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The classical androgens, testosterone and dihydrotestosterone, together with dehydroepiandrosterone, the precusrsor to all androgens, are generally included in diagnostic steroid evaluations of androgen excess and deficiency disorders and monitored in androgen replacement and androgen suppressive therapies. The C11-oxy androgens also contribute to androgen excess disorders and are still often excluded from clinical and research-based steroids analysis. The contribution of the C11-oxy androgens to the androgen pool has not been considered in androgen deficiency. An exploratory investigation into circulating adrenal and gonadal steroid hormones in men was undertaken as neither the classical androgens nor the C11-oxy androgens have been evaluated in the context of concurrent measurement of all adrenal steroid hormones. Serum androgens, mineralocorticoids, glucocorticoids, progesterones and androgens were assessed in 70 healthy young men using ultra high performance supercritical fluid chromatography and tandem mass spectrometry. Testosterone, 24.5 nmol/L was the most prominent androgen detected in all participants while dihydrotestosterone, 1.23 nmol/L, was only detected in 25% of the participants. The 11-oxy androgens were present in most of the participants with 11-hydroxyandrostenedione, 3.37 nmol, in 98.5%, 11-ketoandrostenedione 0.764 in 77%, 11-hydroxytestosterone, 0.567 in 96% and 11-ketotestosterone: 0.440 in 63%. A third of the participants with normal testosterone and comparable 11-ketotestosterone, had significantly lower dehydroepiandrosterone (p < 0.001). In these males 11-hydroxyandrostenedione (p < 0.001), 11-ketoandrostenedione (p < 0.01) and 11-hydroxytestosterone (p < 0.006) were decreased. Glucocorticoids were also lower: cortisol (p < 0.001), corticosterone (p < 0.001), cortisone (p < 0.006) 11-dehydrocorticosterone (p < 0.001) as well as cortisol:cortisone (p < 0.001). The presence of dehydroepiandrosterone was associated with 16-hydroxyprogesterone (p < 0.001), which was also significantly lower. Adrenal and gonadal steroid analysis showed unexpected steroid heterogeneity in normal young men. Testosterone constitutes 78% of the circulating free androgens with the 11-oxy androgens abundantly present in all participants significantly contributing 22%. In addition, a subset of men were identified with low circulating dehydroepiandrosterone who showed altered adrenal steroids with decreased glucocorticoids and decreased C11-oxy androgens. Analysis of the classical and 11-oxy androgens with the additional measurement of dehydroepiandrosterone and 16-hydroxyprogesterone may allow better diagnostic accuracy in androgen excess or deficiency., (© 2024. The Author(s).)

Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly(I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36γ compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36γ despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a > 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36γ colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL-36γ, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36γ + /LC3b-II + EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses in antigen presenting cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Rationale: The instability of androst-5-ene-3,7-dione structures under acidic conditions is known. The formation of arimistane from 7-oxo-DHEA, influenced by the conditions of sample extraction, and mainly derivatization reaction and gas chromatography (GC) injector temperature, was described earlier, potentially leading to misinterpretation of results. By using a liquid chromatography (LC)-mass spectrometry (MS) (LC-MS) we investigated the stability of the 7-oxo-DHEA in two different solvents (methanol and dimethyl sulfoxide [DMSO]), and the arimistane formation after the application common analytical procedures. Additionally, in vitro and in vivo studies of 7-oxo-DHEA were performed., Methods: The stability of 7-oxo-DHEA was studied in solutions after 60 days storage at -20°C. In vitro studies were performed by incubating 7-oxo-DHEA with human liver microsomes (HLMs). Healthy volunteers collected urine samples before and after the administration of a single dose of 7-oxo-DHEA. Analyses were performed using high-performance LC (HPLC) coupled to a triple quadrupole mass spectrometer (MS/MS) and GC combustion isotope ratio mass spectrometry (GC-C-IRMS) following HPLC purification., Results: 7-oxo-DHEA was stable after 60 days in DMSO while a protic solvent as methanol promotes the degradation of 7-oxo-DHEA to arimistane. HLM incubations showed no formation of arimistane and the sample preparation only influenced the degradation of 7-oxo-DHEA when solvolysis was applied. After the administration study the presence of arimistane also after the hydrolysis with β-glucuronidase (Escherichia coli) was observed while using β-glucuronidase/arylsulfatase (Helix pomatia) showed the presence of arimistane already in blank samples collected before administration., Conclusions: Our results confirm arimistane as a valuable diagnostic marker of 7-oxo-DHEA administration, but also indicate that its formation is due to degradation processes rather than to metabolic biotransformation reactions., (© 2021 John Wiley & Sons, Ltd.)

Oxygen causes white matter damage in preterm infants and male sex is a major risk factor for poor neurological outcome, which speculates the role of steroid hormones in sex-based differences. Preterm birth is accompanied by a drop in 17β-estradiol (E2) and progesterone along with increased levels of fetal zone steroids (FZS). We performed a sex-based analysis on the FZS concentration differences in urine samples collected from preterm and term infants. We show that, in preterm urine samples, the total concentration of FZS, and in particular the 16α-OH-DHEA concentration, is significantly higher in ill female infants as compared to males. Since we previously identified Nup133 as a novel target protein affected by hyperoxia, here we studied the effect of FZS, allopregnanolone (Allo) and E2 on differentiation and Nup133 signaling using mouse-derived primary oligodendrocyte progenitor cells (OPCs). We show that the steroids could reverse the effect of hyperoxia-mediated downregulation of Nup133 in cultured male OPCs. The addition of FZS and E2 protected cells from oxidative stress. However, E2, in presence of 16α-OH-DHEA, showed a negative effect on male cells. These results assert the importance of sex-based differences and their potential implications in preterm stress response.

BNN20, a C17-spiroepoxy derivative of the neurosteroid dehydroepiandrosterone, has been shown to exhibit strong neuroprotective properties but its role in glial populations has not been assessed. Our aim was to investigate the effect of BNN20 on glial populations by using in vitro and in vivo approaches, taking advantage of the well-established lysophosphatidylcholine (LPC)-induced focal demyelination mouse model. Our in vivo studies, performed in male mice, showed that BNN20 treatment leads to an increased number of mature oligodendrocytes (OLs) in this model. It diminishes astrocytic accumulation during the demyelination phase leading to a faster remyelination process, while it does not affect oligodendrocyte precursor cell recruitment or microglia/macrophage accumulation. Additionally, our in vitro studies showed that BNN20 acts directly to OLs and enhances their maturation even after they were treated with LPC. This beneficial effect of BNN20 is mediated, primarily, through the neurotrophin receptor TrkA. In addition, BNN20 reduces microglial activation and their transition to their pro-inflammatory state upon lipopolysaccharides stimulation in vitro. Taken together our results suggest that BNN20 could serve as an important molecule to develop blood-brain barrier-permeable synthetic agonists of neurotrophin receptors that could reduce inflammation, protect and increase the number of functional OLs by promoting their differentiation/maturation., (© 2021 Wiley Periodicals LLC.)

