Metabolites Of Dehydroepiandrosterone Research Articles

Dehydroepiandrosterone and Its Metabolite 5-Androstenediol: New Therapeutic Targets and Possibilities for Clinical Application.

Dehydroepiandrosterone and its sulfate are the most abundant steroids in humans. The metabolism of dehydroepiandrosterone can differ significantly depending on the organ or tissue and the subtype of steroid receptors expressed in it. For dehydroepiandrosterone, as a precursor of all steroid hormones, intracrine hormonal activity is inherent. This unique feature could be beneficial for the medicinal application, especially for the local treatment of various pathologies. At present, the clinical use of dehydroepiandrosterone is limited by its Intrarosa® (Quebec city, QC, Canada) prasterone) 6.5 mg vaginal suppositories for the treatment of vaginal atrophy and dyspareunia, while the dehydroepiandrosterone synthetic derivatives Triplex, BNN 27, and Fluasterone have the investigational status for the treatment of various diseases. Here, we discuss the molecular targets of dehydroepiandrosterone, which open future prospects to expand its indications for use. Dehydroepiandrosterone, as an oral drug, is surmised to have promise in the treatment of osteoporosis, cachexia, and sarcopenia, as does 10% unguent for skin and muscle regeneration. Also, 5-androstenediol, a metabolite of dehydroepiandrosterone, is a promising candidate for the treatment of acute radiation syndrome and as an immunostimulating agent during radiopharmaceutical therapy. The design and synthesis of new 5-androstenediol derivatives with increased bioavailability may lead to the appearance of highly effective cytoprotectors on the pharmaceutical market. The argumentations for new clinical applications of these steroids and novel insights into their mechanisms of action are discussed.

Profile of key metabolites and identification of HMGCS1-DHEA pathway in porcine Sertoli cells treated by Vitamin C

Vitamin C (Ascorbic acid, AA), as vital micro-nutrient, plays an essential role for male animal reproduction. Previously, we showed that vitamin C reprogrammed the transcriptome and proteome to change phenotypes of porcine immature Sertoli cells (iSCs). Here, we used LC-MS-based non-targeted metabolomics to further investigate the metabolic effects of vitamin C on porcine iSCs. The results identified 43 significantly differential metabolites (DMs) (16 up and 27 down) as induced by vitamin C (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) treatment of porcine iSCs, which were mainly enriched in steroid related and protein related metabolic pathways. ELISA (Enzyme-Linked ImmunoSorbent Assay) showed that significantly differential metabolites of Dehydroepiandrosterone (DHEA) (involved in steroid hormone biosynthesis) and Desmosterol (involved in steroid degradation) were significantly increased, which were partially consistent with metabolomic results. Further integrative analysis of metabolomics, transcriptomics and proteomics data identified the strong correlation between the key differential metabolite of Dehydroepiandrosterone and 6 differentially expressed genes (DEGs)/proteins (DEPs) (HMGCS1, P4HA1, STON2, LOXL2, EMILIN2 and CCN3). Further experiments validated that HMGCS1 could positively regulate Dehydroepiandrosterone level. These data indicate that vitamin C could modulate the metabolism profile, and HMGCS1-DHEA could be the pathway to mediate effects exerted by vitamin C on porcine iSCs.

Gas chromatography-mass spectrometry based steroid metabolomics in women with different phenotypes of polycystic ovarian syndrome and normal body weight

Objective: to study the steroid metabolomics in women with normal body weight and various PCOS phenotypes by gas chromatography-mass spectrometry (GC-MS). Materials and methods: forty-eight(48)women with PCOS aged 25±0,3 yearswith a BMI less than 25 kg/m2 were examined. The control group (CG) consisted of twenty-five (25) healthy women aged 26±0,6 years with a BMI of 23 (21-24) kg/m2. Immunoassays were used to determine the levels of hormones in serum. Urinary steroid profiles (USP) were studied by GC-MS method. Statistical data processing was performed using the software system STATISTICA for WINDOWS (ver. 10). Results: the article provides an analysis of the metabolism of androgens, glucocorticoids and progestogens in women with different phenotypes of polycystic ovary syndromeaccording to gas chromatography-mass spectrometry. Summary: the urinary excretion of androstenedione metabolites was increased in PCOS patients with androgen excess and anovulation (A and B phenotypes), dehydroepiandrosterone metabolites - in PCOS patients with androgen excess (A, B and C phenotypes). PCOS women with phenotype C showed raised urinary excretion of 11-oxo-pregnanetriol, pregnanetriol and 17-hydroxypregnanolone, a decrease in the ratios of the sum of tetrahydro derivatives of cortisol and cortisone to these progestogens, as well as determination of tetrahydro-21-deoxycorticol and nonclassical 5-ene-pregnenes according to GC-MS data. In fact, it indicated to deficiency of the 21-hydroxylase enzyme in these patients. It was found PCOS patients with androgen excess (A, B and C phenotypes) had the signs of insufficient 3β-hydroxysteroid dehydrogenase activity. PCOS women with phenotype A were revealed deficiency of 11β-hydroxysteroid dehydrogenase (type 1).

