The Effects of the Steroids 5-Androstenediol and Dehydroepiandrosterone and Their Synthetic Derivatives on the Viability of K562, HeLa, and Wi-38 Cells and the Luminol-Stimulated Chemiluminescence of Peripheral Blood Mononuclear Cells from Healthy Volunteers.

In order to evaluate the role of substituents at 3-C and 17-C in the cytotoxic and cytoprotective actions of DHEA and 5-AED molecules, their derivatives were synthesized by esterification using the corresponding acid anhydrides or acid chlorides. As a result, seven compounds were obtained: four DHEA derivatives (DHEA 3-propionate, DHEA 3-butanoate, DHEA 3-acetate, DHEA 3-methylsulfonate) and three 5-AED derivatives (5-AED 3-butanoate, 5-AED 3,17-dipropionate, 5-AED 3,17-dibutanoate). All of these compounds showed micromolar cytotoxic activity toward HeLa and K562 human cancer cells. The maximum cytostatic effect during long-term incubation for five days with HeLa and K562 cells was demonstrated by the propionic esters of the steroids: DHEA 3-propionate and 5-AED 3,17-dipropionate. These compounds stimulated the growth of normal Wi-38 cells by 30-50%, which indicates their cytoprotective properties toward noncancerous cells. The synthesized steroid derivatives exhibited antioxidant activity by reducing the production of reactive oxygen species (ROS) by peripheral blood mononuclear cells from healthy volunteers, as demonstrated in a luminol-stimulated chemiluminescence assay. The highest antioxidant effects were shown for the propionate ester of the steroid DHEA. DHEA 3-propionate inhibited luminol-stimulated chemiluminescence by 73% compared to the control, DHEA, which inhibited it only by 15%. These data show the promise of propionic substituents at 3-C and 17-C in steroid molecules for the creation of immunostimulatory and cytoprotective substances with antioxidant properties.

The potential value of 5-androstenediol in countering acute radiation syndrome.

Moderate-to-high doses of ionizing irradiation can lead to potentially life-threatening morbidities and increase mortality risk. In preclinical testing, 5-androstenediol has been shown to be effective in protecting against hematopoietic acute radiation syndrome. This agent is important for innate immunity, serves to modulate cell cycle progression, reduces radiation-induced apoptosis, and regulates DNA repair. The drug has been evaluated clinically for its pharmacokinetics and safety. The United States Food and Drug Administration granted investigational new drug status to its injectable depot formulation (NEUMUNE). Its safety and efficacy profiles make it an attractive candidate for further development as a radiation countermeasure.

Ameliorative effects of androstenediol against acetic acid-induced colitis in male wistar rats via inhibiting TLR4-mediated PI3K/Akt and NF-κB pathways through estrogen receptor ß activation.

5-androstenediol (ADIOL) functions as a selective estrogen receptor ß (ERß) ligand with a protective effect against many diseases. So, we conducted a novel insight into its role in acetic acid (AA)-induced colitis and investigated its effect on TLR4-Mediated PI3K/Akt and NF-κB Pathways and the potential role of ERß as contributing mechanisms. METHODS: Rats were randomized into 5 Groups; Control, Colitis, Colitis + mesalazine (MLZ), Colitis + ADIOL, and Colitis + ADIOL + PHTPP (ER-ß antagonist). The colitis was induced through a rectal enema of acetic acid (AA) on the 8th day. At the end of treatment, colons were collected for macroscopic assessment. Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor kappa b (NF-κB), toll-like receptor (TLR4), and phosphorylated Protein kinase B (pAKT) were measured. Besides, Gene expression of interleukin-1beta (IL-1ß), metalloproteases 9 (Mmp9), inositol 3 phosphate kinase (PI3K), Neutrophil gelatinase-associated lipocalin (NGAL), ERß and NLRP6 were assessed. Histopathological and immunohistochemical studies were also investigated. RESULTS: Compared to the untreated AA group, the disease activity index (DAI) and macroscopic assessment indicators significantly decreased with ADIOL injections. Indeed, ADIOL significantly decreased colonic tissue levels of MDA, TLR4, pAKT, and NF-κB immunostainig while increased SOD activity and ß catenin immunostainig. ADIOL mitigated the high genetic expressions of IL1ß, NGAL, MMP9, and PI3K while increased ERß and NLRP6 gene expression. Also, the pathological changes detected in AA groups were markedly ameliorated with ADIOL. The specific ERß antagonist, PHTPP, largely diminished these protective effects of ADIOL. CONCLUSION: ADIOL could be beneficial against AA-induced colitis mostly through activating ERß.

