularly in women, in terms of hydration, epidermal thickness, sebum production, and pigmentation. A number of biological indices confirmed the lack of harmful consequences of this 50 mg/day DHEA administration over one year, also indicating that this kind of replacement therapy normalized some effects of aging, but does not create "supermen/women" (doping).

Dehydroepiandrosterone (DHEA) and its sulphate ester (DHEAS) are neuroactive and are both imported into the brain from the circulation and produced in the nervous system. These neurosteroids have neurotrophic and excitatory effects, and further study is needed to delineate their physiological and pathological functions.

The term "neurosteroids" applies to those steroids that are both formed in the nervous system from sterol precursors, and accumulate in the nervous system, at least in part, independently of peripheral steroidogenic glands secretion. Neurosteroids that are active on the central nervous system include, mainly, pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters (PREG-S and DHEA-S), as well as the reduced metabolite of progesterone, 3 alpha,5 alpha-TH PROG also called allopregnanolone. These neuroactive neurosteroids alter neuronal excitability by modulating the activity of several neurotransmitter receptors and thus can influence behavior. PREG-S decreases the sleeping time in rats anesthetized with a barbiturate, which is consistent with its antagonist action on the GABAA receptor (GABAA-R). Allopregnanolone is anxiolytic in rats tested in a conflict paradigm, through an interaction at a site specific for the benzodiazepine (BZ) receptor inverse agonist RO15-4513 and/or at the picrotoxinin site on GABAA-R. The contribution of the amygdala, a key region involved in the control of anxiety, is also demonstrated for the anxiolytic action of allopregnanolone. An anti-agressive effect of DHEA can be observed in castrated male mice who become agressive in the presence of lactating females. This inhibition of agressiveness by DHEA is associated to a selective decrease in the brain of PREG-S, which may, in turn, trigger an increase of endogenous GABAergic tone. Finally, cognitive performances of aged rats tested in the Morris water maze and the Y-maze can be correlated with individual concentrations of PREG-S in the hippocampus, i.e. poor performance in both tasks with low levels of PREG-S. Remarkably, the memory deficits are significantly improved, albeit transiently, by an intra-hippocampal injection of PREG-S in impaired aged rats. Promnesiant PREG-S may then reinforce some neurotransmitter systems that can decline with age. This brief review provides evidence of the pharmacology and physiological correlates of neurosteroids involved in behavioral phenomena. However, neurobiological mechanisms of behavioral effects of neurosteroids await further investigation.

The physiological importance and therapeutical interest of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are still controversial. Panhypopituitarism is characterized by the absence of secretion of adrenal and gonadal steroids and thus the production of their metabolites. The conversion of DHEA given orally into delta 5 derivatives, androgens, androgen metabolites, and estrogens was studied in ten patients with complete panhypopituitarism. Sex steroid therapy was withdrawn for at least 2 months. Each patient received, at 1-month intervals and in a random order, two single oral doses of DHEA (50 mg and 200 mg) and placebo. During each treatment, urine samples were collected for 24 h, and blood samples were drawn at hourly intervals for 8 h. In patients with pituitary deficiency, plasma DHEA and DHEAS were not detectable and increased, with the 50 mg dose, up to levels observed in young adults. The administration of 200 mg of DHEA induced an increase of both steroids to supraphysiological plasma levels. A small increase of delta 5-androstenediol was observed. In contrast, the increase of plasma delta 4-androstenedione was important and dose dependent. DHEA was also converted into the potent sex steroid testosterone (T). The administration of a 50 mg dose of DHEA restored plasma T to levels similar to those observed in young women. The 200 mg dose induced an important increase of plasma T, slightly below the levels observed in normal men. The increase of plasma dihydrotestosterone levels was small at both doses of DHEA, in contrast with the large conversion of DHEA into androsterone glucuronide and androstanediol glucuronide. Finally, DHEA administration induced a significant and dose dependent increase of plasma estrogens and particularly of estradiol. In conclusion, this short term study demonstrates that: 1) panhypopituitarism is a model of interest to study the metabolism of DHEA; 2) in the absence of pituitary hormones and of adrenal and gonadal steroids, DHEA given orally is mainly converted into delta 4 derivatives, which in turn are strongly metabolized into 5 alpha-3keto-reduced steroids; 3) a significant increase of sex active hormones was observed in plasma after 200 and even 50 mg of DHEA. Thus, biotransformation of DHEA into potent androgens and estrogens may explain several of the reported beneficial actions of this steroid in aging people.

N-Methyl-D-aspartate (NMDA, 200 microM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the high-affinity sigma inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of > or = 100 nM. Neither 3 alpha-hydroxy-5 alpha-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H]NE overflow. The sigma antagonists haloperidol and 1-[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine, although inactive by themselves, completely prevented the effects of DHEA S, PREG S, and 1,3-di(2-tolyl)guanidine on NMDA-evoked [3H]NE release. Progesterone (100 nM) mimicked the antagonistic effect of haloperidol and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-piperazine. These results indicate that the tested steroid sulfate esters differentially affected the NMDA response in vitro and suggest that DHEA S acts as a sigma agonist, that PREG S acts as a sigma inverse agonist, and that progesterone may act as a sigma antagonist. Pertussis toxin, which inactivates the Gi/o types of guanine nucleotide-binding protein (Gi/o protein) function, suppresses both effects of DHEA S and PREG S. Since sigma 1 but not sigma 2 receptors are coupled to Gi/o proteins, the present results suggest that DHEA S and PREG S control the NMDA response via sigma 1 receptors.

