BACKGROUND

GABAA receptors are an important site of action of endogenous neurosteroids and an important mediator of several behavioral effects of alcohol. This study examined the effects of alcohol on plasma steroid hormone concentrations on the hypothesis that the endocrine effects mediate some of the subjective effects of alcohol.

METHODS

Thirty-two healthy subjects (17 men) with no history of a substance use disorder participated in this human laboratory study. All subjects consumed three standard drinks of grain alcohol. Subjective measures and blood samples for steroid concentrations were collected at baseline and 40 min after alcohol consumption.

RESULTS

Alcohol increased self-reported stimulation, alcohol liking, and desire for more alcohol. Alcohol also increased pregnenolone (PREG) and dehydroepiandrosterone (DHEA) concentrations, while it decreased progesterone (PROG) and allopregnanolone (ALLO) concentrations, as well as ALLO/PREG and PROG/PREG ratios. In men, the change in PREG concentration was significantly correlated with alcohol liking, while the alcohol-induced change in ALLO concentration correlated significantly with both alcohol liking and desire for more alcohol.

CONCLUSIONS

These findings provide preliminary support for the hypothesis that endogenous neurosteroids mediate some of the subjective effects of alcohol. Efforts to replicate these findings should aim to specify more clearly the nature and time course of the effects.

OBJECTIVE

PSC has characteristics of an (auto)immune-mediated disease: however, few studies have evaluated corticosteroid therapy for this disorder.

METHODS

We performed an 8-wk double-blind randomized pilot study to assess the effects of additional treatment with 9 mg budesonide (n = 6) versus 3 mg budesonide (n = 6) versus 10 mg prednisone (n = 6) in patients who had been treated with UDCA (mean dose, 12 mg/kg/day) for at least 5 months without achieving biochemical remission. Pruritus and fatigue were evaluated using visual analog scales. Serum liver biochemistry was measured every 4 wk. At entry and at the end of the trial, adrenocorticotrophic hormone (ACTH) and dehydroepiandrosterone (DHEA) were measured to assess effects on the pituitary-adrenal axis. Duodenal bile was collected for assessment of biliary corticosteroid activity.

RESULTS

Pruritus decreased significantly more in the prednisone group compared to both the 3-mg and the 9-mg budesonide groups (p < 0.05). Alkaline phosphatase (mean: -23.4%; p = 0.03) and IgG (mean: -16.2%; p = 0.04) decreased in the prednisone group, whereas bilirubin, gamma-glutamyl transferase, aspartate aminotransferase, and alanine aminotransferase did not change significantly. No significant clinical or liver biochemical changes were observed in the 3-mg and 9-mg budesonide groups. Significantly larger drops in serum ACTH were found in the 10-mg prednisone group (-40.7%; p = 0.04) and 9-mg budesonide group (-36.6%; p = 0.02) compared to the 3-mg budesonide group (+ 19.0%). No significant differences in percentage change in baseline values for DHEA between the three treatment arms were found. Mononuclear cell proliferation assays did not demonstrate corticosteroid activity in bile. Autoimmune hepatitis was observed in one case (9 mg budesonide) when corticosteroids were tapered off.

CONCLUSIONS

The results of this pilot study suggest only minor beneficial short-term effects of prednisone but not budesonide on symptoms and serum liver tests in UDCA-treated PSC patients.

Reduced pregnane neurosteroids such as allopregnanolone and pregnanolone are potent neuromodulators able to affect a number of membrane receptors, including gamma-aminobutyric acid (GABA)(A), N-methyl-D-aspartate (NMDA), 5-hydroxytryptamine (5-HT)(3), and sigma(1) receptors. The present study used a drug discrimination procedure to assess further the receptor effects of pregnanolone in vivo. Rats were trained to discriminate 5 mg/kg pregnanolone from saline in a two-lever operant task maintained by food reinforcement. The opiate agonist morphine and the negative GABA(A) modulator dehydroepiandrosterone sulfate did not substitute for pregnanolone. All of the GABA(A) positive modulators tested (allopregnanolone, epipregnanolone, androsterone, pentobarbital, midazolam, and zolpidem) dose dependently substituted for pregnanolone. The direct GABA-site agonists 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol and muscimol failed to substitute for pregnanolone. Ethanol and the sigma(1) receptor agonist SKF 10047 fully substituted for pregnanolone, and the NMDA antagonist MK-801 partially substituted for pregnanolone. The 5-HT(3) antagonist tropisetron did not substitute at any dose tested. The 5-HT(3) agonist SR 57227A reached full substitution, whereas the other 5-HT(3) agonist tested, m-chlorophenylbiguanide, produced partial substitution. These results suggest that positive GABA(A) modulation, but not direct agonism, confers a discriminative stimulus effect similar to pregnanolone. Additionally, antagonism of NMDA receptors and activation of 5-HT(3) and sigma(1) receptors modulate stimulus effects similar to the pregnanolone cue. Overall, the data suggest that pregnanolone produces discriminative stimulus effects representative of a wide-spectrum sedative hypnotic.