The protective and therapeutic effects of 5-androstene3ß, 17ß-diol (ADIOL) in abdominal post-operative adhesions in rat: Suppressing TLR4/NFκB/HMGB1/TGF1 ß/α SMA pathway.

Neurosteroid, 5-androstenediol (ADIOL) had been experimentally applied to protect against many diseases as it had anti-oxidant, anti-inflammatory, and anti-apoptotic effects. In our study, we investigate its role in abdominal postoperative adhesion (APA) formations. Our results demonstrate that ADIOL alleviates APA formation after induction by cecal abrasion (CA) model in the male rat. Interestingly, per administration of ADIOL before APA induction leads to inhibit oxidative stress by increasing superoxide dismutase (SOD) and decreasing Malondialdehyde (MAD) levels to a similar level to the sham group, in addition inhibiting inflammatory pathway by decreasing toll-like receptor 4 (TLR4), nuclear factor kappa-B (NFκB), and High mobility group box 1 (HMGB1) to a similar level to the sham group, furthermore decreasing Transforming growth factor beta 1 (TGFß1) and alpha Smooth muscle -actin (α SMA) levels to similar levels in the sham group. While administration of ADIOL after APA induction lead to decrease adhesions formation by decreasing oxidative stress (↓MDA and ↑SOD levels), inflammatory markers (↓TLR4, ↓NFκB, and ↓HMGB1levels), and collagen deposition by (↓TGF1 ß and↓α SMA levels) is the highly significant manner to those levels in CA model but also significant to those levels in the sham group. Concluded that, pre-administration of ADIOL before APA induction was more effective than its administration after adhesions formations.

Sex differences in serum levels of 5α-androstane-3ß, 17ß-diol, and androstenediol in the young adults: A liquid chromatography-tandem mass spectrometry study.

Animal experiments have consistently shown that estrogen receptor ß (ERß)-selective ligands have antidepressant and anxiolytic effects. In humans, endogenous ligands for ERß include 5α-androstane-3ß, 17ß-diol (3ßAdiol) and androstenediol (Δ5-diol). We determined, for the first time, the exact serum levels of 3ßAdiol and Δ5-diol in young healthy volunteers using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We investigated the effect of the menstrual cycle on the levels of these steroids in women; then, we performed a gender comparison. Blood samples were collected from 48 subjects: 23 women (mean age = 28.4±7.8 years) and 25 men (mean age = 31.4±7.8 years). We collected the blood samples of women at three time-points in the menstrual cycle: the early follicular phase, ovulatory or mid-cycle phase, and mid-luteal phase. A total of 92 blood samples were analyzed using LC-MS/MS. The levels of two well-studied steroids, namely dehydroepiandrosterone (DHEA) and 17ß-estradiol (E2), were simultaneously measured. Depression rating scale (Hamilton Rating Scale for Depression, Beck Depression Inventory-II and Quick Inventory of Depressive Symptomatology) scores were also recorded at the time of blood sampling. Significant differences in the levels of 3ßAdiol and E2 and in the depression rating scale scores were observed over the duration of the menstrual cycle of the women. The levels of 3ßAdiol and Δ5-diol were significantly lower in women than in men. E2 levels were higher in women than in men, and DHEA levels did not differ significantly between men and women. Further, women had higher scores than men on the Hamilton Rating Scale for Depression. Sex differences in depressive symptoms can be explained by 3ßAdiol and Δ5-diol levels, and the effect of the menstrual cycle on mood can be explained by 3ßAdiol and E2 levels, not by Δ5-diol level.

Hyperandrogenism? Increased 17, 20-Lyase Activity? A Metanalysis and Systematic Review of Altered Androgens in Boys and Girls with Autism.