Background: The serum prostate-specific antigen is the most widespread biomarker for prostate disease. Its low specificity for prostatic malignancies is a matter of concern and the reason why new biomarkers for screening purposes are needed. The correlation between altered production of the main steroids and prostate carcinoma (PCa) occurrence is historically known. The purpose of this study is to evaluate the modifications of a comprehensive urinary endogenous steroidal profile (USP) induced by PCa, by multivariate statistical methods., Methods: A total of 283 Italian subjects were included in the study, 139 controls and 144 PCa-affected patients. The USP, including 17 steroids and five urinary steroidal ratios, was quantitatively evaluated using gas chromatography coupled with single quadrupole mass spectrometry (GC-MS). The data were interpreted using a chemometric, multivariate approach (intrinsically more sensible to alterations with respect to traditional statistics) and a model for the discrimination of cancer-affected profiles was built., Results: Two multivariate classification models were calculated, the former including three steroids with the highest statistical significance (e.g. testosterone, etiocholanolone and 7β-OH-DHEA) and PSA values, the latter considering the three steroids' levels only. Both models yielded high sensitivity and specificity scores near to 70%, resulting significantly higher than PSA alone., Conclusions: Three USP steroids resulted significantly altered in our PCa population. These preliminary results, combined with the simplicity and low-cost of the analysis, open to further investigation of the potential role of this restricted USP in PCa diagnosis.

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus-mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.

BNN-20 is a synthetic microneurotrophin, long-term (P1-P21) administration of which exerts potent neuroprotective effect on the "weaver" mouse, a genetic model of progressive, nigrostriatal dopaminergic degeneration. The present study complements and expands our previous work, providing evidence that BNN-20 fully protects the dopaminergic neurons even when administration begins at a late stage of dopaminergic degeneration (>40%). Since neuroinflammation plays a critical role in Parkinson's disease, we investigated the possible anti-neuroinflammatory mechanisms underlying the pharmacological action of BNN-20. The latter was shown to be microglia-mediated, at least in part. Indeed, BNN-20 induced a partial, but significant, reversal of microglia hyperactivation, observed in the untreated "weaver" mouse. Furthermore, it induced a shift in microglia polarization towards the neuroprotective M2 phenotype, suggesting a possible beneficial shifting of microglia activity. This observation was further supported by morphometric measurements. Moreover, BDNF levels, which were severely reduced in the "weaver" mouse midbrain, were restored to normal even after short-term BNN-20 administration. Experiments in "weaver"/NGL (dual GFP/luciferase-NF-κВ reporter) mice using bioluminescence after a short BNN-20 treatment (P60-P74), have shown that the increase of BDNF production was specifically mediated through the TrkB-PI3K-Akt-NF-κB signaling pathway. Interestingly, long-term BNN-20 treatment (P14-P60) significantly increased dopamine levels in the "weaver" striatum, which seems to be associated with the improved motor activity observed in the treated mutant animals. In conclusion, our findings suggest that BNN-20 may serve as a lead molecule for new therapeutic compounds for Parkinson's disease, combining strong anti-neuroinflammatory and neuroprotective properties, leading to elevated dopamine levels and improved motor activity., (Copyright © 2019. Published by Elsevier Ltd.)

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-κB, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca-/- mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.

Background: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), affecting approximately one third of the world's population. Development of an adequate immune response will determine disease progression or progress to chronic infection. Risk of developing TB among human immunodeficiency virus (HIV)-coinfected patients (HIV-TB) is 20-30 times higher than those without HIV infection, and a synergistic interplay between these two pathogens accelerates the decline in immunological functions. TB treatment in HIV-TB coinfected persons is challenging and it has a prolonged duration, mainly due to the immune system failure to provide an adequate support for the therapy. Therefore, we aimed to study the role of the hormone 7-oxo-dehydroepiandrosterone (7-OD) as a modulator of anti-tuberculosis immune responses in the context of HIV-TB coinfection., Methods: A cross-sectional study was conducted among HIV-TB patients and healthy donors (HD). We characterized the ex vivo phenotype of CD4 + T cells and also evaluated in vitro antigen-specific responses by Mtb stimulation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of 7-OD. We assessed lymphoproliferative activity, cytokine production and master transcription factor profiles., Results: Our results show that HIV-TB patients were not able to generate successful anti-tubercular responses in vitro compared to HD, as reduced IFN-γ/IL-10 and IFN-γ/IL-17A ratios were observed. Interestingly, treatment with 7-OD enhanced Th1 responses by increasing Mtb-induced proliferation and the production of IFN-γ and TNF-α over IL-10 levels. Additionally, in vitro Mtb stimulation augmented the frequency of cells with a regulatory phenotype, while 7-OD reduced the proportion of these subsets and induced an increase in CD4 + T-bet+ (Th1) subpopulation, which is associated with clinical data linked to an improved disease outcome., Conclusions: We conclude that 7-OD modifies the cytokine balance and the phenotype of CD4 + T cells towards a more favorable profile for mycobacteria control. These results provide new data to delineate novel treatment approaches as co-adjuvant for the treatment of TB.

Non-invasive measures of stress are crucial for captive and conservation management programs. The adrenal hormone dehydroepiandrosterone-sulfate (DHEAS) has recently been adopted as a stress marker, but there is little investigation of its relationship to glucocorticoids (GC), well-known indicators of stress. This study examined the influence of age, reproductive state and environment on GC and DHEAS levels in orangutans, to test whether the GC/DHEAS ratio can provide an index of stress response in primates. We measured fecal GC (fGC) and fecal DHEAS (fDHEAS) concentrations in 7 captive orangutans from zoological parks in Japan and 22 wild orangutans from Danum Valley Conservation Area, Malaysia. We found that in a stressful condition (transportation), fDHEAS levels increased 2 days after the fGC response, which occurred 1 day after the stressor. One pregnant female had elevated levels of both hormones, and a higher fGC/fDHEAS ratio than baseline. Females in the first year of lactation had fGC levels and the fGC/fDHEAS ratio significantly higher than both baseline and females in the second and subsequent years of lactation. There was no effect of age on fGC levels, but the fGC/fDHEAS ratio was higher in infants than adults and adolescents. fDHEAS concentrations were lower in infants than juveniles, adolescents and adults, a phenomenon known as adrenarche, shared with humans and other great apes. We suggest that changes in DHEAS during orangutan life history are associated with changes in the dynamics of maintaining homeostasis that vary with age and reproductive state. The GC/DHEAS ratio index is useful to evaluate age-related abilities of responding to stressful challenges.