During the course of gestation the increase of maternal total blood volume and cardiac output may result from two mechanisms acting in concert: 1) the production of several hormones by the fetus and the placenta, and 2) the uteroplacental circulation acting as an arteriovenous shunt. Plasma volume appears to increase as a consequence of renal Na+ reabsorption and water retention, which result from increased aldosterone production via the renin-angiotensin system, as a consequence of placental estrogen production. This estrogen production in turn results from increasing availability of the estrogen substrate dehydroepiandrosterone (chiefly from the fetal adrenal gland). Erythrocyte volume increases as a consequence of the erythropoietic effect of placental chorionic somatomammotropin, progesterone, and perhaps prolactin. In addition, maternal blood volume increases in response to the uteroplacental circulation functioning as a low-resistance circuit. In turn, this increases cardiac output and nutrient delivery for further growth of the products of gestation. Thus there may exist a feedback mechanism whereby fetal growth and, in particular, increasing steroidogenesis by the developing fetal adrenal gland result in the maternal cardiovascular adaptations to pregnancy that optimize further fetal development. In certain complications of pregnancy the less-than-normal maternal blood volume increase may result from failure of these mechanisms, while in turn contributing to the further genesis of these disorders.

Dehydroepiandrosterone (DHEA) may be beneficial in cardiovascular health, but mechanisms of DHEA action in the cardiovascular system are unclear. We have therefore 1) determined DHEA effects on the proliferation of cultured endothelial cells (EC), 2) compared effects of DHEA with estradiol (E) and testosterone (T), and 3) examined DHEA effects on subcellular messengers. We have in addition examined effects of DHEA (100 mg/d, 3 months) in 36 healthy postmenopausal women on blood pressure, lipids, and endothelial function, assessed noninvasively in large vessels by flow-mediated dilation of the brachial artery during reactive hyperemia, and in small vessels by laser Doppler velocimetry with iontophoresis of acetylcholine. DHEA, E, and T all increased EC proliferation; the effect of E was abolished by the estrogen receptor antagonist ICI 182,780, and that of T was abolished by the androgen receptor antagonist flutamide; neither blocked the effect of DHEA. In vitro, DHEA increased EC expression of endothelial nitric oxide synthase and activity of extracellular signal-regulated kinase 1/2. In vivo, DHEA increased flow-mediated dilation and laser Doppler velocimetry and reduced total plasma cholesterol. Thus, DHEA increases EC proliferation in vitro by mechanism(s) independently of either androgen receptor or estrogen receptor and in vivo enhances large and small vessel EC function in postmenopausal women.

Men (n = 31), women estrogen-users (n = 14), and women estrogen non-users (n = 41), whose average age was 72.1 +/- 5.6 years, were tested with a battery of psychological tests measuring verbal memory, visual memory, concentration and attention, language fluency and semantic memory, and mood. Plasma levels of testosterone (T), estradiol (E2), cortisol (CRT) and dehydroepiandrosterone-sulfate (DHEAS) were assessed by radioimmunoassay. The ratio of DHEAS to CRT was calculated to determine it's relationship to memory functioning. The men had higher T and DHEAS levels than both groups of women. Women estrogen-users had higher E2 levels than both men and estrogen non-users and the men had higher E2 levels and a higher DHEAS/CRT ratio than the estrogen non-users. There were no group differences in CRT levels. Men and estrogen-users had higher total (p < .01) and forward (p < .001) digit span scores compared with non-users. Women estrogen-users also had higher backward digit span scores than non-users (p < .05), while both groups of women performed better than men on category retrieval (p < .01). The implications of these findings with respect to hormonal influences on memory in elderly men and women are discussed.