Introduction: There is increasing evidence that steroid hormone levels and, especially, androgen levels are elevated in autism. An overactivity of 17, 20-lyase with a higher production of the testosterone precursors dehydroepiandrosterone (DHEA) and androstenedione/androstenediol seems especially present in autism. Methods: An encompassing literature analysis was performed, searching for altered androgens in children with autism and using preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines. Included were all studies published before 31 March 2021 found using the following electronic databases: PubMed, Google Scholar, Cochrane Library, Scopus, and TRIP. Eight studies with boys and three studies with girls where steroid hormone measurements were performed from either plasma, urine, or saliva were found and analyzed. Analyses were performed for DHEA(-S/-C), androstenedione/androstenediol, and testosterone. Effect sizes were calculated for each parameter between mean concentrations for children with autism versus healthy controls. Results: Higher levels of androgens in autism were detected, with the majority of calculated effect sizes being larger than one. Conclusions: We found higher levels of the main testosterone precursors DHEA, androstenedione, and androstenediol, likely causing an additionally higher level of testosterone, and an increased 17, 20-lyase activity is therefore implied. Medications already used in PCOS such as metformin might be considered to treat hyperandrogenism in autism following further research.

Correlations of androstenediol with reproductive hormones and cortisol according to stages during the menopausal transition in Japanese women.

Associations of androstenediol, which has both androgenic and estrogenic activities, with circulating reproductive hormones and stress hormone in women during the menopausal transition may be different depending on the menopausal stage. The aim of this study was to determine the changes in circulating androstenediol during the menopausal transition in Japanese women and the associations of androstenediol with estrogen, androgen and cortisol for each stage of the menopausal transition. We divided the 104 subjects into 6 stages by menstrual regularity and follicle-stimulating hormone level: mid reproductive stage, late reproductive stage, early menopausal transition, late menopausal transition, very early postmenopause and early postmenopause. Levels of dehydroepiandrosterone sulfate (DHEAS), estradiol, estrone, testosterone (T), free T, androstenedione and cortisol were measured. Serum androstenediol concentration was measured by using liquid chromatography mass spectrometry. There were no significant differences in androstenediol levels among the 6 stages. Levels of DHEA-S and testosterone showed significant and positive correlations with androstenediol in all stages. Estradiol levels showed negative correlations with androstenediol levels in the late menopausal transition and very early postmenopause (r=-0.452, p = 0.052 and r=-0.617, p = 0.006, respectively). Cortisol levels showed significant and positive correlations with androstenediol levels in the mid and late reproductive stages (r = 0.719, p = 0.003 and r = 0.808, p < 0.001, respectively).The associations of androstenediol with estradiol and cortisol were different depending on the stage of the menopausal transition. Androstenediol may play a compensatory role for estrogen deficiency from late menopausal transition to very early postmenopause.

Storage stability of five steroids and in dried blood spots for newborn screening and retrospective diagnosis of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a severe inherited disorder of cortisol biosynthesis that is potentially lethal or can seriously affect quality of life. For the first time, we aimed to assess the stability of 21-deoxycortisol (21Deox), 11-deoxycortisol (11Deox), 4-androstenedione (4AD), 17-hydroxyprogesterone (17OHP) and cortisol (Cort), diagnostic for CAH, in dried blood spots (DBSs) during a 1 year storage at different temperatures. Spiked DBS samples were stored at room temperature, 4 °C, -20 °C or -70 °C, respectively and analyzed in triplicates using liquid chromatography-tandem mass spectrometry at Weeks 0, 1, 2, 3 and 4, Month 6 and Year 1. Analyte levels within ±15% vs the baseline were considered stable. Our observations show that 21Deox, 4AD and 17OHP were not significantly changed for 1 year even at room temperature at either analyte levels. In contrast, Cort required storage at 4 °C, -20 °C or -70 °C for long-term stability, being significantly decreased at room temperature from Month 6 (p<0.01) in both the 30(60) nM and the 90(180) nM samples. 11Deox was significantly decreased at room temperature at Year 1 (p<0.01) and only in the 30(60) nM samples. Thus, all biomarkers were stable for up to 1 year at 4 °C, -20 °C or -70 °C and at least for 4 weeks at room temperature. These findings have implications for analyses of stored DBS samples in 2nd-tier assays in newborn screening and for retrospective CAH studies.

5-Androstenediol prevents radiation injury in mice by promoting NF-κB signaling and inhibiting AIM2 inflammasome activation.

