Major Adrenal Steroid Research Articles

Profiling of anabolic androgenic steroids and selective androgen receptor modulators for interference with adrenal steroidogenesis

Anabolic-androgenic steroids (AAS) are testosterone derivatives developed for steroid-replacement and treatment of debilitating conditions. They are widely used by athletes in elite sports and bodybuilding due to their muscle-building and performance-enhancing properties. Excessive AAS use is associated with cardiovascular diseases, mood changes, endocrine and metabolic disorders; however, the underlying mechanisms remain unknown. Selective androgen receptor modulators (SARMs) aim to reduce adverse androgenic effects, while maximizing anabolic effects. This study assessed potential steroidogenic disturbances of 19 AAS and 3 SARMs in human adrenocortical carcinoma H295R cells, comparing basal and forskolin-activated states by mass spectrometry-based quantification of nine major adrenal steroids. Mesterolone, mestanolone and methenolone increased mineralocorticoid but decreased adrenal androgen production, indicating CYP17A1 dysfunction. Cell-free activity assays failed to detect direct CYP17A1 inhibition, supported by molecular modeling. The mRNA expression levels of 3β-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2 were unaffected, suggesting indirect inhibition involving post-translational modification and/or impaired protein stability. Clostebol and oxymetholone decreased corticosteroid but increased dehydroepiandrosterone biosynthesis in H295R cells, suggesting CYP21A2 inhibition, sustained by molecular modeling. These AAS did not affect the expression of key steroidogenic genes. None of the SARMs tested interfered with steroidogenesis. The chosen approach allowed the grouping of AAS according to their steroidogenic-disrupting effects and provided initial mechanistic information. Mesterolone, mestanolone and methenolone potentially promote hypertension and cardiovascular diseases via excessive mineralocorticoid biosynthesis. Clostebol and oxymetholone might cause metabolic disturbances by suppressing corticosteroid production, resulting in adrenal hyperplasia. The non-steroidal SARMs exhibit an improved safety profile and represent a preferred therapeutic option.

Changes in plasma concentrations of progesterone, dehydroepiandrosterone and corticosterone in response to acute stress of capture, handling and restraint in two subspecies of white-crowned sparrows

The aim of this study was to determine circulating patterns of the three major adrenal steroids in blood in response to stress during acute restraint handling in two subspecies of white-crowned sparrow. Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) are long distance migrants that breed at high latitudes and Nuttall's white-crowned sparrows (Zonotrichia leucophrys nuttalli) are residents of coastal California. Column partition chromatography was developed to separate progesterone, dehydroepiandrosterone (DHEA), and corticosterone from a small plasma sample. Each of these steroids has the capability to modulate the stress response through various mechanisms. For example, progesterone is bound to corticosterone binding globulin (CBG) with a higher affinity than corticosterone. If plasma levels of progesterone rise during acute stress, then this could displace corticosterone from CBG and increase the amount of biologically active, free, corticosterone in blood. Dehydroepiandrosterone (DHEA) has been implicated to have many anti-stress properties with the potential to mitigate some of the actions of corticosterone. Results indicate that progesterone levels in both subspecies are elevated in response to acute stress handling. DHEA levels declined in Gambel's but did not change in Nuttall's. Thus DHEA does not follow the same secretory pattern as in mammals. Corticosterone levels were elevated in response to acute stress handling in both subspecies. This study provides new insight into an integrated stress response among three steroids.

Hormone Supplementation During Aging: How Much and When?

Circulating levels of dehydroepiandrosterone, a major adrenal steroid, show a marked age-related decrease in both humans and nonhuman primates. Because this decrease has been implicated in age-related cognitive decline, we administered supplementary dehydroepiandrosterone to perimenopausal rhesus macaques (Macaca mulatta) to test for cognitive benefits. Although recognition memory improved, there was no benefit to spatial working memory. To address the limitations of this study we developed a hormone supplementation regimen in aged male macaques that more accurately replicates the 24-hr androgen profiles of young animals. We hypothesize that this more comprehensive physiological hormone replacement paradigm will enhance cognitive function in the elderly.