The influence of endogenous androgens on atherosclerotic disease in women is unknown. In this study involving 101 pre- and post-menopausal females, we evaluated the relationship between serum androgen levels and both carotid artery intimal-medial thickness (IMT) and major cardiovascular risk factors. In addition to evaluation of blood pressure, body mass index, and waist-to-hip ratio, serum dehydroepiandrosterone sulfate (DHEA-S), androstenedione (A), total testosterone (TTS), free testosterone (FTS), insulin, cholesterol (total and high density lipoproteins), triglycerides, and glucose were measured. All women underwent carotid ultrasonography. Spearman correlation coefficients showed that serum DHEA-S and A levels were negatively related (P < 0.03-0.0004) to several IMT measures. Higher tertiles of DHEA-S, A, and FTS corresponded to significantly lower measures of carotid thickness. DHEA-S, and all androgens were inversely related to age (P < 0.03 or less), showing no unfavorable association with major cardiovascular risk factors. In contrast, serum DHEA-S was negatively associated with WHR (P < 0.02), while A was negatively associated with body mass index (P < 0.02). Stepwise multiple regression analysis indicated that A and FTS showed an inverse association with IMT measures (P < 0.05-0.001). In conclusion, our data indicate that in women serum DHEA-S and androgens decline with age and that normal hormonal levels are not associated with major cardiovascular risk factors. They also show that higher DHEA-S and androgen concentrations are related to lower carotid wall thickness; for A this association is independent of cardiovascular risk factors. Our results suggest that, in the physiological range, DHEA-S and androgens in women are correlated with lower risk of carotid artery atherosclerosis.

Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.

In the golden hamster, there are marked sex differences in the Harderian gland. Male glands (which are heavier than female glands) possess two cell forms (Type I and Type II cells); female glands only exhibit the former. Female (but not male) glands contain large amounts of porphyrin, which are readily visible as solid depositions within the lumina. The weight, histology and porphyrin content of the Harderian gland was examined in intact adult male hamsters and in male hamsters castrated for 1,2 or 8 months. Castration resulted in a significant reduction in the weight of the gland, the disappearance of Type II cells, and the presence in the gland of solid porphyrin accretions. The levels of copro- and (especially) protoporphyrin were greatly increased. These changes were more marked with time after castration. When the ability of diverse androgens (testosterone, 5alpha-dihydrotestosterone, androst-4-ene-3,17-dione (androstenedione), dehydroepiandrosterone and androsterone) to prevent these changes was tested, testosterone and androstenedione maintained glandular weight. All the androgens maintained normal frequencies of Type II cells and all except dehydroepiandrosterone prevented deposition of porphyrin. The potencies of the various androgens in maintaining normal Harderian gland morphology and activity are compared with their effects on other somatic variables and sexual behaviour.

Ergogenic drugs are substances that are used to enhance athletic performance. These drugs include illicit substances as well as compounds that are marketed as nutritional supplements. Many such drugs have been used widely by professional and elite athletes for several decades. However, in recent years, research indicates that younger athletes are increasingly experimenting with these drugs to improve both appearance and athletic abilities. Ergogenic drugs that are commonly used by youths today include anabolic-androgenic steroids, steroid precursors (androstenedione and dehydroepiandrosterone), growth hormone, creatine, and ephedra alkaloids. Reviewing the literature to date, it is clear that children are exposed to these substances at younger ages than in years past, with use starting as early as middle school. Anabolic steroids and creatine do offer potential gains in body mass and strength but risk adverse effects to multiple organ systems. Steroid precursors, growth hormone, and ephedra alkaloids have not been proven to enhance any athletic measures, whereas they do impart many risks to their users. To combat this drug abuse, there have been recent changes in the legal status of several substances, changes in the rules of youth athletics including drug testing of high school students, and educational initiatives designed for the young athlete. This article summarizes the current literature regarding these ergogenic substances and details their use, effects, risks, and legal standing.