In the present study, the therapeutic effects of 5-androstenediol on radiation-induced myeloid suppression and tissue damage in mice and the possible mechanism were explored. The mice were subjected to whole-body irradiation, and 5-androstenediol was administered subcutaneously at different times and doses. The evaluation of the survival rate showed that the administration of 5-androstenediol every three days post-irradiation was the most effective in decreasing the death of the mice. Additionally, 5-androstenediol dose-dependently reduced the death caused by 9 Gy radiation. The pharmacological mechanism was investigated by blood analysis, western blot analysis, immunofluorescence and immunohistochemistry. 5-Androstenediol significantly ameliorated myeloid suppression, as demonstrated by elevated levels of total white blood cells, including neutrophils and platelets, in the peripheral blood. By H&E staining, we found that radiation-induced myeloid suppression in the bone marrow and spleen, as well as tissue damage in the lung and colon, was significantly ameliorated by treatment with 5-androstenediol. Immunohistochemistry showed elevated phosphorylation of p65 in the bone marrow and spleen, indicating the activation of NF-κB signaling. Moreover, 5-androstenediol markedly hampered the radiation-induced activation of caspase-1 and GSDMD in the colon by decreasing the interaction between AIM2 and ASC. Taken together, our results suggest that, by promoting NF-κB signaling and inhibiting inflammasome-mediated pyroptosis, 5-androstenediol can be used as a radioprotective drug.

Alcohol consumption and serum metabolite concentrations in young women.

PURPOSE: Alcohol consumption is an established breast cancer risk factor, though further research is needed to advance our understanding of the mechanism underlying the association. We used global metabolomics profiling to identify serum metabolites and metabolic pathways that could potentially mediate the alcohol-breast cancer association. METHODS: A cross-sectional analysis of reported alcohol consumption and serum metabolite concentrations was conducted among 211 healthy women 25-29 years old who participated in the Dietary Intervention Study in Children 2006 Follow-Up Study (DISC06). Alcohol-metabolite associations were evaluated using multivariable linear mixed-effects regression. RESULTS: Alcohol was significantly (FDR p < 0.05) associated with several serum metabolites after adjustment for diet composition and other potential confounders. The amino acid sarcosine, the omega-3 fatty acid eicosapentaenoate, and the steroid 4-androsten-3beta,17beta-diol monosulfate were positively associated with alcohol intake, while the gamma-tocopherol metabolite gamma-carboxyethyl hydroxychroman (CEHC) was inversely associated. Positive associations of alcohol with 2-methylcitrate and 4-androsten-3beta,17beta-diol disulfate were borderline significant (FDR p < 0.10). Metabolite set enrichment analysis identified steroids and the glycine pathway as having more members associated with alcohol consumption than expected by chance. CONCLUSIONS: Most of the metabolites associated with alcohol in the current analysis participate in pathways hypothesized to mediate the alcohol-breast cancer association including hormonal, one-carbon metabolism, and oxidative stress pathways, but they could also affect risk via alternative pathways. Independent replication of alcohol-metabolite associations and prospective evaluation of confirmed associations with breast cancer risk are needed.

The Non-Aromatic Δ5-Androstenediol Derivative of Dehydroepiandrosterone Acts as an Estrogen Agonist in Neonatal Rat Osteoblasts through an Estrogen Receptor α-related Mechanism.

Purpose: It has been proposed that DHEA influences bone formation through, bioconversion to 17ß-estradiol; however, DHEA is converted to Δ5-androstenediol (Δ5-Adiol), a metabolite with estrogenic potential involved in diverse biological process. To gain new insight into the role of Δ5-Adiol in bone cells, we examined DHEA and Δ5-Adiol effects in neonatal rat and human hFOB1.19 osteoblasts. Methods: Osteoblast activity was assessed by analyzing proliferation, alkaline phosphatase activity, and expression of OSX and ALPL. We also examined binding affinities for osteoblast-ER and transcriptional activation of human (h)ERα, hERß or hAR in U2-OS cells. Results: The most striking finding was that Δ5-Adiol had greater stimulatory effect than DHEA on rat osteoblast proliferation and differentiation, as well as ALPL expression in human osteoblasts. Interestingly, the Δ5-Adiol or DHEA-induced effects were not precluded with letrozole or trilostane, consistent with bioconversion of DHEA to Δ5-Adiol due to elevated expression of Hsd17b1 in neonatal rat osteoblasts, suggesting a high level of 17ß-hydroxysteroid dehydrogenase type 1 activity. Conversely, Δ5-Adiol and DHEA-induced proliferative effects were inhibited with ICI 182780 alone or combined with trilostane, which correlates with the higher binding affinity of Δ5-Adiol for ER compared to DHEA. Furthermore, Δ5-Adiol showed a greater relative agonist activity for hERα than for hERß or hAR. Conclusion: This study is the first to show that a bioactive DHEA derivative stimulates E2-dependent osteoblast activities, including proliferation and differentiation in rat and human osteoblasts, through ERα-related mechanisms.