7-keto-DHEA (3β-hydroxy-androst-5-ene-7,17-dione) is included in section S1 of the World Antidoping Agency (WADA) List of Prohibited Substances. The detection of its misuse in sports needs special attention, since it is naturally present in urine samples. The main goal of this study is to investigate the in vivo metabolism of 7-keto-DHEA after a single administration to healthy volunteers and to better describe the relationship between arimistane (androst-5-ene-7,17-dione) and 7-keto-DHEA after the application of the common routine procedures to detect anabolic steroids in WADA accredited antidoping laboratories. Free, glucuro-, and sulpho-conjugated steroids extracted from urine samples obtained before and after the administration of 7-keto-DHEA were analyzed by different gas chromatographic (GC)-mass spectrometric (MS) techniques. Gas chromatography coupled to tandem MS to study the effect on the endogenous steroid profile, coupled to isotope ratio mass spectrometry (IRMS) to investigate the potential formation of androgens derived from DHEA and coupled to high resolution accurate mass spectrometry (HRMS) to investigate new diagnostic metabolites. The analysis by IRMS confirmed that there is no formation of DHEA from 7-keto-DHEA. Ten proposed metabolites, not previously reported, were described. These include reduced and hydroxylated structures that are not considered part of the steroid profile in antidoping analyses. They showed considerable responses in all fractions analyzed. Some deoxidation reactions (including arimistane formation) were found and most probably can be linked to the sample preparation or instrumental analysis. This is important when interpreting the results after the application of procedures to detect steroids in urine currently used in antidoping laboratories. 7-keto-DHEA metabolism in humans for antidoping purposes was studied and unexpected results were found. This could lead to a misinterpretation of the data, depending on the procedure applied and the analytical instrumentation used., (© 2019 John Wiley & Sons, Ltd.)

DHEA and DHEAS are neuroactive neurosteroids that interact with several major receptor systems in the brain, including sigma (σ), glutamate, and GABA-A receptors. It has been recognized as early as 1952, that the loss of DHEA/DHEAS in adult life is associated with neuropsychiatric disorders (eg schizophrenia, depression). However, the mechanistic role for DHEA/DHEAS in any of these domains remains speculative, not the least because the presence of these androgens in the adrenal gland and brain is largely confined to humans and only some non-human primates. DHEA and DHEAS are dynamically regulated from before birth and before the onset of puberty, and therefore an understanding of the synthesis, regulation, and functions of this important androgen pathway warrants attention. Here, we draw attention to the possible modulating influence of DHEA/DHEAS in early brain development from fetal life to the remarkable increase of these steroids in early childhood - the adrenarche. We propose that the pre-pubertal DHEA/DHEAS surge plays a key role in modulating early brain development, perhaps by prolonging brain plasticity during childhood to allow the pre-adolescent brain to adapt and re-wire in response to new, and ever-changing social challenges. Nonetheless, the aetiology of neurodevelopmental phenomena in relation to DHEA/DHEAS synthesis and action cannot be easily studied in humans due to the obvious ethical restrictions on mechanistic studies, the uncertainty of predicting the future mental characteristics of individuals, and the difficulty of conducting retrospective investigations based on pre-birth and/or neonatal complications. We discuss new opportunities for animal studies to resolve these important questions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Beauveria bassiana is an entomopathogenic fungus used as a biological control agent. It is a well-known biocatalyst for the transformation of steroid compounds. Hydroxylations at the 7α or 11α position and oxidation to D-homo lactones are described in the literature. In our study, we examined the diversity of metabolism of five different B. bassiana strains and compared them to already known pathways. According to the literature, 7α and 11α-hydroxy derivatives as well as 3β,11α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one have been observed. Here we describe new DHEA metabolic pathways and two products not described before: 3β-hydroxy-17a-oxa-D-homo-androst-5-en-7,17-dione and 3β,11α-dihydroxyandrost-5-en-7,17-dione. We also used for the first time another species from this genus, Beauveria caledonica, for steroid transformation. DHEA was hydroxylated at the 7α, 7β and 11α positions and then reactions of oxidation and reduction leading to 3β,11α-dihydroxyandrost-5-en-7,17-dione were observed. All tested strains from the Beauveria genus effectively transformed the steroid substrate using several different enzymes, resulting in cascade transformation.

The aim of the present study was to determine the cytotoxic effects of a series of novel dehydroepiandrosterone derivatives containing triazole at the C 16 position on human cancer cells. The cancer cells used in the present study were A549, Hela, HepG-2, BEL7402, MCF-7, and HCT116. Several of the synthesised compounds exhibited potent antiproliferative effects. The most promising compound was ( E )-3-hydroxy-16-((1-(4-iodophenyl)-1 H -1,2,3-triazole-4-yl)methylene)-10,13-dimet-hyl-1,3,4,7,8,9,10,11,12,13,15,16-dodecahydro-2 H -cyclopenta[a]phenanthren-17(14)-one (compound 2n ), which showed considerably high antiproliferative activity in the HepG-2 cell line, with an IC 50 value of 9.10 µM, and considerably high activity against the MCF-7 cell line, with an IC 50 value of 9.18 µM. Flow cytometry assays demonstrated that compound 2n exerted antiproliferative effects by arresting cells in the G2 phase of the cell cycle and inducing apoptosis.

An estimated one third of the world's population is affected by latent tuberculosis (TB), which once active represents a leading cause of death among infectious diseases. Human immunodeficiency virus (HIV) infection is a main predisposing factor to TB reactivation. Individuals HIV-TB co-infected develop a chronic state of inflammation associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation. This results in a hormonal imbalance, disturbing the physiological levels of cortisol and dehydroepiandrosterone (DHEA). DHEA and its oxygenated metabolites androstenediol (AED), androstenetriol (AET) and 7-oxo-DHEA are immunomodulatory compounds that may regulate physiopathology in HIV-TB co-infection. In order to study possible changes in plasma levels of these hormones, we developed an approach based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). To our knowledge, this represents the first report of their simultaneous measurement in HIV-TB individuals and the comparison with healthy donors, obtaining statistically higher plasma levels of DHEA, AET and 7-oxo-DHEA in patients. Moreover, we found that concentrations of 7-oxo-DHEA positively correlated with absolute CD4+ T cell counts, nadir CD4+ T cell values and with individuals who presented TB restricted to the lungs. This research contributes to understanding the role of these hormones in HIV-TB and emphasizes the importance of deepening their study in this context.

In the clinical laboratories, dehydroepiandrostenedione (DHEA) is usually quantified by immunoassay-based methods, which are often affected by cross-reactivity with endogenous interferences, such as 4-androsten-3β-ol-17-one. The interfering compounds lead to a poor accuracy of the measurements, mainly at a low concentration level. The present paper describes a validated method based on tandem mass spectrometry coupled to liquid chromatography, for the accurate quantification of DHEA in serum. The peculiarity of this method is the use of calibrators and quality controls prepared by adding measured amounts of DHEA-D 5 , a stable isotope-labeled analogue of DHEA, to real serum from healthy subjects. DHEA-D 5 is used in place of DHEA, which is usually present in unstripped serum at physiological levels, as it has the same basic structure, provides an equivalent instrumental response, and can be easily distinguish by DHEA by mass spectrometry due to its different m/z value. The method proved to be sensitive, with a LLOD of 0.09 ng/mL and a LLOQ of 0.23 ng/mL, and selective, with overall performances that allow its use on a routine basis.