Opioid therapy is one of the most effective forms of analgesia currently in use. In the past few decades, the use of opioids as a long-term treatment for chronic pain has increased dramatically. Accompanying this upsurge in the use of long-term opioid therapy has been an increase in the occurrence of opioid associated endocrinopathy, most commonly manifested as an androgen deficiency and therefore referred to as opioid associated androgen deficiency (OPIAD). This syndrome is characterized by the presence of inappropriately low levels of gonadotropins (follicle stimulating hormone and luteinizing hormone) leading to inadequate production of sex hormones, particularly testosterone. Symptoms that may manifest in patients with OPIAD include reduced libido, erectile dysfunction, fatigue, hot flashes, and depression. Physical findings may include reduced facial and body hair, anemia, decreased muscle mass, weight gain, and osteopenia or osteoporosis. Additionally, both men and women with OPIAD may suffer from infertility. While the literature regarding OPIAD remains limited, it is apparent that OPIAD is becoming increasingly prevalent among chronic opioid consumers but often goes unrecognized. OPIAD can have a significant negative impact on the the quality of life of opioid users, and clinicians should anticipate the potential for its occurrence whenever long-term opioid prescribing is undertaken. Once diagnosed, treatment for OPIAD may be offered utilizing a number of androgen replacement therapy options including a variety of testosterone preparations and, for female patients with OPIAD, dehydroepiandrosterone (DHEA) supplementation. Follow-up evaluation of patients receiving androgen replacement therapy should include a review of any unresolved symptoms of hypogonadism, laboratory evaluation, and surveillance for potential adverse effects of androgen replacement therapy including prostate disease in males.:

Over 50% of patients with the polycystic ovary syndrome (PCOS) demonstrate excess levels of adrenal androgens (AAs), particularly dehydroepiandrosterone sulfate (DHS). Nonetheless, the mechanism for the AA excess remains unclear. It has been noted that in PCOS the pituitary and ovarian responses to their respective trophic factors (i.e. GnRH and LH, respectively) are exaggerated. Similarly, we have postulated that excess AAs in PCOS arises from dysfunction of the hypothalamic-pituitary-adrenal axis, due to 1) exaggerated pituitary secretion of ACTH in response to hypothalamic CRH, 2) excess sensitivity/responsivity of AAs to ACTH stimulation, or 3) both. To test this hypothesis we studied 12 PCOS patients with AA excess (HI-DHS; DHS,>> 8.1 mumol/L or 3000 ng/mL), 12 PCOS patients without AA excess (LO-DHS; DHS, < 7.5 mumol/L or 2750 ng/mL), and 11 controls (normal subjects). Each subject underwent an acute 90-min ovine CRH stimulation test (1 microgram/kg) and an 8-h incremental i.v. stimulation with ACTH-(1-24) at doses ranging from 20-2880 ng/1.5 m2.h) with a final bolus of 0.25 mg. All patient groups had similar mean body mass indexes and ages, and both tests were performed in the morning during the follicular phase (days 3-10) of the same menstrual cycle, separated by 48-96 h. During the acute ovine CRH stimulation test, no significant differences in the net maximal response (i.e. change from baseline to peak level) for ACTH, dehydroepiandrosterone (DHA), androstenedione (A4), or cortisol (F) or for the DHA/ACTH, A4/ACTH, or F/ACTH ratios was observed. Nonetheless, the net response of DHA/F and the areas under the curve (AUCs) for DHA and DHA/F indicated a greater response for HI-DHS vs. LO-DHS or normal subjects. The AUC for A4 and A4/F and the delta A4/delta F ratio (delta = net maximum change) indicated that HI-DHS and LO-DHS had similar responses, which were greater than that of the normal subjects, although the difference between LO-DHS patients and normal subjects reached significance only for the AUC of the A4 response. No difference in the sensitivity (i.e. threshold or minimal stimulatory dose) to ACTH was noted between the groups for any of the steroids measured. Nonetheless, the average dose of ACTH-(1-24) required for a threshold response was higher for DHA than for F and A4 in all groups. No difference in mean responsivity (slope of response to incremental ACTH stimulation) was observed for DHA and F between study groups, whereas the responsivity of A4 was higher in HI-DHS patients than in normal or LO-DHS women. The net maximal and the overall (i.e. AUC) responses of DHA were greater for HI-DHS than for normal or LO-DHS women. The response of A4 and the delta A4/delta F ratio were greater for HI-DHS patients than for LO-DHS patients or normal subjects. Alternatively, HI-DHS and LO-DHS patients had similar overall responses (i.e. AUC) for A4 or A4/F, although both were greater than those of normal subjects. The relative differences in response to incremental ACTH stimulation between steroids was consistent for all subject groups studied, i.e. A4>> F or DHA. In conclusion, our data suggest that AA excess in PCOS patients is related to an exaggerated secretory response of the adrenal cortex for DHA and A4, but not to an altered pituitary responsivity to CRH or to increased sensitivity of these AAs to ACTH stimulation. Whether the increased responsivity to ACTH for these steroids is secondary to increased zonae reticularis mass or to differences in P450c17 alpha activity, particularly of the delta 4 pathway, remains to be determined.