Neurosteroid Hydroxylase CYP7B: VIVID REPORTER ACTIVITY IN DENTATE GYRUS OF GENE-TARGETED MICE AND ABOLITION OF A WIDESPREAD PATHWAY OF STEROID AND OXYSTEROL HYDROXYLATION

The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7alpha-hydroxylation of oxysterols and 3beta-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7alpha-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7alpha, exemplified by prominent 6alpha-hydroxylation of 5alpha-androstane-3beta,17beta-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (beta-galactosidase) under Cyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7alpha-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7alpha-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6alpha-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7b gene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues.

Ontogeny of the neurosteroid enzyme Cyp7b in the mouse

The function of the major adrenal steroid dehydroepiandrosterone (DHEA) is not known. It has been reported to improve learning and memory in mice and can exert neuroprotective and trophic effects, particularly in the hippocampus. We recently described a cytochrome P450 (Cyp7b), that catalyses the 7α-hydroxylation of DHEA and related steroids and sterols. In this paper, we have used mRNA in situ hybridisation to map the ontogeny of cyp7b in the foetal and adult mouse. Cyp7b mRNA is highly expressed throughout from embryonal (E) day 12.5 (the earliest day studied). There is also expression throughout the body, including the spine, thymus, developing kidneys, lungs and urogenital region. Widespread expression becomes more restricted towards birth: in newborn mice expression is largely limited to the hippocampus, with some expression being detected in kidney. The overall decline in mRNA, and increasing restriction to the hippocampus, is reflected in the DHEA hydroxylation activity of brain homogenates. This pattern of cyp7b mRNA expression in specific organs could be consistent with a protective role in foetal development, with highest expression seen when the foetus is most vulnerable to steroid excess (i.e.) early gestation.

Obesity, blood pressure, and renal sodium handling.

A substantial body of work has been recently directed toward elucidation of the relationships between body weight, blood pressure, and renal and vascular function. In this review only a few of the myriad studies are highlighted in order to survey areas of current investigation and controversy. Epidemiologic studies have confirmed the blood pressure-body weight relationship but suggest a link to insulin resistance. The majority of investigative work in the mechanism and abnormalities in obesity and their link to blood pressure have focused on the kidney as the primary organ of interest. In addition to alterations in renal blood flow and function, the sympathetic nervous system has also been implicated. The role of the major adrenal steroid dehydroepiandrosterone (DHEA) has been examined, as have the roles of bradykinin and leptin, the exiting new polypeptide thought to play a role in obesity.

The actions of exogenous dehydroepiandrosterone in experimental animals and humans.

Dehydroepiandrosterone (DHEA) is the major adrenal steroid of young adults; however, its physiologic functions, if any, are not known. The purpose of this review is to evaluate the current literature in which DHEA was administered to either humans or experimental animals to discern what these functions might be. Reports are divided into five areas: neurologic, immunologic, cardiovascular, oncologic, and metabolic. Particular attention is paid to the dosage and route of administration. This type of analysis shows that at the lowest doses, DHEA has effects on neurologic and immunologic tissues, suggesting that these two sites may be physiologic targets. DHEA also affects cardiologic and metabolic functions as well as tumor growth, but such actions require higher doses and may reflect 'pharmacologic' activities. It is proposed that DHEA's pattern of activity represents a new class of steroid hormones, the "Regnantoids." Further progress in the endocrinology of this family of steroids may only come when synthetic, long-acting analogs of DHEA are available for in vitro studies to allow correlations between hormone action and receptor binding.

Multiple signal transduction systems regulate angiotensin II type 1 (AT1) receptor mRNA expression in bovine adrenocortical cells.