Smoking during pregnancy presents health risks for both the mother and her child. In this study we followed changes in the production of steroid hormones in pregnant smokers. We focused on changes in steroidogenesis in the blood of mothers in their 37(th) week of pregnancy and in mixed cord blood from their newborns. The study included 88 healthy women with physiological pregnancies (17 active smokers and 71 non-smokers). We separately analyzed hormonal changes associated with smoking according to the sex of newborns. In women with male fetuses, we found higher levels of serum cortisone, dehydroepiandrosterone (DHEA), 7alpha-OH-DHEA, 17-OH pregnenolone, testosterone, and androstenedione in smokers at the 37(th) week compared to non-smokers. In women with female fetuses, we found lower serum levels of 7beta-OH-DHEA and higher androstenedione in smokers at the 37(th) week. We found significantly higher levels of testosterone in newborn males of smokers and higher levels of 7alpha-OH-DHEA in female newborns of smokers. Smoking during pregnancy induces changes in the production of steroids in both the mother and her child. These changes are different for different fetal sexes, with more pronounced changes in mothers carrying male newborns as well as in the newborn males themselves.

Neurotrophic factors are among the most promising treatments aiming at slowing or stopping and even reversing Parkinson's disease (PD). However, in most cases, they cannot readily cross the human blood-brain-barrier (BBB). Herein, we propose as a therapeutic for PD the small molecule 17-beta-spiro-[5-androsten-17,2'-oxiran]-3beta-ol (BNN-20), a synthetic analogue of DHEA, which crosses the BBB and is deprived of endocrine side-effects. Using the "weaver" mouse, a genetic model of PD, which exhibits progressive dopaminergic neurodegeneration in the Substantia Nigra (SN), we have shown that long-term administration (P1-P21) of BNN-20 almost fully protected the dopaminergic neurons and their terminals, via i) a strong anti-apoptotic effect, probably mediated through the Tropomyosin receptor kinase B (TrkB) neurotrophin receptor's PI3K-Akt-NF-κB signaling pathway, ii) by exerting an efficient antioxidant effect, iii) by inducing significant anti-inflammatory activity and iv) by restoring Brain-Derived Neurotrophic Factor (BDNF) levels. By intercrossing "weaver" with NGL mice (dual GFP/luciferase-NF-κΒ reporter mice, NF-κΒ.GFP.Luc), we obtained Weaver/NGL mice that express the NF-κB reporter in all somatic cells. Acute BNN-20 administration to Weaver/NGL mice induced a strong NF-κB-dependent transcriptional response in the brain as detected by bioluminescence imaging, which was abolished by co-administration of the TrkB inhibitor ANA-12. This indicates that BNN-20 exerts its beneficial action (at least in part) through the TrkB-PI3K-Akt-NF-κB signaling pathway. These results could be of clinical relevance, as they suggest BNN-20 as an important neuroprotective agent acting through the TrkB neurotrophin receptor pathway, mimicking the action of the endogenous neurotrophin BDNF. Thus BNN-20 could be proposed for treatment of PD., (Copyright © 2017. Published by Elsevier Ltd.)

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 17β-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., (Copyright © 2017 Endocrine Society.)

A series of steroidal[17,16-d]pyrimidines derived from dehydroepiandrosterone were designed and prepared by a convenient heterocyclization reaction. The in vitro anticancer activities for these obtained compounds were evaluated against human cancer cell lines (HepG2, Huh-7, and SGC-7901), which demonstrated that some of these heterocyclic pyrimidine derivatives exhibited significantly good cytotoxic activities against all tested cell lines compared with 5-fluorouracil (5-FU), especially, compound 3b exhibited high potential growth inhibitory activities against all tested cell lines with the IC 50 values of 5.41 ± 1.34, 5.65 ± 1.02 and 10.64 ± 1.49 μM, respectively, which might be used as promising lead scaffold for discovery of novel anticancer agents.

Numerous steroids are essential plant, animal, and human hormones. The medical and industrial applications of these hormones require the identification of new synthetic routes, including biotransformations. The metabolic fate of a steroid can be complicated; it may be transformed into a variety of substituted derivatives. This may be because a steroid molecule can adopt several possible orientations in the binding pocket of a receptor or an enzyme. The present study, based on docking and molecular dynamics, shows that it is indeed possible for a steroid molecule to bind to a receptor binding site in two or more orientations (normal, head-to-tail reversed, upside down). Three steroids were considered: progesterone, dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. Two proteins were employed as hosts: the human mineralocorticoid receptor and a bacterial Baeyer-Villiger monooxygenase. When the steroids were in nonstandard orientations, the estimated binding strength was found to be only moderately diminished and the network of hydrogen bonds between the steroid and the host was preserved.

Metabolism of steroids in healthy and unhealthy human organs is the subject of extensive clinical and biomedical studies. For this kind of investigations, it is essential that the reference samples of new derivatives of natural, physiologically active steroids (especially those difficult to achieve in the chemical synthesis) become available. This study demonstrated for the first time transformation of 7-oxo-DHEA-a natural metabolite of DHEA, using Syncephalastrum racemosum cells. The single-pulse fermentation of substrate produced two new hydroxy metabolites: 1β,3β-dihydroxy-androst-5-en-7,17-dione and 3β,12β-dihydroxy-androst-5-en-7,17-dione, along with the earlier reported 3β,9α-dihydroxy-androst-5-en-7,17-dione and 3β,17β-dihydroxy-androst-5-en-7-one. Simultaneously, the same metabolites, together with small quantities of 7α- and 7β-hydroxy-DHEA, as well as the products of their reduction at the C-17 were obtained after transformation of DHEA under pulse-feeding of the substrate. The observed reactions suggested that this micro-organism contains enzymes exhibiting similar activity to those present in human cells. Thus, the resulting compounds can be considered as potential components of the eukaryotic, including human, metabolome., (© 2016 John Wiley & Sons A/S.)

In this study, we investigated the in vitro effect of 16-formyl-17-methoxy dehydroepiandrosterone derivatives on the activity of 5α-reductase type 2 (5α-R2) obtained from human prostate. The activity of different concentrations of these derivatives was determined for the conversion of labelled testosterone to dihydrotestosterone. The results indicated that an aliphatic ester moiety at the C-3 position of these derivatives increases their in vitro potency as inhibitors of 5α-R2 activity compared to finasteride®, which is considered to be a potent inhibitor of 5α-R2. In this case, the augmentation of the lipophilicity of these dehydroepiandrosterone derivatives increased their potency as inhibitors of 5α-R2. However, the presence of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings as the cycloaliphatic ester moiety at C-3 of the formyl methoxy dehydroepiandrosterone scaffold did not inhibit the activity of this enzyme. This may be due to the presence of steric factors between the enzyme and the spatial structure of these derivatives.