How is Western Lowland Gorilla (Gorilla gorilla gorilla) Behavior and Physiology Impacted by 360 Degree Visitor Viewing Access?

The effect visitors may have on the welfare of professionally managed animals is vital to consider. The present study utilized an experimental approach to examine how 360° visitor viewing access (i.e., viewing from all sides of the habitat) impacts the behavior and physiology of Western lowland gorillas (Gorilla gorilla gorilla, N = 7). Data collection included behavioral observations and obtaining fecal samples to examine physiological indicators of welfare. The study included a baseline period observing the gorillas’ behavior and physiology during the typical 360° visitor viewing access and a treatment period examining the same indicators but reducing viewing access by approximately 70%. Behavioral states, behavioral events, and hormone data were compared across conditions using Wilcoxon signed-rank tests. Restricted viewing did not impact social or solitary grooming (as a state behavior), social or solitary play, foraging, stereotypies, locomotion, inactivity, or behavioral diversity. However, restricted viewing significantly reduced solitary grooming events, fecal glucocorticoid metabolites, and the ratio of glucocorticoid metabolites to dehydroepiandrosterone metabolites. These results suggest that 360° visitor viewing access may have an impact on the welfare of gorillas. However, further research is necessary to thoroughly understand the influence of allowing 360° visitor viewing on behavior and its possible interaction with variables of the zoo environment.

Behavioral diversity as a potential positive indicator of animal welfare in bottlenose dolphins.

Accredited zoological facilities are committed to fully understanding the behavioral, mental, and physical needs of each species to continuously improve the welfare of the animals under their professional care and detect when welfare has diminished. In order to accomplish this goal, internally consistent and externally valid indicators of animal welfare are necessary to advance our understanding of the current welfare status of individual animals. Historically, efforts have focused on monitoring visible or observable signs of poor health or problem behavior, but lack of signs or problems does not necessarily demonstrate that an individual animal is thriving. The current study examined fecal hormone metabolite levels and behavior for two species of bottlenose dolphins (Tursiops truncatus and Tursiops aduncus) from 25 different accredited zoological facilities. At the time of the study, all facilities were accredited by the Alliance of Marine Mammal Parks and Aquariums and/or the Association of Zoos and Aquariums. This was part of the multi-institutional study ‘Towards understanding of the welfare of cetaceans in zoos and aquariums” commonly referred to as the Cetacean Welfare Study. Behavioral diversity was calculated using the Shannon Diversity Index on species-appropriate behavioral events. Behavioral diversity was compared to the fecal metabolites of cortisol, aldosterone, and the ratio of cortisol to dehydroepiandrosterone (DHEA) as well as the stereotypic behavior of route tracing. Similar to previous studies on other species, there was a significant inverse relationship between behavioral diversity and both fecal cortisol metabolites and route tracing. Additionally, a significant inverse relationship also exists between behavioral diversity and the ratio of fecal cortisol to DHEA metabolites. Behavioral diversity and fecal aldosterone metabolites were not associated. Additional research is still needed to validate behavioral diversity as an indicator of positive animal welfare for bottlenose dolphins and across species. However, based on current results, facilities could utilize behavioral diversity combined with other measures of welfare to more comprehensively evaluate the welfare of bottlenose dolphins.

Gas chromatography-mass spectrometry-based metabolic profiling of androgens, progestins and glucocorticoids in women with polycystic ovary syndrome