Regulation of AT1 receptor mRNA expression is an important determinant of angiotensin II-induced steroidogenesis. We have PCR-amplified the bovine adrenal AT1 receptor coding region using primers designed from the published bovine AT1 receptor sequence. This has been used as a probe on Northern blots to detect changes in the levels of AT1 receptor mRNA in primary cultures of bovine zona fasciculata cells in response to activation of several different signal transduction mechanisms in addition to two major adrenal steroid products, cortisol and aldosterone. AT1 receptor mRNA decreased in response to 6hr AII (10 nM) treatment, but returned to basal levels following 48h AII treatment. This effect was mimicked by the phorbol ester PMA (1 microM) and the calcium ionophore A23187 (1 microM), both singly and in combination. Activation of the cAMP pathway by ACTH (1 nM) and 8-bromo-cAMP (0.1 microM) also decreased AT1 receptor mRNA levels. In contrast, both IGF-1 (10 ng/ml) and potassium ions (12 mM) increased the levels of AT1 receptor mRNA. Finally, cortisol (10 microM) but not aldosterone (100 nM) decreased AT1 receptor mRNA. We conclude that the regulation of AT1 receptor mRNA in bovine zona fasciculata cells could involve several different signal transduction systems in addition to adrenocortical steroids themselves.

Gonadal Steroid Hormone Receptors and Sex Differences in the Hypothalamo-Pituitary-Adrenal Axis

The rapid activation of stress-responsive neuroendocrine systems is a basic reaction of animals to perturbations in their environment. One well-established response is that of the hypothalamo-pituitary-adrenal (HPA) axis. In rats, corticosterone is the major adrenal steroid secreted and is released in direct response to adrenocorticotropin (ACTH) secreted from the anterior pituitary gland. ACTH in turn is regulated by the hypothalamic factor, corticotropin-releasing hormone. A sex difference exists in the response of the HPA axis to stress, with females reacting more robustly than males. It has been demonstrated that in both sexes, products of the HPA axis inhibit reproductive function. Conversely, the sex differences in HPA function are in part due to differences in the circulating gonadal steroid hormone milieu. It appears that testosterone can act to inhibit HPA function, whereas estrogen can enhance HPA function. One mechanism by which androgens and estrogens modulate stress responses is through the binding to their cognate receptors in the central nervous system. The distribution and regulation of androgen and estrogen receptors within the CNS suggest possible sites and mechanisms by which gonadal steroid hormones can influence stress responses. In the case of androgens, data suggest that the control of the hypothalamic paraventricular nucleus is mediated trans-synaptically. For estrogen, modulation of the HPA axis may be due to changes in glucocorticoid receptor-mediated negative feedback mechanisms. The results of a variety of studies suggest that gonadal steroid hormones, particularly testosterone, modulate HPA activity in an attempt to prevent the deleterious effects of HPA activation on reproductive function.

The NCI-H295 cell line: a pluripotent model for human adrenocortical studies

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones has, in the past, required the isolation of cells from each of the adrenocortical zones. Indeed, the lack of a human adrenocortical cell line retaining the ability to produce any of the major adrenal steroid products has slowed studies on normal and abnormal adrenal function. This obstacle has now been largely overcome with the availability of H295 cells, which represents the first adrenocortical cell line to maintain the ability, under specified conditions, to produce all the adrenocortical steroids (i.e., mineralocorticoids, glucocorticoids, and adrenal androgens). Thus, H295 cells appear to act as pluripotent adrenocortical cells capable of being directed to produce each of the zone-specific steroids. The H295 cell line should prove to be of value in studying the molecular and biochemical mechanisms controlling adrenal Steroidogenesis.

Daily variation of corticosterone and thyroid hormones in broiler cockerels.

An experiment was conducted to monitor fluctuations in the major adrenal steroid, corticosterone (B), and two iodinated thyroid hormones, triiodothyronine (T3) and thyroxine (T4), over the course of a 24-hr day in broiler cockerels. A circadian variation was observed in B concentrations with the highest found at 0130 hr (23 hr light: 1 hr darkness; photophase beginning at 0100 hr). No circadian variation was observed for either T3 or T4.