The promiscuous estrogen receptor: Evolution of physiological estrogens and response to phytochemicals and endocrine disruptors.

Many actions of estradiol (E2), the principal physiological estrogen in vertebrates, are mediated by estrogen receptor-α (ERα) and ERß. An important physiological feature of vertebrate ERs is their promiscuous response to several physiological steroids, including estradiol (E2), Δ5-androstenediol, 5α-androstanediol, and 27-hydroxycholesterol. A novel structural characteristic of Δ5-androstenediol, 5α-androstanediol, and 27-hydroxycholesterol is the presence of a C19 methyl group, which precludes the presence of an aromatic A ring with a C3 phenolic group that is a defining property of E2. The structural diversity of these estrogens can explain the response of the ER to synthetic chemicals such as bisphenol A and DDT, which disrupt estrogen physiology in vertebrates, and the estrogenic activity of a variety of plant-derived chemicals such as genistein, coumestrol, and resveratrol. Diversity in the A ring of physiological estrogens also expands potential structures of industrial chemicals that can act as endocrine disruptors. Compared to E2, synthesis of 27-hydroxycholesterol and Δ5-androstenediol is simpler, leading us, based on parsimony, to propose that one or both of these steroids or a related metabolite was a physiological estrogen early in the evolution of the ER, with E2 assuming this role later as the canonical estrogen. In addition to the well-studied role of the ER in reproductive physiology, the ER also is an important transcription factor in non-reproductive tissues such as the cardiovascular system, kidney, bone, and brain. Some of these ER actions in non-reproductive tissues appeared early in vertebrate evolution, long before the emergence of mammals.

Potential neuroprotective effect of androst-5-ene-3ß, 17ß-diol (ADIOL) on the striatum, and substantia nigra in Parkinson's disease rat model.

Parkinson's disease (PD) is a progressive neurodegenerative disorder with behavioral and motor abnormalities. Androst-5-ene-3ß, 17ß-diol (ADIOL), an estrogen receptor (ER) ß agonist, was found to mediate a transrepressive mechanism that selectively modulates the extent of neuroinflammation and, in turn, neurodegeneration. In consensus, ERß polymorphism was more frequently detected in early-onset PD patients. Thus, in an approach to elucidate the role of ERß agonists on PD, our study was designed to investigate the possible neuroprotective effect of ADIOL, in three dose levels (0.35, 3.5, 35 mg/kg/day), against rotenone (ROT)-induced PD rat model. Amelioration in striatal dopamine (DA), nuclear factor-kappa B (NF-κB), and the expression of down-stream inflammatory mediators, as well as apoptotic markers were observed in the striatum and substantia nigra (SN) upon pre-treatment with the three doses of ADIOL. Similarly, light microscopy (LM) examination revealed declined degeneration of neurons upon pretreatment with ADIOL. Significant improvement in nigral tyrosine hydroxylase (TH) and reduction of nigral α-synuclein densities were also detected after ADIOL pre-treatment with better results frequently achieved with the middle dose (3.5 mg/kg/day). The middle dose of ADIOL showed behavioral improvement, with elevation in the ATP level, which was emphasized by the improvement in mitochondrial integrity observed upon electron microscopy (EM) examination. In conclusion, the present study confirmed for the first time the ability of ADIOL to protect against neuroinflammation and, in turn, neurodegeneration process and motor dysfunction in PD animal model, which was more obviously observed with the middle dose.

Sexual steroids in serum and prostatic tissue of human non-cancerous prostate (STERPROSER trial).