Hypothesis/aims of study. Polycystic ovary syndrome (PCOS) is a common disease. Depending on the diagnostic criteria, the disease is seen in 10-20% of women of reproductive age and accounts for 70-80% of all forms of hyperandrogenic syndrome. PCOS is a heterogeneous condition of multifactorial etiology characterized by various clinical, endocrine and metabolic disorders. Therefore, it is important to clarify the specific features of steroid hormone biosynthesis and metabolism and steroidogenesis enzyme activity, as well as to search for new laboratory criteria for early diagnosis and prompt treatment. The aim of this study was to perform metabolic profiling of androgens, progestins and glucocorticoids using gas chromatography-mass spectrometry (GC-MS) in obese and non-obese women with PCOS.
 Study design, materials and methods. We examined 53 women of reproductive age diagnosed with PCOS. The first group included 30 women aged 22 to 29 years with normal body weight. The second group comprised 23 obese patients aged 25 to 33 years with an average body mass index (BMI) of 35.3 0.4 kg/m2. The control group consisted of 25 healthy women aged 26 0.6 years having a normal BMI without clinical and biochemical signs of hyperandrogenism. Immunoassay methods were used to determine the serum levels of luteinizing hormone, follicle-stimulating hormone, free testosterone, 17-hydroxyprogesterone, and sex hormone-binding globulin. A glucose tolerance test was performed to determine glucose and insulin levels before and after load. Urine steroid profiles were studied by GC-MS with the optimization of the sample preparation schedule. Statistical data processing was performed using the STATISTICA for WINDOWS software system (version 10). The main quantitative characteristics of the patients are presented as the median (Me), the 25th percentile and the 75th percentile (Q25Q75). To compare the results obtained in the study groups, the nonparametric Mann-Whitney test was used. The 95% confidence interval was considered statistically significant.
 Results. The article presents a metabolomics analysis of androgens, glucocorticoid hormones and progestins in women with PCOS compared to the control group. It was revealed that non-obese patients with PCOS had increased urinary excretion of androstenedione metabolites, dehydroepiandrosterone and its metabolites, 17-hydroxypregnanolone, pregnantriol, and 5-ene-pregnenes, while obese patients with PCOS had increased that of androsterone and dehydroepiandrosterone metabolites (16-oxo-androstenediol and androstenediol-17) compared to the control group findings. Decreased ratios of cortisol and cortisone tetrahydro metabolite amount to the levels of 11-oxo-pregnanetriol, pregnanetriol and 17-hydroxypregnenolone, when compared to the control group, was obtained in non-obese patients with PCOS, which indicates 21-hydroxylase deficiency. In obese patients with PCOS, four signs of increased 5-reductase activity were obtained, and in PCOS patients with a normal BMI, three signs were obtained, which indicates varying 5-reductase activity in PCOS patients depending on the BMI.
 Conclusion. Quantitative evaluation of androgen and progestin metabolites, as well as 5- and 5-metabolites of androstenedione and glucocorticoids in the study of urine steroid profiles by GC-MS method opens new opportunities for PCOS diagnostics.

DHEA in bone: the role in osteoporosis and fracture healing.

Dehydroepiandrosterone (DHEA) is a metabolic intermediate in the biosynthesis of estrogens and androgens with a past clouded in controversy and bold claims. It was once touted as a wonder drug, a fountain of youth that could cure all ailments. However, in the 1980s DHEA was banned by the FDA given a lack of documented health benefits and long-term use data. DHEA had a revival in 1994 when it was released for open market sale as a nutritional supplement under the Dietary Supplement Health and Safety Act. Since that time, there has been encouraging research on the hormone, including randomized controlled trials and subsequent meta-analyses on various conditions that DHEA may benefit. Bone health has been of particular interest, as many of the metabolites of DHEA are known to be involved in bone homeostasis, specifically estrogen and testosterone. Studies demonstrate a significant association between DHEA and increased bone mineral density, likely due to DHEA's ability to increase osteoblast activity and insulin like growth factor 1 (IGF-1) expression. Interestingly, IGF-1 is also known to improve fracture healing, though DHEA, a potent stimulator of IGF-1, has never been tested in this scenario. The aim of this review is to discuss the history and mechanisms of DHEA as they relate to the skeletal system, and to evaluate if DHEA has any role in treating fractures.

Prasterone: A Review in Vulvovaginal Atrophy.

Vulvovaginal atrophy (VVA) is a progressive condition commonly seen in postmenopausal women. The cessation of ovarian estrogen secretion and a fall in serum levels of dehydroepiandrosterone (DHEA), the remaining source of estrogens and androgens, are thought to promote the development of VVA in this population. Intravaginal prasterone (Intrarosa®) is a synthetic form of DHEA indicated for the treatment of VVA in postmenopausal women presenting with moderate to severe symptoms in the EU; prasterone is also approved in the USA for the treatment of dyspareunia due to menopause. Approval for the treatment of VVA was based on the results of the phase III ERC-231 and -238 trials in which intravaginal prasterone 6.5mg/day significantly improved the signs and symptoms of VVA (as assessed by the percentage of parabasal and superficial cells, vaginal pH and the severity of dyspareunia) compared with placebo. The beneficial effects of prasterone were also evident during 52weeks' treatment in the phase III ERC-230 safety trial. Prasterone was generally well tolerated, with the most common treatment-emergent adverse event being application site discharge. During 52weeks of treatment with prasterone, changes in serum concentrations of estrogenic and androgenic metabolites of DHEA increased from baseline but remained within the normal postmenopausal ranges. Thus, intravaginal prasterone is an effective and generally well-tolerated option for the treatment of VVA in postmenopausal women.