To assess the effects of age on both the pituitary ACTH response to corticotropin-releasing hormone (CRH) and the secretory responses of cortisol (F) and dehydroepiandrosterone (DHEA) to endogenous rises in ACTH, we measured evening basal and ovine CRH (oCRH; 1 mu/kg)-stimulated plasma concentrations of ACTH,F, and DHEA in 49 healthy men, aged 21-86 yr. By analysis of variance, we found no change with age in either the basal concentration of ACTH or the magnitude of the peak ACTH response to oCRH. Older men had higher basal F levels (P less than 0.05), while basal plasma levels of CBG and ratios of F to CBG did not vary significantly with age (P greater than 0.1). We also found no significant increase with age in the magnitude of the peak F response to oCRH (P greater than 0.2), although peak F responses occurred significantly earlier (P less than 0.03) in the older men. Basal plasma levels of DHEA decreased significantly with age (P less than 0.001), as did the magnitude of peak DHEA responses to endogenous ACTH rises (P less than 0.01). There was no alteration in the timing of the peak DHEA response with age (P greater than 0.7). We conclude that while ACTH and F responses to evening injections of oCRH are well maintained in healthy aging men, that of DHEA is discordantly decreased. The present findings are compatible with the hypotheses that there is a diminished sensitivity of ACTH secretion to negative feedback regulation by glucocorticoids in older men, and there is an ACTH-independent age-related diminution in adrenal androgen secretion.

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.

Experiments were designed to investigate the influence of estrous cycle and gender of the rat on the effects of a gamma-aminobutyric acid type A (GABA(A)) receptor active neurosteroid, 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), the benzodiazepine, triazolam, and a GABA(A) receptor antagonistic neurosteroid, delta5-androsten-3beta-ol-17-one sulfate (dehydroepiandrosterone sulfate), on food intake and elevated plus-maze learning behaviors. Allopregnanolone (0.25 mg/kg, s.c.) and triazolam (0.25 mg/kg, i.p.) produced a hyperphagic effect, while dehydroepiandrosterone sulfate (5 mg/kg, s.c.) elicited an anorectic effect. However, allopregnanolone was more potent in diestrous females, whereas triazolam exhibited significantly higher hyperphagic potency in estrus females. The extent of anorexia following dehydroepiandrosterone sulfate was alike in male and female rats. The triazolam- and allopregnanolone-induced hyperphagic effect was blocked by bicuculline (1 mg/kg, i.p.), a selective GABA(A) receptor antagonist. In contrast to triazolam, the hyperphagic effect of allopregnanolone was insensitive to flumazenil (5 mg/kg, i.p.), a benzodiazepine antagonist. Vehicle-treated diestrous rats displayed moderately higher latencies in the elevated plus-maze learning task than estrus or proestrus females. Although allopregnanolone and triazolam elicited equipotent learning deficits in plus-maze learning in male and female rats, the magnitude of impairment-induced by triazolam was significantly higher in diestrous females than proestrus females. Dehydroepiandrosterone sulfate enhanced memory performance only in male rats. Although the use of the elevated plus-maze as a learning paradigm with benzodiazepines and neurosteroids may be sensitive to changes in anxiety, the differential data suggest that neurosteroid-induced effects are at least partly specific to learning behavior. These results confirm the role of estrous cycle and sex of rats in modifying the potency of neurosteroids and benzodiazepines on food consumption and learning and memory processes.