BACKGROUND: The specific involvement of the sex steroids in the growth of the prostatic tissue remains unclear. Sex steroid concentrations in plasma and in fresh surgical samples of benign central prostate were correlated to prostate volume. METHODS: Monocentric prospective study performed between September 2014 and January 2017. Age, obesity parameters, and both serum and intraprostatic concentrations of sex steroids were collected complying with the latest Endocrine Society guidelines and the steroids assessed by GC/MS. Statistical calculations were adjusted for age and body mass index (BMI). RESULTS: Thirty-two patients, equally divided between normal- and high-volume prostate groups, were included in the analysis. High-volume prostate patients were older, heavier and had higher BMI. Comparison adjusted for age and BMI showed higher DHT concentrations in high-volume prostate. Both normal- and high-volume prostate tissues concentrate sex steroids in a similar way. Comparison of enzymatic activity surrogate marker ratios within tissue highlighted similar TT/E1 and TT/E2 ratios, and higher DHT/E1 ratio and lower DHT/PSA ratio in the high-volume prostates. CONCLUSIONS: STERPROSER trial provides evidence for higher DHT concentration in highvolume prostates, that could reflect either higher 5-alpha reductase expression or lower expression of downstream metabolizing enzymes such as 3a-hydoxysteroid dehydrogenase.

Protective Effects of Fetal Zone Steroids Are Comparable to Estradiol in Hyperoxia-Induced Cell Death of Immature Glia.

Impaired neurodevelopment in preterm infants is caused by prematurity itself; however, hypoxia/ischemia, inflammation, and hyperoxia contribute to the extent of impairment. Because preterm birth is accompanied by a dramatic decrease in 17ß-estradiol (E2) and progesterone, preliminary clinical studies have been carried out to substitute these steroids in preterm infants; however, they failed to confirm significantly improved neurologic outcomes. We therefore hypothesized that the persistently high postnatal production of fetal zone steroids [mainly dehydroepiandrosterone (DHEA)] until term could interfere with E2-mediated protection. We investigated whether E2 could reduce hyperoxia-mediated apoptosis in three immature glial cell types and detected the involved receptors. Thereafter, we investigated protection by the fetal zone steroids DHEA, 16α-hydroxy-DHEA, and androstenediol. For DHEA, the involved receptors were evaluated. We examined aromatases, which convert fetal zone steroids into more estrogenic compounds. Finally, cotreatment was compared against single hormone treatment to investigate synergism. In all cell types, E2 and fetal zone steroids resulted in significant dose-dependent protection, whereas the mediating receptors differed. The neuroprotection by fetal zone steroids highly depended on the cell type-specific expression of aromatases, the receptor repertoire, and the potency of the fetal zone steroids toward these receptors. No synergism in fetal zone steroid and E2 cotreatment was detected in two of three cell types. Therefore, E2 supplementation may not be beneficial with respect to neuroprotection because fetal zone steroids circulate in persistently high concentrations until term in preterm infants. Hence, a refined experimental model for preterm infants is required to investigate potential treatments.

Simultaneous quantitation of nine hydroxy-androgens and their conjugates in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

Castration resistant prostate cancer (CRPC), the fatal form of prostate cancer, remains androgen dependent despite castrate levels of circulating testosterone (T) and 5α-dihydrotestosterone (DHT). To investigate mechanisms by which the tumor can synthesize its own androgens and develop resistance to abiraterone acetate and enzalutamide, methods to measure a complete androgen profile are imperative. Here, we report the development and validation of a stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometric (SID-LC-ESI-MS/MS) method to quantify nine human hydroxy-androgens as picolinates, simultaneously with requisite specificity and sensitivity. In the established method, the fragmentation patterns of all nine hydroxy-androgen picolinates were identified, and [13C3]-5α-androstane-3α, 17ß-diol and [13C3]-5α-androstane-3ß, 17ß-diol used as internal standards were synthesized enzymatically. Intra-day and inter-day precision and accuracy corresponds to the U.S. Food and Drug Administration Criteria for Bioanalytical Method Validation. The lower limit of quantitation (LLOQ) of nine hydroxy-androgens is 1.0pg to 2.5pg on column. Diols which have been infrequently measured: 5-androstene-3ß, 17ß-diol and 5α-androstane-3α, 17ß-diol can be determined in serum at values as low as 1.0pg on column. The method also permits the quantitation of conjugated hydroxy-androgens following enzymatic digestion. While direct detection of steroid conjugates by electrospray-ionization tandem mass spectrometry has advantages the detection of unconjugated and conjugated steroids would require separate methods for each set of analytes. Our method was applied to pooled serum from male and female donors to provide reference values for both unconjugated and conjugated hydroxy-androgens. This method will allow us to interrogate the involvement of the conversion of 5-androstene-3ß, 17ß-diol to T, the backdoor pathway involving the conversion of 5α-androstane-3α, 17ß-diol to DHT and the inactivation of DHT to 5α-androstane-3ß, 17ß-diol in advanced prostate cancer.