Inhibition of ICa,L by DHEA in Aortic Smooth Muscle Cells: Voltage‐dependency, Modulation by GPCR Signaling and Inhibition of Glucose‐6‐phosphate Dehydrogenase

Dehydroepiandrosterone (DHEA) is a hormone abundantly released from adrenal cortex as DHEA‐sulfate (DHEA‐S). DHEA‐S is released also in brain. DHEA and epiandrosterone (EPI), a metabolite of DHEA, produces relaxation of high‐K+ induced contraction of aortic smooth muscle (ASM). EPI increases voltage‐dependent inactivation (voltage‐dependent inhibition, VDI) of ICa,L in ventricular myocytes. Whole‐cell ICa,L was recorded as IBa from ASM cells (ASMCs) of A7r5 cell line and enzymatically isolated bovine coronary ASMCs to study mechanism and modulation of DHEA‐induced inhibition of ICa,L. DHEA produced VDI; acceleration of time course of inactivation, increase of inhibition by depolarization of holding potential (HP) and leftward shift of steady state inactivation curve; f∞, 2s‐V, obtained by 2 s conditioning pulse and f∞, HP‐V as a function HP. In addition, DHEA produced voltage‐independent inhibition (VIDI) represented by inhibition of peak amplitude (ICa,L,peak) of ICa,L elicited by pulses from hyperpolarized HPs. Window ICa,L (IWD) supplies Ca2+ in the E‐C coupling of ASM. IWD obtained from I–V of ICa,L,peak and f∞, HP‐V fitted well with recorded IWD and they were strongly inhibited by DHEA. Efficacy of DHEA‐induced VDI was reduced by rightward shift of f∞, 2s‐V curve produced by depletion of cholesterol by MβCD. Blocking of GPCR signaling by intracellular dialysis of GDP‐β‐S also shifted f∞, 2s‐V to the right and it produced decrease of DHEA‐induced inhibition of ICa,L at depolarized HP (VDI). DHEA and EPI are inhibitors of glucose‐phosphate‐dehydrogenase (G6PD), a redox‐related pentose‐phosphate pathway enzyme. 6‐Aminonicotinamide (6‐AN), another inhibitor of G6PD produced VIDI without inducing VDI. DHEA‐S that does not inhibit G6PD induced small but significant VDI at an order higher concentration of DHEA without generating VIDI. GPCR signaling and membrane cholesterol are indirectly involved in the modulation of DHEA‐induced VDI by affecting basal voltage‐dependent inactivation. The occurrence of VIDI by 6‐AN indicates that G6PD inhibition underlies DHEA‐induced VIDI. DHEA‐induced inhibition of IWD by VDI and VIDI contributes for DHEA‐induced relaxation of ASM.Support or Funding InformationNational Heart, Lung, and Blood Institute Grant RO1 HL‐085352 to S. A. Gupte.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Androst-5-ene-3β,7α/β,17β-triols, their plasma levels and dependence on the hypothalamic–pituitary–adrenal axis

Androst-5-ene-triols are metabolites of dehydroepiandrosterone, the most abundant steroid hormone in human circulation. Many observations in rodents have demonstrated the anti-inflammatory and immune modulating activity of 7β-hydroxy-androst-5-enes, and on the basis of these experiments androst-5-ene-3β,7β,17β-triol is considered as a potential agent in the treatment of autoimmune diseases. In contrast to the fairly abundant information on the levels and effects of androst-5-ene-triols in experimental animals and of their the pharmacological perspective, little is known about androst-5-ene-3β,7α/β,17β-triols circulating in human blood, their regulation by the hypothalamo–pituitary–adrenal axis, or their daily concentration variability. Here we provide some data on androst-5-ene-3β,7α/β,17β-triol concentrations under various conditions in men and women.

Dehydroepiandrosterone Research: Past, Current, and Future.