The goal of this work was a good-yielding chemical synthesis of a metandienone metabolite which is of interest in doping analysis. 20βOH-NorMD (IUPAC: 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-triene-3-one) has been identified as a long-term urinary metabolite which can be detected and attributed to metandienone up to almost 3weeks after exposure. The chemical synthesis of its epimer 20αOH-NorMD has been described before, as was an enzymatic synthesis of 20βOH-NorMD, but no chemical synthesis was published., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The antiviral activity of dehydroepiandrosterone (DHEA) and 21 synthetic derivatives against influenza A virus (IAV) replication was investigated in vitro in cell culture. Our results revealed that three DHEA analogues were potent inhibitors of IAV multiplication in MDCK cells and mainly blocked the post-attachment stage of viral infection. Among these derivatives, one containing a 2-OH-Ph moiety (3i) exhibited the best inhibitory effects against H1N1 and H3N2 IAV in a dose-dependent manner. Moreover, treatment with compound 3i decreased progeny virus yields, viral RNA synthesis and protein expression. Orally administered compound 3i at 25 or 50 mg/kg/day for 5 days protected mice from lethal A/FM/1/47 (H1N1) challenge by reducing the viral titers in the lungs and promoting survival of infected mice. Our results suggest that DHEA-dihydrazone derivatives may provide promising lead scaffolds for further design and synthesis of potential antiviral agents.

Dietary supplement effects and drug interactions can lead to significant adverse health events, thus potentially impacting the safe delivery of oral healthcare. This study sought to determine the frequency of, and factors impacting, dietary supplement use among 209 dental patients and whether the design of a medical history questionnaire influences reporting of supplement use. Patients were randomly allocated to 1 of 2 groups in which they completed either a standard medical history questionnaire (n = 107) or the same questionnaire with an additional item about dietary supplement use (n = 102). All patients were then administered a survey with questions about their demographics, their use and knowledge of dietary supplements, and the person or persons who recommended dietary supplement use to the patient. While 62% of the total population (130/209) reported supplement use, specific prompting nearly doubled the number of supplements reported (mean with prompting: 1.53; mean without prompting: 0.76; P < 0.0001). Patients younger than 30 years of age reported significantly less dietary supplement use than all other age groups except the 30-40 age group (P = 0.0003). An estimated 70% of all respondents were not aware of potentially detrimental side effects of dietary supplement use or possible interactions with conventional drug therapies. Since patients tended to report a greater use of dietary supplements when specifically asked about their use on a medical history questionnaire, a checklist or set of designated questions may be a suitable first step toward gathering this essential information.

Microneurotrophins (MNT's) are small molecule derivatives of dehydroepiandrosterone (DHEA) and do not have significant interactions with sex steroid receptors. MNT's retain high-affinity binding to protein tyrosine kinase (Trk) receptors and can mimic many pleiotropic actions of neurotrophin (NT) proteins on neurons. MNT's offer therapeutic potential for diseases such as amyotrophic lateral sclerosis (ALS) where motor neurons (MN) degenerate. MNT's cross artificial membranes mimicking the blood-brain barrier, are not major substrates for ABC (ATP-binding cassette) transporters and are metabolized rapidly by mouse but more slowly by human hepatocytes. A lead MNT (BNN27) and its mono-oxidation metabolites enter mouse brain rapidly. RNA-sequencing measured gene expression profiles of human H9eSC-(embryonic stem cell)-derived or CTL (control) subject iPSC-(induced pluripotential stem cell)-derived MN's exposed to NT proteins or MNT molecules. Expression ratios (relative to DMSO (dimethylsulfoxide) vehicle) were calculated, and the resulting top 500 gene lists were analyzed for Gene Ontology (GO) grouping using DAVID (Database for Annotation, Visualization and Integrated Discovery). The MNT's BNN20, BNN23, and BNN27 showed overlap of GO terms with NGF (nerve growth factor) and BDNF (brain-derived neurotrophic factor) in the H9eSC-derived MN's. In the iPSC-derived MN's two (BNN20, BNN27) showed overlap of GO terms with NGF or BDNF. Each NT protein had GO terms that did not overlap with any MNT in the MN cell lines., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR) is a serious microvascular complication of diabetes and a major cause of blindness in the developing world. Early diabetic retinopathy is characterized by a loss of pericytes and vascular endothelial cells, a breakdown of the blood-retinal barrier, vascular dysfunction and vascular-neuroinflammation. However, optimal treatment options and related mechanisms are still unclear. MicroRNA-126 (miR-126) plays a potential role in the pathogenesis in DR, which may regulate VEGF, Ang-1 and VCAM-1 expressions. This study investigated the therapeutic effects and mechanisms of Niaspan treatment of DR in diabetes (DM) rats. DM rats exhibits significantly decreased miR-126 and tight junction Claudin-5/Occludin/ZO-1 genes expression, and increased Blood retinal-barrier (BRB) breakdown, retinal apoptosis and VEGF/VEGFR, as well as VCAM-1/CD45 expressions in the retina compared to normal control group. Niaspan treatment significantly improved clinical and histopathological outcomes; decreased the expressions of VEGF/VEGFR, VCAM-1/CD45, apoptosis and BRB breakdown, significantly increased tight junction proteins and Ang-1/Tie-2 expressions, as well as increased retinal miR-126 expression compared to non-treatment diabetic rats. These data are the first to show that Niaspan treatment ameliorates DR through its repair vascular and inhibits inflammatory effects, and also suggest that the miR-126 pathway may contribute to Niaspan treatment induced benefit effects.

The circulating levels of 16β hydroxydehydroepiandrosterone (16β OH-DHEA) are at the limit of detection (less than 10 pg/mL), unlike the serum concentrations of 16α-hydroxydehydroepiandrosterone (16α OH-DHEA, 10-300 pg/mL) in premenopausal, postmenopausal and male serum. A major reason could be the rapid conversion of 16β OH-DHEA to 5-androstene-3beta, 17beta-diol 16 one (3β, 17β-diol 16-oxo) in serum due to the stereospecific structure of 16β OH-DHEA. In ultrapure H2O, there is no apparent conversion observed while 16β OH-DHEA (10 ng/mL) spiked in stripped or unstripped serum is quickly converted to 3β, 17β-diol 16-oxo at room temperature. During this conversion, a further converted product was observed with a difference in molecular weight of 16 Da from that of 16β OH-DHEA and 3β, 17β-diol 16-oxo, which could be their hydroxylation product, i.e. triol-ketone. Under basic conditions, further conversion occurs. The present data can explain the practically undetectable concentration of serum 16β OH-DHEA while 3β, 17β-diol 16-oxo is at the level of less than 50 pg/mL. Serum concentrations of (0.0-9.9 pg/mL for 16β OH-DHEA, 8.9-50.7 pg/mL for 3β, 17β-diol 16-oxo and 10.0-285.0 pg/mL for 16α OH-DHEA are measured in sera of premenopausal, postmenopausal women and men over 50 years of age., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As an extension of previously conducted studies on developing an anti-Alzheimer's disease agent, denosomin (1-deoxy-24-norsominone, an artificial inducer of neurite elongation), derivatives were designed and synthesized based on the hypothesis that our denosomin would exhibit axonal extension activity via a 1,25D(3)-membrane-associated, rapid response steroid-binding protein (1,25D(3)-MARRS) pathway. The biological assay revealed that the hybridization of characteristic δ-lactone in denosomin and the triene moiety in VD(3) was effective to enhance the nerve re-extension activity in amyloid β (Aβ)-damaged neurons.