Separation of the major adrenal steroids by reversed-phase high-performance liquid chromatography

The major adrenal steroids were separated by multistep gradient elution with a reversed-phase high-performance liquid chromatography system, employing water and 1-propanol as solvents. With this solvent system, a wide range of 21 5-ene-3β-ol and 4-ene-3-one steroids can be resolved in a single chromatogram, which was not possible with previously published gradient solvent systems. In particular, intermediate steroids of the biosynthetic pathway, 17α-hydroxyprogesterone, 17α-hydroxypregnenolone, and dehydroepiandrosterone, were separated with baseline or sufficient resolution to allow accurate quantitation. Using the 1-propanol-water gradient, the separations of 5-ene and 4-ene steroids were compared on different octadecylsilyl packings. Optimum resolution was obtained with a fully covered, spherical particle. The 1-propanol-water gradient was compared to a previously published methanol-water gradient in the analysis of steroidogenesis by adrenocortical cell cultures. HPLC analysis of the steroid production was quantitatively the same with both gradient solvent systems. However, qualitatively, the methanol-water gradient system did not resolve the above-mentioned intermediate steroids.

In vivo utilization of cholesteryl esters in low density lipoprotein for steroidogenesis in rhesus monkeys

To determine whether cholesteryl linoleate in circulating low density lipoprotein (LDL) is a precursor for adrenal, placental and ovarian steroid synthesis in vivo and is utilized in preference to unesterified cholesterol, rhesus monkeys ( Macaca mulatta) were injected during the luteal phase of the menstrual cycle and during midgestation with [ 14C]-cholesterol and LDL-labeled with [ 3H]-cholesteryl linoleate. During the first 30 min following injection, [ 3H]-cholesteryl linoleate and [ 14C]-cholesterol disappeared rapidly from the circulation of both groups of monkeys; however, between 1 and 6 h sterol levels remained nearly constant. During the entire time pregnant animals exhibited substantially lower plasma values of both sterols. After a delay [ 14C]-cholesteryl linoleate and [ 3H]-cholesterol appeared in the circulation of all animals. In nonpregnant monkeys [ 3H]-androstenedione (a major adrenal steroid) was abundant in the plasma, comprising 96% of the [ 3H]-steroids isolated. Small amounts of radioactivity were also noted in thin layer chromatogram regions occupied by 17-hydroxyprogesterone, dehydroepiandrosterone and progesterone standards. [ 14C]-Androstenedione and [ 14C]-progesterone were not detected. In contrast, plasma from pregnant monkeys contained little [ 3H]-androstenedione but relatively more [ 3H]-progesterone (a placental hormone) and unidentified metabolites. The results were interpreted to mean that circulating cholesteryl linoleate in LDL can be metabolized by the adrenal and placenta, while unassociated cholesterol is not readily available for steroid synthesis.

Reversed- and normal-phase high-performance liquid chromatography of 18-hydroxylated steroids and their derivatives : Comparison of selectivity, efficiency and recovery from biological samples

The chromatographic behaviour of adrenal 18-hydroxysteroids, and a series of derivatives thereof, has been studied in reversed-phase and normal-phase high-performance liquid chromatography (HPLC). Both 18-hydroxycorticosterone and 18-hydroxy-11-deoxycorticosterone exhibited a marked loss of chromatographic efficiency when separated on incompletely-covered C 18 reversed-phase packings with methanol-water gradient elution. Maximum-coverage reversed-phase supports (as determined by methyl red adsorption) showed no such effect, nor was it seen when aprotic solvents, such as dioxane, were used. This phenomenon is unique among the wide range of adrenal and testicular steroids that we have studied and affords a useful test, applicable under aqueous conditions, of coverage by the alkylsilane reversed phase, a factor of considerable importance in the successful resolution of complex mixtures of adrenal steroids by reversed-phase HPLC. The retention times of the 20-methoxy, 20-ethoxy, 21-acetoxy, etiolactone, 11β, 18-ether and dimeric derivatives of the naturally-occurring 18-hydroxysteroids have been determined in relation to major adrenal steroids. Procedures for extraction and reversed-phase HPLC of 18-hydroxysteroids from tissues without the formation of these less polar forms, which can complicate their separation by other chromatographic techniques, are illustrated with respect to a human malignant adrenocortical tumour which caused hypermineralocorticism.