The discovery of "oestrus-producing" hormones was a major research breakthrough in biochemistry and pharmacology during the early part of the 20th century. The elucidation of the molecular weight and chemical structure of major oxidative metabolites of dehydroepiandrosterone (DHEA) led to the award of the Nobel Prize in 1939 to Adolf Frederick Johann Butenandt and Leopold Ruzicka. Considered a bulk androgen in the circulation, DHEA and its sulfated metabolite DHEA-S can be taken up by most tissues where the sterols are metabolized to active androgenic and estrogenic compounds needed for growth and development. Butenandt's interactions with the German pharmaceutical company Schering led to production of gram quantities of these steroids and other chemically modified compounds of this class. Sharing chemical expertise allowed Butenandt's laboratory at the Kaiser Wilhelm Institute to isolate and synthesize many steroid compounds in the elucidation of the pathway leading from cholesterol to testosterone and estrogen derivatives. As a major pharmaceutical company worldwide, Schering AG sought these new biological sterols as pharmacological agents for endocrine-related diseases, and the European medical community tested these compounds in women for conditions such as postmenopausal depression, and in men for increasing muscle mass. Since it was noted that circulating DHEA-S levels decline as a function of age, experimental pathology experiments in animals were performed to determine how DHEA may protect against cancer, diabetes, aging, obesity, immune function, bone density, depression, adrenal insufficiency, inflammatory bowel disease, diminished sexual function/libido, AIDS/HIV, chronic obstructive pulmonary disease, coronary artery disease, chronic fatigue syndrome, and metabolic syndrome. While the mechanisms by which DHEA ameliorates these conditions in animal models have been elusive to define, even less is known about its role in human disease, other than as a precursor to other sterols, e.g., testosterone and estradiol. Our groups have shown that DHEA and many of its oxidative metabolites serve as a low-affinity ligands for hepatic nuclear receptors, such as the pregnane X receptor, the constitutive androstane receptor, and estrogen receptors α/β (ERα/ERβ) as well as G protein-coupled ER (GPER1). This chapter highlights the founding research on DHEA from a historical perspective, provides an overview of DHEA biosynthesis and metabolism, briefly summarizes the early work on the beneficial effects attributed to DHEA in animals, and summarizes the human trials addressing the action of DHEA as a therapeutic agent. In general, most human studies involve weak correlations of circulating levels of DHEA and disease outcomes. Some support for DHEA as a therapeutic compound has been demonstrated for postmenopausal women, in vitro fertilization, and several autoimmune disorders, and adverse health effects, such as, acne, embryo virilization during pregnancy, and possible endocrine-dependent cancers.

Androgen excess is due to elevated 11-oxygenated androgens in treated children with congenital adrenal hyperplasia

Adrenal androgen excess is the hallmark of classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Recently, 11-oxygenated C19 steroids, a class of highly active adrenal-derived androgens, have been described in patients with CAH.The aim of our study was to elucidate the significance of 11-oxygenated androgens in children with CAH.We retrospectively analysed 190 daily urinary excretion rates of glucocorticoid-, 17α-hydroxyprogesterone (17OHP)-, and androgen metabolites determined by gas chromatography-mass spectrometry of 99 children aged 3.0–10.9 years with classic CAH on hydrocortisone and fludrocortisone treatment. Daily urinary steroid metabolite excretions were transformed into z-scores using references of healthy children. Androgen metabolite z-scores were separately calculated for androsterone (AN), the major urinary metabolite of androstenedione (A4), testosterone and 5α-dihydrotestosterone, for urinary metabolites of dehydroepiandrosterone (DHEA), and for 11β-hydroxyandrosterone (11OHAN), the major urinary metabolite of adrenal-derived 11-oxygenated androgens. Multivariate regression analysis was applied to analyse the precursors of 11OHAN synthesis.11OHAN, cortisol-, and 17OHP metabolite z-scores were elevated in treated children with CAH, whereas AN- and DHEA metabolite z-scores were normalized or suppressed. Multivariate regression analysis revealed that 11OHAN excretion was strongest associated with 21-deoxycortisol (β = 0.379; P =.0006), followed by A4 (β = 0.280; P = .0008)) and 17OHP (β = 0.243; P = .04) metabolite excretion.Androgen excess in treated children with CAH is solely due to elevated 11-oxygenated androgens that derive in addition to the known conversion from A4 also by direct conversion from 21-deoxycortisol. 11-Oxygenated androgens may represent better biomarkers of adrenal androgen status and treatment response than conventional androgens.