The present study investigates the physiological significance of dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, androstenedione (Delta(4)), dihydrotestosterone (DHT), estrone (E1), and E2 on recombinant human FSH- (rhFSH) resistant type 4 follicles obtained from immature mice. Type 4 follicles of a diameter of 100-120 microm with one or two granulosa cell layers around the oocyte and an intact basal lamina with theca cells were isolated from the ovaries of 11-d-old BDF-1 mice and cultured with medium alone (control) or with dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, Delta(4), DHT, E1, or E2 at concentrations ranging from 1 x 10(-11) to 1 x 10(-7) M for 4 d. We examined the mean diameters of type 4 follicles, levels of immunoreactive (IR)-inhibin, and E2 and progesterone in the culture media on day 4. In addition, we evaluated follicular cell proliferation by immunofluorescence staining with 5-bromo-2'-deoxyuridine. All tested androgens significantly increased the diameter of type 4 follicles in a dose-dependent manner without the production of IR-inhibin and E2. The nuclei of granulosa cells in type 4 follicles cultured with all tested androgens exhibited intense 5-bromo-2'-deoxyuridine-positive staining, compared with those of controls. In contrast, neither E1 nor E2 had any stimulatory effects. The stimulatory effects of T, Delta(4), or DHT were inhibited by an AR antagonist in a dose-related fashion but not by an aromatase inhibitor. Furthermore, all tested androgens had a synergistic effect with rhFSH on follicular growth and the production of IR-inhibin and E2. These results demonstrated that neither adrenal nor ovarian androgens are arteriogenic but that they stimulate type 4 follicles unresponsive to rhFSH and augment the responsiveness of these follicles to rhFSH.

In 1979, the first prostate cancer patient was treated with a GnRH agonist at the Laval University Medical Center in Quebec City, Canada, thus rapidly leading to the worldwide replacement of surgical castration and high doses of estrogens. The discovery of medical castration with GnRH agonists was soon followed by fundamental changes in the endocrine therapy of prostate cancer. Most importantly, the excellent tolerance accompanying the treatment with GnRH agonists has been a key factor that permitted a series of studies demonstrating a major reduction in the death rate from prostate cancer ranging from 31 to 87% at 5 yr of follow-up in patients with localized or locally advanced prostate cancer. In fact, a one third reduction in prostate cancer deaths has been calculated in the metaanalysis of all available studies. The general acceptance of this discovery by patients and physicians is illustrated by world sales above 3.0 billion U.S. dollars in 2003. Although extremely efficient in achieving complete medical castration and well tolerated, with no other side effects than the expected hypoandrogenicity, GnRH agonists should not be administered alone. In fact, shortly after discovery of the castration effects of GnRH agonists, we observed that approximately 50% of androgens remain in the prostate after castration, thus leading to the recognition of the role of adrenal dehydroepiandrosterone as an important source of the androgens synthesized locally in the prostate and in many peripheral target tissues. We therefore developed combined androgen blockade (CAB), whereby the androgens of both testicular and adrenal origins are blocked simultaneously at start of treatment with the combination of a GnRH agonist to block the testis and a pure antiandrogen to block the action of the androgens produced locally. CAB, first used in advanced metastatic disease, has been the first treatment shown to prolong life in prostate cancer. Most interestingly, in 2002, we made the observation that CAB alone given continuously for 6.5 yr or more leads to cure of the disease in at least 90% of cases, thus suggesting that androgen blockade combining a GnRH agonist and a pure antiandrogen could well be the most efficient treatment of localized prostate cancer, and thus offering the possibility of practically eliminating death from prostate cancer.

Neurosteroids such as dehydroepiandrosterone (DHEA), pregnenolone and 17beta-estradiol are synthesized by cytochrome P450s from endogenous cholesterol. We previously reported a new cytochrome P450 enzyme, CYP7B, highly expressed in rat and mouse brain that metabolizes DHEA and related steroids by hydroxylation at the 7alpha position. Such 7-hydroxylation can enhance DHEA bioactivity in vivo. Here we show that the reaction is conserved across mammalian species: in addition to mouse and rat, DHEA hydroxylation activity was present in brain extracts from sheep, marmoset and human. Northern blotting using a human CYP7B complementary deoxyribonucleic acid (cDNA) probe confirmed the presence of CYP7B mRNA in marmoset and human hippocampus; CYP7B mRNA was present in marmoset cerebellum and brainstem, with lower levels in hypothalamus and cortex. In situ hybridization to human brain revealed higher levels of CYP7B mRNA in the hippocampus than in cerebellum, cortex, or other brain regions. We also measured CYP7B expression in Alzheimer's disease (AD). CYP7B mRNA was significantly decreased (approximately 50% decline; P<0.05) in dentate neurons from AD subjects compared with controls. A decline in CYP7B activity may contribute the loss of effects of DHEA with ageing and perhaps to the pathophysiology of AD.