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 50 μM) that revealed to be non-cytotoxic in these experimental conditions. We measured hSCs metabolites consumption/production by (1)H 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 50 μM) 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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Thirteen novel furoxan-based nitric oxide (NO) releasing hybrids (14a-e, 15a-e, 17b-d) of 16,17-pyrazo-annulated steroidal derivatives were synthesized and evaluated against the MDA-MB-231, HCC1806, SKOV-3, DU145, and HUVEC cell lines for their in vitro anti-proliferative activity. Most of the compounds displayed potent anti-proliferative effects. Among them, 17c exhibited the best activity with IC50 values of 20-1.4nM against four cell lines (MDA-MB-231, SKOV-3, DU145, and HUVEC), and 1.03μM against a tamoxifen resistant breast cancer cell line (HCC1806). Furthermore, five compounds (14a, 15a, 17b-d) were selected to screen for VEGF inhibitory activity. Compounds 15a, 17b,c showed obviously better activity than 2-Methoxyestradiol (2-ME) on reducing levels of VEGF secreted by MDA-MB-231 cell line. In a Capillary-like Tube Formation Assay, compounds 17b,c exhibited a significant suppression of the tubule formation in the concentration of 1.75nM and 58nM, respectively. The preliminary SAR showed that steroidal scaffolds with a linker in 3-position were favorable moieties to evidently increase the bioactivities of these hybrids. Overall, these results implied that 17c merited to be further investigated as a promising anti-cancer candidate., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Normal pressure hydrocephalus (NPH) is a treatable neurological disorder affecting elderly people with the prevalence increasing with age. NPH is caused by abnormal cerebrospinal fluid (CSF) reabsorption and manifested as a balance impairment, urinary incontinence and dementia development. These symptoms are potentially reversible if recognized early. Diagnosis of NPH is difficult and can be easily mistaken for other neurodegenerative disorders, which makes NPH one of the major misdiagnosed diseases worldwide. The aim of the study was to find out the appropriate combination of indicators, based on CSF steroids, which would contribute to a clearer NPH diagnosis. The levels of CSF cortisol, cortisone, dehydroepiandrosterone (DHEA), 7α-OH-DHEA, 7β-OH-DHEA, 7-oxo-DHEA, 16α-OH-DHEA and aldosterone (all LC-MS/MS) were determined in our patients (n=30; NPH, 65-80 years) and controls (n=10; 65-80 years). The model of orthogonal projections to latent structures (OPLS) was constructed to predict NPH. Cortisone, 7α-OH-DHEA, 7β-OH-DHEA, 7-oxo-DHEA, aldosterone, 7α-OH-DHEA /DHEA, 7-oxo-DHEA/7α-OH-DHEA, 7β-OH-DHEA/7-oxo-DHEA and 16α-OH-DHEA/DHEA in the CSF were identified as the key predictors and the model discriminated patients from controls with 100% sensitivity and 100% specificity. The suggested model would contribute to early and accurate NPH diagnosis, enabling promptly treatment of the disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: The occurrence of hyperglycemia in non-diabetics during development of acute coronary ischemia (ACI) indicates latent glucose metabolism disorder, or is a case of newly discovered diabetes mellitus (DM) as a result of stress. Acute coronary syndrome refers to a group of clinical syndromes caused by a sudden circulatory disorder in coronary arteries, resulting in the corresponding myocardial ischemia. It covers range from unstable angina and myocardial infarction (MI) without Q wave in the electrocardiogram finding (NSTEMI) up to myocardial infarction with Q wave in the electrocardiogram finding (STEMI)., Goal: To determine the incidence of hyperglycemia in non-diabetics immediately after the occurrence of acute coronary ischemia and assess its risk factors., Results: The sample included 80 respondents. Men dominated with a total prevalence of 77.5%. The respondent was at mean age of 62.8±13.8 years. During the first measurement, immediately after hospital admission, 50% of respondents had increased blood glucose value and during the second measurement 62%. Hypertension as a risk factor has 54% and 56% smoking. The incidence of stress diabetes after ACI does not depend on the diagnosis of hypertension, χ(2)=0.050; p=0.823. The differences of mean values (median) BMI between examined persons with/without stress DM are not statistically significant p=0.402. Independent t-test showed that there was no statistically significant difference in the average values of HDL and LDL in patients with stress diabetes than in patients without diabetes stress after ACI p>0.05. For each year of age odds ratio for "stress diabetes" increases by 7% and 95% CI is 2% -12%., Conclusion: The incidence of stress diabetes ACI is not dependent on the working diagnosis (MI or angina pectoris). As risk factors we set hypertension and current smoking. There were no statistically significant associations between active smoking and hypertension as a risk factor in relation to occurrence of stress diabetes.

Estriol biosynthesis in human placenta requires the uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT)-4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [(3)H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hours but was inhibited in the presence of 50 μM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.

Dehydroepiandrosterone (DHEA) and its 7-oxo- and 7-hydroxy-metabolites occurring in the brain are considered neurosteroids. Metabolism of the latter is catalysed by 11β-hydroxysteroid dehydrogenase (11β-HSD) which also interconverts cortisol and cortisone. The concurrent metabolic reaction to DHEA 7-hydroxylation is the formation of 16α-hydroxy-DHEA. The LC-MS/MS method using triple stage quadrupole-mass spectrometer was developed for simultaneous quantification of free DHEA, 7α-hydroxy-DHEA, 7β-hydroxy-DHEA, 7-oxo-DHEA, 16α-hydroxy-DHEA, cortisol and cortisone in human plasma and cerebrospinal fluid (CSF). The method employs 500 μL of human plasma and 3000 μL of CSF extracted with diethyl ether and derivatized with 2-hydrazinopyridine. It has been validated in terms of sensitivity, precision and recovery. In plasma, the following values were obtained: limit of detection: 2-50p g/mL; limit of quantification: 5-140 pg/mL; within-day precision 0.58-14.58%; between-day precision: 1.24-13.89% and recovery: 85-113.2%). For CSF, the values of limit of detection: 2-28 pg/mL; limit of quantification: 6-94 pg/mL; within-day precision; 0.63-5.48%; between-day precision: 0.88-14.59% and recovery: 85.1-109.4% were acquired. Medians and concentration ranges of detected steroids in plasma and CSF are given in subjects with excluded normal pressure hydrocephalus (n=37; 65-80 years). The method enables simultaneous quantification of steroids important for the estimation of 11β-HSD activity in human plasma and CSF. It will be helpful in better understanding various degenerative diseases development and progression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To compare E-selectin, resistin and reactive oxygen species (ROS) levels in serum and follicular fluid (FF) of subfertile women undergoing Controlled Ovarian Hyperstimulation (COH) during IVF/ICSI cycles, using GnRH-agonist and -antagonist protocols., Methods: In this prospective cohort study, 85 subfertile women undergoing IVF/ICSI were included. Participants underwent the GnRH-agonist and -antagonist protocols; and blood samples were collected at three time points: basic (at start of COH), on the day of hCG and at oocyte retrieval (OR); and from the FF from the first follicle aspirate. Clinical and IVF cycle characteristics, were compared between groups, together with the levels of E-selectin, resistin and ROS in serum and FF, through ELISA. Their prognostic value on pregnancy outcomes was examined., Result(s): Examining molecules levels are increasing in serum, from start of COH until OR, irrespectively of the protocol used; FF levels at OR were similar to those in serum at that day. Resistin FF levels were lower in GnRH agonists, compared with the antagonist protocol. Resistin levels at start of COH were associated with clinical pregnancy rates, and this remained significant following adjustment for age, BMI and IVF protocol used, while values of >13.5 ng/ml were associated with a six times greater odd of a pregnancy., Conclusion: E-selectin, resistin and ROS levels are increasing during COH, reaching their highest values at OR, with comparable values measured in the FF at that time. Resistin values >13.5 ng/ml are linked with a 6-fold increase on the odds of a pregnancy.