The effect of dehydroepiandrosterone on inflammatory response of astroglial cells

An increased interest in neuroinflammation is conditioned by its involvement in various pathological processes in the brain. Astrocytes play an important role in neuroinflammation, participating in its regulation, throwing out a large number of signaling molecules. Steroid compounds, actively produced by astrocytes, are of interest with regards to the regulation of inflammatory processes in the central nervous system. In the present work the effect of dehydroepiandrosterone (DHEA) on astroglial cells (cultured primary rat astrocytes) in a model of inflammation was studied. The inflammatory response was stimulated with lipopolysaccharide (LPS). Expression levels of pro-inflammatory factor TNFα, antinflammatory interleukin IL-10, and both pro- and antiinflammatory protein COX-2 were measured. The expression of IL-10, COX-2, and TNFα mRNA was determined by real-time PCR, COX-2 protein level by immunoblotting method, TNFα and IL-10 release by enzyme immunoassay. The effect of short-term (30 min) and long-term (24 h) exposure to DHEA was evaluated. It was shown that DHEA potentiates LPS-stimulated (1) increase in the IL-10 mRNA level; (2) IL-10 release; (3) does not affect TNFα level, and (4) exerts a weak pulsating bidirectional effect on COX-2. Using trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase, a key enzyme of DHEA metabolism, it was shown that DHEA metabolites make the main contribution to its effect. Thus, DHEA is of interest as a stimulant of anti-inflammatory processes in the brain.

DHEA‐induced Voltage‐dependent Inhibition of ICa,L Is Augmented by Cholesterol Depletion and Down‐regulated by GPCR Blockade in Aortic Smooth Muscle Cells

Dehydroepiandrosterone (DHEA) is a steroid hormone synthesized from cholesterol and abundantly secreted as DHEA sulfate from the adrenal gland. DHEA produces relaxation of systemic and pulmonary arterial smooth muscles (ASM) and epiandrosterone, a metabolite of DHEA, causes voltage‐dependent inhibition (VDI) of ICa,L in ventricular myocytes. Here, we characterized DHEA‐induced VDI of ICa,L recorded with Ba2+ as a charge carrier by whole cell clamp and studied underlying mechanism and modulation by signal transduction in ASM cells (ASMCs) from bovine coronary artery and A7r5 cells from rat aorta. DHEA decreased peak amplitude of ICa,L rapidly, reversibly and dose‐dependently and accelerated time course of current decay decreasing the ratio of current amplitude at 500 ms (r500) to peak amplitude and shifted steady‐state inactivation curve (f∞‐V) to the left. DHEA‐induced inhibition of ICa,L became prominent by depolarization of holding potential; e.g. at −30 mV, the inhibition was 29% at 10 μM and 89% at 100 μM. Blockade of GPCR by dialysis of GDP βS shifted both f∞‐V and I–V relationships to the depolarizing direction, and decreased 10 μM‐DHEA‐induced VDI obtained at HP of −40 mV by 40%. Depletion of membrane cholesterol by methyl‐b‐cyclodextrin increased r500 and shifted f∞‐V to the right. Conversely, loading PEG‐cholesterol, a non‐ionic amphiphile, to the membrane decreased r500 and shifted f∞‐V to the left. In the cholesterol‐depleted cells, DHEA produced more potent decrease of r500 and larger negative shift of f∞‐V than in control. Therefore, DHEA‐induced VDI of ICa,L in ASMCs is due, at least partly, to cholesterol‐like action of DHEA on CaV1.2 in the membrane lipid bilayer. GPCR‐mediated signaling regulates voltage‐dependence of basal ICa,L and indirectly augments DHEA‐induced VDI of ICa,L in ASMCs.

Synthesis of 3α-deuterated 7α-hydroxy-DHEA and 7-oxo-DHEA and application in LC-MS/MS plasma analysis

7-Oxygenated metabolites of dehydroepiandrosterone (DHEA) are known for their neuroprotective and immunomodulatory properties. These neuroactive steroids are currently predominately analysed by mass spectrometry, for which the use of internal deuterated standards is necessary. The aim of this study was to synthesize the deuterated derivatives of 7α-hydroxy-DHEA and 7-oxo-DHEA and test them in liquid chromatography–tandem mass spectrometry (LC-MS/MS) in order to enhance the performance characteristics of this method. Here we report the synthesis of 3α deuterium-labelled 7α-hydroxy-DHEA and 7-oxo-DHEA. Deuterium was introduced into the 3α position by reduction of the corresponding 3-ketone with a protected 17-carbonyl group using NaBD4. Our new procedure allows the easier synthesis of deuterated steroid labelled compounds. The use of these deuterated steroids enabled us to improve the human plasma LC-MS/MS analysis of 7α-hydroxy-DHEA and 7-oxo-DHEA in terms of sensitivity, precision and recovery.