Molecular Mechanisms of Androstenediol in the Regulation of the Proliferative Process of Human Endometrial Cells.

Proliferation in endometria of women with polycystic ovarian syndrome (PCOS) is increased, similar to the biosynthesis of androstenediol (estrogenic metabolite). As previously shown, in human endometrial cells, androstenediol increases CYCLIN D1 levels and KI67 and decreases P27 content. The objective of the present investigation was to determine the mechanisms by which androstenediol promotes endometrial cell-cycle progression. Estrogen receptor α (ERα) activation and changes in CYCLIN D1 and P27 levels were evaluated by Western blot in T-HESC and St-T1b endometrial cell lines, using receptor antagonists; activation of PI3K-protein kinase B (AKT) and mitogen-activated protein kinases-extracellular signal-regulated kinases (MAPK-ERK)1/2 pathways was evaluated using PI3K, MAPK/ERK kinase (MEK)1/2, and RNA-polymerase II inhibitors. The data showed that androstenediol treatment significantly increases CYCLIN D1 and decreases P27 levels through ERα activation ( P < .05). In addition, an increase in AKT/ERK1/2 phosphorylations was determined ( P < .05). In the presence of RNA-polymerase II inhibitor, phosphorylation of AKT/ERK1/2 decreased ( P < .05), meaning that endometrial cells need transcriptional activity to activate the kinases involved. It was also observed that PI3K action is required for P27 and CYCLIN D1 changes. Therefore, the action of androstenediol in endometria depends on PI3K-AKT and MAPK-ERK1/2 pathways activation, together with cell transcriptional machinery. This could be of clinical significance, as in pathologies such as PCOS, increased endometrial levels of androstenediol together with a high prevalence of endometrial hyperplasia and adenocarcinoma have been reported.

Age-dependent Increases in Adrenal Cytochrome b5 and Serum 5-Androstenediol-3-sulfate.

CONTEXT: Adrenal production of dehydroepiandrosterone sulfate (DHEA-S) increases throughout childhood owing to expansion of the zona reticularis (ZR). ZR features cells with a steroidogenic phenotype distinct from that of the adjacent zona fasciculata, with higher expression of cytochrome b5 type A (CYB5A) and steroid sulfotransferase type 2A1 but decreased 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2). In addition to DHEA-S, three adrenal Δ5-steroid sulfates could provide additional tools to define adrenal maturation. OBJECTIVE: This study sought to simultaneously measure serum levels of four adrenal Δ5-steroid sulfates, pregnenolone sulfate (Preg-S), 17α-hydroxypregnenolone sulfate (17OHPreg-S), DHEA-S, and 5-androstenediol-3-sulfate (Adiol-S) as a function of age and relate their production to the age-dependent adrenal localization of CYB5A. PARTICIPANTS AND METHODS: Δ5-steroid sulfates were quantified by liquid chromatography-tandem mass spectrometry in sera from 247 normal children (129 males,118 females) age 1.5-18 y and 42 adults (20 males, 22 females). Immunofluorescence localized HSD3B2 and CYB5A in normal adrenal glands from subjects age 2-35 y. Finally, HAC15 adrenocortical cells were transduced with lentiviral short hairpin RNA to suppress CYB5A expression. RESULTS: Of the Δ5-steroid sulfates quantified, DHEA-S was most abundant. Adiol-S increased in parallel with DHEA-S. Steroid ratios (17OHPreg-S/DHEA-S) suggested increases in 17,20-lyase activity during childhood. Immunofluorescence analysis showed age-related increases in ZR CYB5A immunoreactivity. Furthermore, silencing CYB5A in HAC15 adrenocortical cells significantly reduced DHEA-S and Adiol-S production. CONCLUSION: Adiol-S shows a similar age-related increase to that of DHEA-S. This likely results from the childhood expansion of CYB5A-expressing ZR, which enhances 17,20-lyase activity and the production of DHEA-S and Adiol-S.