Rhodococcus equi infection is increasing in regions with high HIV prevalence worldwide. The microbiological features and clinical mimicry of tuberculosis infection pose diagnostic challenges in high-tuberculosis-incidence settings. We present two HIV-associated cases of R. equi infection from Vietnam and discuss the unique diagnostic challenges in such settings., (Copyright © 2015, Le et al.)

The transformation of dehydroepiandrosterone by Spicaria fumoso-rosea VKM F-881 produced 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, 3beta,7alpha-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one, and 3beta,7beta-dihydroxy- 17a-oxa-D-homo-androst-5-en-17-one. The yield of the main product-3beta,7beta-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one-was 49.5-72 mol % at substrate loadings of 5-20 g/L. Lactone formation proceeded through 7alpha- and 7beta-hydroxy derivatives of dehydroepiandrosterone. The structure of the products was determined by mass spectrometry, 1H-NMR spectroscopy, and 13C-NMR spectroscopy. The proposed microbiological method for producing steroid lactones opens prospects for the syn- thesis of novel steroid compounds.

Dehydroepiandrosterone (DHEA) and its sulfate bound form (DHEAS) are important steroids of mainly adrenal origin. They are produced also in gonads and in the brain. Dehydroepiandrosterone easily crosses the brain-blood barrier and in part is also produced locally in the brain tissue. In the brain, DHEA exerts its effects after conversion to either testosterone and dihydrotestosterone or estradiol via androgen and estrogen receptors present in the most parts of the human brain, through mainly non-genomic mechanisms, or eventually indirectly via the effects of its metabolites formed locally in the brain. As a neuroactive hormone, DHEA in co-operation with other hormones and transmitters significantly affects some aspects of human mood, and modifies some features of human emotions and behavior. It has been reported that its administration can increase feelings of well-being and is useful in ameliorating atypical depressive disorders. It has neuroprotective and antiglucocorticoid activity and modifies immune reactions, and some authors have also reported its role in degenerative brain diseases. Here we present a short overview of the possible actions of dehydroepiandrosterone and its sulfate in the brain, calling attention to various mechanisms of their action as neurosteroids and to prospects for the knowledge of their role in brain disorders., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a-i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4 g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4 g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f-h), could have anticancer properties., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are small RNA molecules that play important roles in gene regulation and translational repression. The mechanisms that facilitate miRNA target binding and recognition have been extensively studied in recent years. However, it is still not well known how the miRNA regulation is affected by the location and the flanking sequences of miRNA target sites. In this study, we systematically quantify the contribution of a wide spectrum of target sites on miRNA-mediated gene expression regulation. Our study investigates target sites located in four different gene regions, including 3' untranslated regions, coding sequences, 5' untranslated regions and promoter regions. We have also introduced four additional non-canonical types of seed matches beyond the canonical seed matches, and included them in our study. Computational analysis of quantitative proteomic data has demonstrated that target sites located in different regions impact the miRNA-mediated repression differently but synergistically. In addition, we have shown the synergistic effects among non-canonical seed matches and canonical ones that enhance the miRNA regulatory effects. Further systematic analysis on the site accessibility near the target regions and the secondary structure of the mRNA sequences have demonstrated substantial variations among target sites of different locations and of different types of seed matches, suggesting the mRNA secondary structure could explain some of the difference in the miRNA regulatory effects impacted by these different target sites. Our study implies miRNAs might regulate their targets under different mechanisms when target sites vary in both their locations and the types of seed matches they contain.