A validated LC–MS/MS method for the sensitive quantitation of serum 7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone using a novel derivatization reagent

7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone (7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA) are oxidized metabolites of dehydroepiandrosterone (DHEA). Their concentrations are low in the circulation, especially in postmenopausal women, thus resulting in a considerable challenge for their reliable measurement. A sensitive and accurate LC–MS/MS method has been developed using a simple sample preparation procedure and a novel derivatization with 1-amino-4-methyl piperazine (MP). The derivatized metabolites are stable in high water content reagents. A 10pg/mL (0.2pg on column) for the low limit of quantitation (LLOQ) has been achieved for all three compounds. A proper choice of multiple reaction monitoring (MRM) transitions provides good specificity. The excess amount of reagent can be removed from the sample during the derivatization process. Within the calibration range of 10–2000pg/mL, a good linearity was obtained with R>0.99 where the weighing factor is 1/X while the bias and coefficient of variance (CV) are within 8% for all levels of QCs and calibration curves. This method has been fully validated according to the FDA guidelines, where the results of the matrix effect meet the acceptance criteria while freeze–thaw stability, short and long term stability in matrix and solution as well as post-processed sample stability meet the requirements. With this method, the concentrations of 7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA were measured in premenopausal and postmenopausal serum. The average concentration of 7α OH-DHEA is equivalent to that of 7β OH-DHEA in both types of sera.

DHEA-induced modulation of renal gluconeogenesis, insulin sensitivity and plasma lipid profile in the control- and dexamethasone-treated rabbits. Metabolic studies.

In view of antidiabetic and antiglucocorticoid effects of dehydroepiandrosterone (DHEA) both invitro and invivo studies were undertaken: (i) to elucidate the mechanism of action of both dexamethasone phosphate (dexP) and DHEA on glucose synthesis in primary cultured rabbit kidney-cortex tubules and (ii) to investigate the influence of DHEA on glucose synthesis, insulin sensitivity and plasma lipid profile in the control- and dexP-treated rabbits. Data show, that in cultured kidney-cortex tubules dexP significantly stimulated gluconeogenesis by increasing flux through fructose-1,6-bisphosphatase (FBPase). DexP-induced effects were dependent only upon glucocorticoid receptor. DHEA decreased glucose synthesis via inhibition of glucose-6-phosphatase (G6Pase) and suppressed the dexP-induced stimulation of renal gluconeogenesis. Studies with the use of inhibitors of DHEA metabolism in cultured renal tubules showed for the first time that DHEA directly affects renal gluconeogenesis. However, in view of analysis of glucocorticoids and DHEA metabolites levels in urine, it seems likely, that testosterone may also contribute to DHEA-evoked effects. In dexP-treated rabbits, plasma glucose level was not altered despite increased renal and hepatic FBPase and G6Pase activities, while a significant elevation of both plasma insulin and HOMA-IR was accompanied by a decline of ISI index. It thus appears that increased insulin levels were required to maintain normoglycaemia and to compensate the insulin resistance. DHEA alone affected neither plasma glucose nor lipid levels, while it increased insulin sensitivity and diminished both renal and hepatic G6Pase activities. Surprisingly, DHEA co-administrated with dexP did not alter insulin sensitivity, while it partially suppressed the dexP-induced elevation of renal G6Pase activity and plasma cholesterol and triglyceride contents. As (i) gluconeogenic pathway in rabbit is similar to that in human, and (ii) DHEA counteracts several dexP-evoked effects, it seems likely, that its supplementation might be beneficial to patients treated with glucocorticoids.

Dehydroepiandrosterone and 7-oxo-dehydroepiandrosterone in male reproductive health: Implications of differential regulation of human Sertoli cells metabolic profile