Ninety-four institutionalized subjects (mean age 82.9 years; 13 men and 81 women) were assessed for serum dehydroepiandrosterone sulfate (DHEAS) levels. The mean value was 457 ng/ml (SD = 426) in this cross-sectional study. There was a significant decrease in DHEAS levels with age. There was no difference among demented patients with Alzheimer's disease (n = 46), other dementia cases (n = 16), and other individuals (n = 32). The subjects treated for arterial hypertension had significantly lower DHEAS levels. No correlation was found between DHEAS and cortisol values. After age adjustment, no association was observed between DHEAS and chronic diseases, medications, several biochemical or hematological parameters, or 3-year mortality.

To assess the significance of the serum dehydroepiandrosterone sulfate (DHEAS) concentration as a parameter of the individual hormonal milieu, two different groups of subjects were studied: DHEAS levels were determined 3 times at 6-month intervals in 47 elderly hospitalized women, aged 71-94 yr (group 1), and 6 times over 2 consecutive weeks in 10 healthy male volunteers, aged 24-30 yr (group 2). For reference, serum cortisol (F) levels were determined concomitantly. In each group and on each sampling occasion, the subjects were ranked according to their DHEAS or F values. The stability over time of the ranking was much higher for DHEAS than for F; estimated concordance coefficients were 92% (group 1) and 88% (group 2) for DHEAS vs. 51% (group 1) and 49% (group 2) for F. We conclude that due to a comparatively low within- to between-subject variability ratio, DHEAS is a highly specific individual marker.

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3-ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha-OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta-HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Two 'neurosteroids', dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the 'twin ion' technique of g.l.c.-m.s. with deuterated substrates. The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA. Under optimal conditions (pH 7.4, 0.5 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 13.8 and 4.4 microM respectively, and the Vmax. values are 322 and 38.8 pmol/min per mg of microsomal protein respectively. Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected. Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation. Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults. Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive. Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain.

Dehydroepiandrosterone (DHA) and 3 beta-methyl-androst-5-en-17-one (CH3-DHA) suppress attacks by castrated male mice towards lactating female intruders (Haug et al. Physiol. Behav. 46:955, 1989). Their effects on the concentrations of DHA, pregnenolone (delta 5P), and their sulfate ester (S) have been investigated in the brain of control and treated mice. A more than 2 fold, significant, decrease of delta 5P-S was the only change common to both steroids. DHA and CH3-DHA effects might be related to the antagonistic action of delta 5P-S on GABAA receptors.

Female mice of an inbred Swiss strain were injected subcutaneously (s.c.) with either testosterone propionate or oil-vehicle on the day of birth. Once adult (8 week-old), they were spayed, and then treated either with dehydroepiandrosterone (80 micrograms/day s.c. for 15 days) or with solvent. The decrease of biting attacks by dehydroepiandrosterone was definitely more prominent in neonatally imprinted females than in controls.

Pregnenolone (delta 5-P) and dehydroepiandrosterone (DHEA) were measured in the limbic system of young adult male rats (M) exposed for several days to the scent of cycling females but in absence of sexual contacts (M/F), and compared to levels obtained in males similarly exposed to other males (M/M). delta 5-P was highest in the olfactory bulbs of M/M, as compared to other regions of the limbic system. It decreased greater than 50% in M/F olfactory bulbs, but was identical in the olfactory tubercles, the amygdalas and the hypothalamus of M/M and M/F, as well as in the plasma, the adrenals and the spleen (taken as a representative non-endocrine organ). In comparison with M/M levels, DHEA selectively increased in the hypothalamus of M/F. These results demonstrate very different steroid concentrations in different regions of the brain, and they reveal their selective and eventually opposite changes upon heterosexual exposure. Therefore, they suggest regulatory mechanisms specific to various parts of the brain which are not directly related to the hormonal levels in the blood, and which could be part of the response to still undefined signals emitted by animals of the other sex.

Dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, I) sulfate (Ia) has been characterized in the anterior and the posterior parts of the brain of adult male rats. Its level (1.58 +/- 0.14 and 4.89 +/- 1.06 ng/g, mean +/- SD, in anterior and posterior brain, respectively) largely exceeded that of I in brain (0.42 +/- 0.10 and 0.12 +/- 0.03 ng/g in anterior and posterior brain, respectively) and of Ia in plasma (0.26 +/- 0.13 ng/ml). Brain Ia level did not seem to depend on adrenal secretion; it was unchanged after administration of corticotropin or dexamethasone for 3 days, and no meaningful change occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. In contrast, stress conditions prevailing 2 days after adrenalectomy plus orchiectomy or after the corresponding sham operation resulted in a significantly increased concentration of Ia in the brain. Changes of Ia level in brain occurred irrespective of changes in corresponding plasma samples. It is proposed that Ia formation or accumulation (or both) in the rat brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system.