Plasma Concentrations of Progesterone and Corticosterone During the Ovulation Cycle of the Hen (Gallus Domesticus)

The observation that injections of either ACTH or corticosterone induced ovulation in the hen has prompted speculation which implicates the adrenal gland in the mechanisms controlling ovulation. In this study, the role of the major adrenal steroid in birds was further examined by measuring the plasma concentration of corticosterone during the 24 hr period preceding midsequence ovipositions and the 28 hr period preceding terminal ovipositions. Progesterone, which is an accurate index of preovulatory ovarian activity, was also measured in the same samples.An increase in plasma progesterone preceded mid-sequence ovipositions which are accompanied by an ovulation. This preovulatory surge in plasma progesterone began approximately 7 hr before ovulation, reached a maximum of 6.9 ng/ml 3 to 2 hr before ovulation, and returned to baseline concentrations at the time of ovulation. A major preovulatory peak of progesterone was not observed during the interval between sequences, although a small but statistically significant rise in progesterone was observed between midnight and 0600 hr on the day of the last oviposition of the sequence. The concentration of corticosterone increased approximately two-fold during the dark portion of the photoperiod regardless of the position of the oviposition in the sequence. Baseline concentrations during the illuminated portion of the photoperiod ranged between 1 and 4 ng of corticosterone per ml of plasma whereas during the period of darkness, they ranged between 2 and 6 ng of corticosterone per ml of plasma. It was concluded, therefore, that the circulating concentration of corticosterone was regulated by a circadian rhythm which operates independently of follicular maturation.

Aldosterone, deoxycorticosterone and corticosterone differences between arteriosclerotic breeder vs nonarteriosclerotic virgin rats

The circulating levels of the major adrenal steroids, corticosterone (Cmpd. B), deoxycorticosterone (DOC), and aldosterone were compared in male and female, arteriosclerotic, breeder and non-arteriosclerotic virgin, Sprague—Dawley rats under resting conditons, i.e., quiescence, and after exposure to a mild stress, i.e., movement from one room to another just prior to autopsy. Under resting conditions, the arteriosclerotic animals had significantly greater circulating levels of Cmpd. B, DOC and aldosterone than the non-arteriosclerotic animals. Both the arteriosclerotic and non-arteriosclerotic animals were able to respond adequately to the mild stress stimulus. However, the female breeder rats which manifest the most severe aortic sclerosis, showed the greatest increase in Cmpd. B and aldosterone in response to the mild stress. Although no statistically significant differences could be found between female breeders with grossly visible aortic sclerosis of clear, minimal, moderate, or severe degree, it was apparent that adrenal steroid responsiveness becomes progressively compromised with increasing severity of arteriosclerosis, e.g., unusually high or low levels of Cmpd. B and aldosterone under both quiescent and mild stress conditions. It is suggested that there may be some connection between abnormal hypothalamic-pituitary-adrenal-gonadal function in repeatedly-bred and the pathogenesis of their naturally-occurring ateriosclerosis.

Adrenal steroidogenesis in the white-crowned sparrow ( Zonotrichia leucophrys)

1. 1. Evidence is presented for corticosterone as the major adrenal steroid produced by adrenal homogenates of the white-crowned sparrow ( Zonotrichia leucophrys) based on chromatographic behaviour and recrystallization. 2. 2. While traces of 17-hydroxycorticosteroid may be produced, its contribution to the total steroid production in this species is small.

Secretion of corticosterone in vitro by normothermic and hibernating ground squirrels

1. 1. Evidence is presented for corticosterone as the major adrenal steroid in the ground squirrel ( Citellus) based on Chromatographic behavior and crystallization data of the free and acetylated product obtained after incubation of 14C-labeled progesterone. 2. 2. A fluorometric analysis of the adrenal secretions after incubation in vitro demonstrated the expected response to ACTH by normothermic animals. 3. 3. The adrenals of hibernating animals did not respond to any level of ACTH employed.