Dehydroepiandrosterone (DHEA) is a precursor of androgen synthesis whose action is partially exerted through its metabolites. 7-Oxo-dehydroepiandrosterone (7-oxo-DHEA) is a common DHEA metabolite, non-convertible to androgens, which constitutes a promising therapeutic strategy for multiple conditions. Sertoli cells (SCs) are responsible for the support of spermatogenesis, having unique metabolic characteristics strongly modulated by androgens. Consequently, disruptions in androgen synthesis compromise SCs function and hence male fertility. We aimed to evaluate the effects of DHEA and 7-oxo-DHEA in human SCs (hSCs) metabolism and oxidative profile. To do so, hSCs were exposed to increasing concentrations of DHEA and 7-oxo-DHEA (0.025, 1 and 50μM) that revealed to be non-cytotoxic in these experimental conditions. We measured hSCs metabolites consumption/production by 1H NMR, the protein expression levels of key players of the glycolytic pathway by Western blot as well as the levels of carbonyl groups, nitration and lipid peroxidation by Slot blot. The obtained data demonstrated that 7-oxo-DHEA is a more potent metabolic modulator than DHEA since it increased hSCs glycolytic flux. DHEA seem to redirect hSCs metabolism to the Krebs cycle, while 7-oxo-DHEA has some inhibitory effect in this path. The highest 7-oxo-DHEA concentrations (1 and 50μM) also increased lactate production, which is of extreme relevance for the successful progression of spermatogenesis in vivo. None of these steroids altered the intracellular oxidative profile of hSCs, illustrating that, at the concentrations used they do not have pro- nor antioxidant actions in hSCs. Our study represents a further step in the establishment of safe doses of DHEA and 7-oxo-DHEA to hSCs, supporting its possible use in hormonal and non-hormonal therapies against male reproductive problems.

ADIOL protects against 3-NP-induced neurotoxicity in rats: Possible impact of its anti-oxidant, anti-inflammatory and anti-apoptotic actions.

Huntington's disease (HD) is a progressive neurodegenerative disorder with a wide spectrum of cognitive, behavioral and motor abnormalities. The mitochondrial toxin 3-nitropropionic acid (3-NP) effectively induces specific behavioral changes and selective striatal lesions similar to that observed in HD. Some neurosteroids, synthesized in neurons and glial cells, previously showed neuroprotective abilities. 5-Androstene-3β-17β-diol (ADIOL) is a major metabolite of dehydroepiandrosterone (DHEA) with previously reported anti-inflammatory, anti-apoptotic and neuroprotective activities. The neuroprotective potential of ADIOL in HD was not previously investigated. Therefore, the present study investigated the neuroprotective effects of ADIOL against 3-NP-induced behavioral changes, oxidative stress, inflammation and apoptosis. Intraperitoneal administration of 3-NP (20mg/kg) for 4 consecutive days in rats caused significant loss in body weight, reduced prepulse inhibition (PPI) of acoustic startle response, locomotor hypoactivity with altered cortical/striatal histological structure, increased cortical/striatal oxidative stress, inflammation and apoptosis. Administration of ADIOL (25mg/kg, s.c.) for two days before 3-NP significantly attenuated the reduction in body weights and PPI, increased locomotor activity and restored cortical/striatal histological structure nearly to normal. Moreover, it displayed anti-oxidant, anti-inflammatory and anti-apoptotic activities as evidenced by the elevation of cortical and striatal reduced glutathione levels, reductions of cortical and striatal malondialdehyde, striatal tumor necrosis factor alpha and interleukin-6 levels. Only a small number of iNOS and caspase-3 positive cells were detected in sections from rats pretreated with ADIOL. This study suggests a potential neuroprotective role of ADIOL against 3-NP-induced Huntington's disease-like manifestations. Such neuroprotection can be attributed to its anti-oxidant, anti-inflammatory and anti-apoptotic activities.

Dehydroepiandrosterone-induces miR-21 transcription in HepG2 cells through estrogen receptor β and androgen receptor

Although oncomiR miR-21 is highly expressed in liver and overexpressed in hepatocellular carcinoma (HCC), its regulation is uncharacterized. We examined the effect of physiologically relevant nanomolar concentrations of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S) on miR-21 expression in HepG2 human hepatoma cells. 10nM DHEA and DHEA-S increase pri-miR-21 transcription in HepG2 cells. Dietary DHEA increased miR-21 in vivo in mouse liver. siRNA and inhibitor studies suggest that DHEA-S requires desulfation for activity and that DHEA-induced pri-miR-21 transcription involves metabolism to androgen and estrogen receptor (AR and ER) ligands. Activation of ERβ and AR by DHEA metabolites androst-5-ene-3,17-dione (ADIONE), androst-5-ene-3β,17β-diol (ADIOL), dihydrotestosterone (DHT), and 5α-androstane-3β,17β-diol (3β-Adiol) increased miR-21 transcription. DHEA-induced miR-21 increased cell proliferation and decreased Pdcd4 protein, a bona fide miR-21. Estradiol (E2) inhibited miR-21 expression via ERα. DHEA increased ERβ and AR recruitment to the miR-21 promoter within the VMP1/TMEM49 gene, with possible significance in hepatocellular carcinoma.