BACKGROUND

Epidemiological studies show a substantially reduced risk of breast cancer in adult daughters of preeclamptic pregnancies, and modest risk reductions have been demonstrated for mothers also. Alterations in pregnancy hormone concentrations, particularly lower in utero exposure to oestrogen, are hypothesized to mediate this association.

METHODS

Pregnancy hormone concentrations were measured in maternal sera collected at hospital admission for labour and delivery from 86 preeclamptic and 86 uncomplicated, singleton pregnancies matched on length of gestation, maternal age, parity, and type of delivery.

RESULTS

Case and control pregnancies were similar in several maternal and pregnancy factors. Serum unconjugated oestradiol, oestrone, and oestriol concentrations were not lower in preeclamptic pregnancies in a matched analysis with adjustment for race and whether blood was collected before or after labour commenced. Serum unconjugated androstenedione (506.3 versus 316.0 ng/dl; P = 0.0007) and testosterone concentrations (214.5 versus 141.9 ng/dl; P = 0.004), however, were significantly higher in preeclamptic compared with control pregnancies, whereas dehydroepiandrosterone (DHEA) and DHEA sulphate did not differ.

CONCLUSIONS

These data do not support the hypothesis that cancer risk in mothers and offspring of preeclamptic pregnancies is explained by exposure to lower maternal blood oestrogen concentrations, but raise the possibility that androgens play a role.

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.

Plasma 17-hydroxyprogesterone (17PROG) hyperresponsiveness to GnRH agonist (nafarelin) testing is typical of polycystic ovary syndrome and other functional ovarian hyperandrogenism (FOH) that does not meet customary criteria for the diagnosis of polycystic ovary syndrome. We have postulated that this results from abnormal regulation of androgen secretion. Whether this dysregulation is the result of a normal physiological response to ovarian hyperstimulation or escape from down-regulation of steroidogenesis is unknown. To distinguish between these possibilities, we have analyzed the ovarian steroid responses to nafarelin for the apparent efficiency of the steroidogenic steps and the apparent dose-response relationships between blood LH and steroid levels. We compared normal women (n = 18) with three groups of hyperandrogenic women (n = 15-19/group): patients with 17PROG hyperresponsiveness with or without elevated LH levels (type 1 and type 2 FOH, respectively) and patients with normal 17PROG responses to nafarelin (nafarelin negative). Subjects were pretreated with dexamethasone to suppress coincidental adrenal contributions to plasma steroid levels. The pattern of steroid secretion was similarly abnormal in both types of FOH, with the high LH group having generally more severe abnormalities in the levels of steroid intermediates. Baseline 17PROG and 17-hydroxypregnenolone and the ratio of 17PROG to androstenedione (AD) were increased (P < 0.05). In addition, the apparent slope of the 17PROG response to LH was significantly increased. Baseline levels of both AD and dehydroepiandrosterone and the AD response to nafarelin were increased, yet the ratio of peak minus baseline (delta) AD/delta 17PROG (another index of 17,20-lyase activity) was subnormal in FOH. The apparent slope of the testosterone (T) response to LH was significantly increased, and indexes of aromatase activity [estradiol (E2)/T and delta estradiol/delta T] were significantly decreased. Nafarelin stimulated plasma E2 in all groups to rise along an apparently similar LH-E2 dose-response slope. We interpret these results as indicating that FOH patients have generalized overactivity of thecal steroidogenesis, but nevertheless compensate so as to maintain a normal dose-response relationship between blood levels of LH and E2. FOH patients, whether they have LH excess or not, seen to form excessive 17PROG and incompletely dampen (down-regulate) thecal cell 17PROG, AD, and T secretion in response to LH stimulation. 17PROG hyperresponsiveness to nafarelin seems to be prominent both because it is formed in excess and because 17,20-lyase efficiency is rate limiting. The T elevation seems to arise mainly from overactive steroidogenesis, but also partly from an additional functional decrease in aromatase efficiency, which is secondary to negative feedback by the substrate-driven tendency toward estrogen excess.(ABSTRACT TRUNCATED AT 400 WORDS)