16α-Hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16α-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16α-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16α-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na(+)-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC-MS-MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16α-OH-DHEAS with an apparent Km of 23.1 ± 5.1 μM and Vmax of 485.0 ± 39.1 pmol/mg protein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with Km of 319.0 ± 59.5 μM and Vmax of 1465.8 ± 118.8 pmol/mg protein/min for SOAT and Km of 51.4 ± 9.9 μM and Vmax of 1423.3 ± 109.6 pmol/mg protein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16α-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium. In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16α-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: Optic nerve inflammation, demyelination, and axonal loss are all prominent features of optic neuritis. While corticosteroids hasten visual recovery in optic neuritis, no treatment improves final visual outcomes. HE3286 (17α-ethynyl-5-androstene-3β,7β,17β-triol), a synthetic derivative of a natural steroid, β-AET (5-androstene-3β,7β,17β-triol), exerts anti-inflammatory effects in several disease models and has purported neuroprotective effects as well. HE3286's ability to suppress optic neuritis was examined in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis., Methods: Experimental autoimmune encephalomyelitis was induced in C57/BL6 mice. Mice were treated daily with intraperitoneal vehicle or 40 mg/kg HE3286. Visual function was assessed by optokinetic responses (OKR) at baseline and every 10 days until euthanasia at 40 days post immunization. Retinas and optic nerves were isolated. Inflammation (hematoxylin and eosin and Iba1 staining), demyelination (Luxol fast blue staining), and axonal loss (neurofilament staining) were assessed in optic nerve sections. Retinal ganglion cells (RGCs) were immunolabeled with Brn3a antibodies to quantify RGC survival., Results: Progressive decreases in OKR occurred in vehicle-treated EAE mice, and HE3286 treatment reduced the level of this vision loss. HE3286 also attenuated the degree of inflammation, demyelination, and axonal loss in EAE optic nerves as compared to nerves from vehicle-treated EAE mice. Retinal ganglion cell loss that occurred in both vehicle- and HE3286-treated EAE mice was reduced in the temporal retinal quadrant of HE3286-treated mice., Conclusions: HE3286 suppresses inflammation, reduces demyelination and axonal loss, and promotes RGC survival during experimental optic neuritis. Importantly, HE3286 treatment also preserves some RGC function. Results suggest that HE3286 is a potential novel treatment for optic neuritis., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Background: Steroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different functionalizations - mainly hydroxylations - at the steroid molecule, they vary highly in their mode of action. The pharmaceutical industry is, therefore, interested in hydroxysteroids as therapeutic agents. The insertion of hydroxyl groups into a steroid core, however, is hardly accomplishable by classical chemical means; that is because microbial steroid hydroxylations are investigated and applied since decades. CYP106A2 is a cytochrome P450 monooxygenase from Bacillus megaterium ATCC 13368, which was first described in the late 1970s and which is capable to hydroxylate a variety of 3-oxo-delta4 steroids at position 15beta. CYP106A2 is a soluble protein, easy to express and to purify in high amounts, which makes this enzyme an interesting target for biotechnological purposes., Results: In this work a focused steroid library was screened in vitro for new CYP106A2 substrates using a reconstituted enzyme assay. Five new substrates were identified, including dehydroepiandrosterone and pregnenolone. NMR-spectroscopy revealed that both steroids are mainly hydroxylated at position 7beta. In order to establish a biotechnological system for the preparative scale production of 7beta-hydroxylated dehydroepiandrosterone, whole-cell conversions with growing and resting cells of B. megaterium ATCC1336 the native host of CYP1062 and also with resting cells of a recombinant B. megaterium MS941 strain overexpressing CYP106A2 have been conducted and conversion rates of 400 muM/h (115 mg/l/h) were obtained. Using the B. megaterium MS941 overexpression strain, the selectivity of the reaction was improved from 0.7 to 0.9 for 7beta-OH-DHEA., Conclusions: In this work we describe CYP106A2 for the first time as a regio-selective hydroxylase for 3-hydroxy-delta5 steroids. DHEA was shown to be converted to 7beta-OH-DHEA which is a highly interesting human metabolite, supposed to act as neuroprotective, anti-inflammatory and immune-modulatory agent. Optimization of the whole-cell system using different B. megaterium strains lead to a conversion of DHEA with B. megaterium showing high selectivity and conversion rates and displaying a volumetric yield of 103 mg/l/h 7beta-OH-DHEA.

Elevated levels of glucocorticoids lead to the development of obesity and metabolic syndrome. Local glucocorticoid levels are regulated through the enzyme 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD 1), an enzyme that regenerates active cortisol from inert cortisone. Increased expression of 11beta-HSD 1 in adipose tissue promotes higher body mass index (BMI), insulin resistance, hypertension, and dyslipidemia. Human 11beta-HSD 1 is also responsible for inter-conversion of 7-hydroxylate metabolites of dehydroepiandrosterone (7-OH-DHEA) to their 7-oxo-form. To better understanding the mechanism of the action, we focused on 7-OH- and 7-oxo-DHEA, and their circulating levels during the reductive treatment in adolescent obese patients. We determined plasma levels of 7alpha-OH-DHEA, 7beta-OH-DHEA, and 7-oxo-DHEA in 55 adolescent patients aged 13.04-15.67 years, BMI greater than 90th percentile. Samples were collected before and after one month of reductive therapy. Circulating levels of 7alpha-OH-DHEA decreased during the reductive therapy from 1.727 (1.614; 1.854, transformed mean with 95 % confidence interval) to 1.530 nmol/l (1.435; 1.637, p<0.05) in girls and from 1.704 (1.583; 1.842) to 1.540 nmol/l (1.435; 1.659, p<0.05) in boys. With regard to the level of 7-oxo-DHEA, a significant reduction from 1.132 (1.044; 1.231) to 0.918 nmol/l (0.844; 1.000, p<0.05) was found after the treatment, but only in boys. No significant difference in 7beta-OH-DHEA levels was observed. In conclusions, diminished levels of 7alpha-OH-DHEA indicate its possible effect on activity of 11beta-HSD 1. Further studies are necessary to clarify whether competitive substrates for 11beta-HSD 1 such as 7alpha-OH-DHEA could inhibit production of glucocorticoids and may be involved in metabolic processes leading to reduction of obesity.

Using dehydroepiandrosterone as the starting material, we have synthesized a series of steroid analogs possessing a D-ring fused with heterocycles which are pyridine, imidazo [2,1-b]thiazoles or substituted thiazole imines. All the final structures are first reported and identified by NMR and MS spectroscopys, the yields of these products are moderate to good and the reaction conditions are mild. The cytotoxicity of the synthesized compounds against EC-109(human esophageal carcinoma), EC-9706(human esophageal carcinoma), MGC-803(human gastric carcinoma) were investigated., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The neuroprotective action of dehydroepiandrosterone (DHEA) in the absence of a known specific receptor has been attributed to its metabolism by different cell types in the brain to various steroids, with a preference to its 7-hydroxylated products. The E(t)C cerebellar granule cell line converts DHEA almost exclusively to 7α-hydroxy-DHEA (7α-OH-DHEA). It has been postulated that DHEA's 7-OH and 7-oxo metabolites can decrease glucocorticoid levels by an interactive mechanism involving 11β-hydroxysteroid dehydrogenase (11β-HSD). In order to study the relationship of 7-hydroxylation of DHEA and glucocorticoid metabolism in intact brain cells, we examined whether E(t)C cerebellar neurons, which are avid producers of 7α-OH-DHEA, could also metabolize glucocorticoids. We report that E(t)C neuronal cells exhibit 11β-HSD1 reductase activity, and are able to convert 11-dehydrocorticosterone into corticosterone, whereas they do not demonstrate 11β-HSD2 dehydrogenase activity. Consequently, E(t)C cells incubated with DHEA did not yield 7-oxo- or 7β-OH-DHEA. Our findings are supported by the reductive environment of E(t)C cells through expression of hexose-6-phosphate dehydrogenase (H6PDH), which fosters 11β-HSD1 reductase activity. To further explore the role of 7α-OH-DHEA in E(t)C neuronal cells, we examined the effect of preventing its formation using the CYP450 inhibitor ketoconazole. Treatment of the cells with this drug decreased the yield of 7α-OH-DHEA by about 75% without the formation of alternate DHEA metabolites, and had minimal effects on glucocorticoid conversion. Likewise, elevated levels of corticosterone, the product of 11β-HSD1, had no effect on the metabolic profile of DHEA. This study shows that in a single population of whole-cells, with a highly reductive environment, 7α-OH-DHEA is unable to block the reducing activity of 11β-HSD1, and that 7-hydroxylation of DHEA does not interfere with the activation of glucocorticoids. Our investigation on the metabolism of DHEA in E(t)C neuronal cells suggest that other alternate mechanisms must be at play to explain the in vivo anti-glucocorticoid properties of DHEA and its 7-OH-metabolites., (Copyright © 2013 Elsevier Ltd. All rights reserved.)