Triads of castrated male mice were chronically administered with either oil vehicle or 280 nmol of dehydroepiandrosterone (D) or pregnenolone (P). They were tested for their attack on a lactating intruder female introduced in their home-cage 2, 24 or 47 hr after their last injection. D significantly reduced male aggressive behavior for at least 24 hr. Other groups of castrated males were daily treated with vehicle or 280 nmol of D, dehydroepiandrosterone sulfate (DS) or androstenediol (ADIOL), a D metabolite with clear-cut oestrogenic properties. D, but neither DS nor ADIOL, significantly reduced their aggressive responses to lactating intruders. Finally, neural levels of D, DS and testosterone (T) were measured in intact and castrated males injected with either vehicle or D. Measurable amounts of D and DS were detected, with DS being the predominant chemical form. D and DS concentrations were unchanged by castration but neural D was increased more than twenty fold in castrates treated with D, whereas DS was unchanged. The concentration of T in the brain of the intact (sham-operated) mouse approached 3 ng/g but fell close to the detection limit after gonadectomy. D treatment caused a slight but significant increase in brain T concentration in castrated mice, although T remained far below the level measured in intact males.

In North American women at low or high risk of developing breast cancer, as assessed by an epidemiologic questionnaire, the plasma concentration of dehydroepiandrosterone sulfate shows a statistically significant circannual variation. In adolescents, in all seasons, circulating dehydroepiandrosterone sulfate is a classifier of the risk of developing breast cancer, a relatively low concentration of this hormone being associated with an increased risk.

Dehydroepiandrosterone, either unconjugated (D) or conjugated to sulfuric acid (DS), has been identified in the brain of male Mice; DS had been previously found in Rat brain. DS amounts in posterior brain, were 0.8-2.0 ng/g in controls, and very much lower in dysmyelinic jimpy and quaking Mice of the same age. Conversely, amounts of D were increased in affected Mice, suggesting an impaired sulfoconjugation. Results may be explained by accumulation of D and DS in brain, unrelated to the endocrine system.

In adrenalectomized and orchidectomized rats, the removal of the steroidogenic endocrine glands is associated with a near-disappearance of corticosterone (B) from plasma and brain; circadian variations of B and of plasma dehydroepiandrosterone (D), characteristic of the intact rat, are no more detected. By contrast, analyses of brain D measurements by the cosinor method demonstrate a persisting circadian rhythm of large amplitude.

The rat brain contains large amounts of pregnenolone (P) and dehydroepiandrosterone (D) arising from local biosynthetic pathways. We have devised a procedure for the measurement of both "neurosteroids" either unconjugated or released from their sulfate (S) or fatty acid (L) esters. The measurements were performed at the acrophase of the circadian variation of neurosteroids, and confirmed the large accumulation of P (25 +/- 8 ng/g, mean +/- SD) and of PS (19 +/- 6 ng/g) and DS (2.1 +/- 0.5 ng/g) in the brain of adult male rats. We found that fatty acid esters constitute the major species of neurosteroids in brain (PL 46 +/- 14, and DL 36 +/- 7 ng/g, in adult males). The levels of P and DS were increased by daily injection of vehicle to intact males, whereas castration, without or with testosterone or estradiol supplementation (2 mg daily for 7 days), did not produce a significant change of neurosteroids concentrations. Measurements of neurosteroids had not been previously reported in cyclic females. The levels of P, PL, and DS were identical in proestrous females and in intact males, whereas PS (26 +/- 6 ng/g) and DL (50 +/- 16 ng/g) were increased in females. Compared to proestrous females, diestrous females had lower levels of PS (19 +/- 6 ng/g), DS (1.7 +/- 0.4 ng/g), and PL (43 +/- 19 ng/g). These differences suggested a modulatory role of ovarian secretions on the metabolism of neurosteroids.

Three experiments were performed. In Experiment I, triads of adult castrated male mice were chronically administered with either oil-vehicle or 80 micrograms/day of dehydroepiandrosterone (D) or 3-beta-methylandrost-5-en-17-one (D-CH3). They were subsequently tested for their attack on a lactating intruder female introduced for 15 min in their home-cage, 2 hr after their last injection. Both D and D-CH3 significantly reduced male aggressive responses (cf. oil-injected category). In Experiment II the effects of D-CH3 dosed at 20, 40 or 80 micrograms/day were measured in the same testing situation. D-CH3 dose-dependently reduced the mice attack probability on lactating intruders with the highest dose being the most effective. Finally, to test in Experiment III that D-CH3 was not demonstrably androgenic, castrated males received daily injections of 20, 40 or 80 micrograms of this synthetic steroid for 4, 8, 16 or 32 days before tissue sampling. At all doses and whatever treatment duration, D-CH3 did not significantly increase the weights of the accessory sex organs.