Testosterone (T) and dehydroepiandrosterone (DHEA) are fat-reducing hormones, even though they exert this effect by different mechanisms. In particular, T inhibits lipid uptake and lipoprotein-lipase (LDL) activity in adipocytes, and stimulates lipolysis by increasing the number of lipolytic beta-adrenergic receptors. An indirect sign of these effects is the decrease of adipocyte leptin production. Lastly, T inhibits differentiation of adipocyte precursor cells. Concerning DHEA, this hormone does not seen to have any of T effects; however, DHEA stimulates resting metabolic rate (RMR) and lipid oxidation, and enhances glucose disposal, by increasing the expression of GLUT-1 and GLUT-4 on fat cell plasma membrane. The insulin-like effect of DHEA would be associated to a decrease of plasma insulin concentrations and, thus, to an increase of the molar ratio between lipolytic hormones and insulin. Noteworthy, the fat-reducing effect of both T and DHEA seems to be more evident at the level of visceral adipose tissue.

Our earlier studies have identified oligomycin sensitivity-conferring protein (OSCP), a subunit of proton F0F1-ATPase/ATP synthase in the mitochondrial inner membranes, as a new estradiol binding protein. This finding suggests that mitochondrial ATPase/ATP synthase could be a potential target for estradiol or compounds with similar structures. Here, we report that estradiol and several other compounds inhibited F0F1-ATPase activity of detergent-solubilized rat brain mitochondrial preparations in a following decreasing order: diethylstilbestrol (half-inhibition concentration, IC50 of 10-25 microM)>> alpha-zearalenol, 4-hydroxyestradiol (1C50 of 55 microM) >2-hydroxyestradiol (IC50 of 110 microM), 17beta-estradiol, 17alpha-estradiol>> beta-zearalanol>> estriol, testosterone, 16alpha-hydroxyestrone>> corticosterone, progesterone, dehydroepiandrosterone, dehydroepiandrosterone 3-sulfate, cholesterol (less than 10% inhibition at 140 microM). On the other hand, Na+, K+ -ATPase of porcine cortex showed different sensitivity to the compounds tested above. At 70 microM, the rank of inhibitory potency in decreasing order was as follows: 2-hydroxyestradiol (IC50 of 70 microM)>> diethylstilbestrol> 4-hydroxyestradiol>> progesterone>> alpha-zearalenol, while other compounds had little effect (less than 5%). The data indicate that the ubiquitous mitochondrial F0F1-ATPase is a specific target site for estradiol and related estrogenic compounds; however, under this in vitro condition, the effect seems to require pharmacological concentrations.

Eleven women (TRW; 64 +/- 4 yrs) and ten men (TRM; 65 +/- 5 yrs) participated in the strength/power training twice a week for 24 weeks. Basal concentrations of serum total and free testosterone, growth hormone (GH), dehydroepiandrosterone sulfate (DHEAS), cortisol and sex hormone binding globulin (SHBG) as well as acute responses of serum total and free testosterone, growth hormone (GH) were measured. Maximal 1RM strength in the squat, chair rise time and muscle fibre distribution and areas of type I and IIa and IIb of the vastus lateralis were also examined. 1RM squat increased in TRW by 26 (SD10)% (p < .001), and in TRM by 35 (7)% (p < .001) and chair rise time improved in both groups (p < .001). Fibre areas increased in type I, (p < .01), IIa (p < .01) and IIb (p < .01) in TRM and type I (p < .05) and IIa (p < .05) in TRW. The proportion of type IIa increased from 31% to 43% (p < .05) in TRW and that of type IIb decreased from 27% to 17% (p < .05) in TRW and from 25% to 17% (p < .05) in TRM. Individual concentrations of testosterone/cortisol ratios correlated (r = 0.63; p < .05) with the individual increases in 1RM strength in TRW. The exercise sessions resulted in acute increases in serum GH in both groups (p < .05) with a further increase (p < .01) up to 10 minutes post-loading in TRM at post-training.