Fetal Zone Steroids and Estrogen Show Sex Specific Effects on Oligodendrocyte Precursor Cells in Response to Oxidative Damage.

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 17ß-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.

Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells.

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.

Arimistane: Degradation product or metabolite of 7-oxo-DHEA?

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 ß-glucuronidase (Escherichia coli) was observed while using ß-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.

The beneficial role of the synthetic microneurotrophin BNN20 in a focal demyelination model.

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.

Prospective evaluation of urinary steroids and prostate carcinoma-induced deviation: preliminary results.

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 7ß-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.

AGER1 downregulation associates with fibrosis in nonalcoholic steatohepatitis and type 2 diabetes.

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.

7-oxo-DHEA enhances impaired M. tuberculosis-specific T cell responses during HIV-TB coinfection.

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.

Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia.

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.

Anti-neuroinflammatory, protective effects of the synthetic microneurotrophin BNN-20 in the advanced dopaminergic neurodegeneration of "weaver" mice.

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.

7-keto-DHEAmetabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field.

7-keto-DHEA (3ß-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.

Developmental changes in the endocrine stress response in orangutans (Pongo pygmaeus).

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.

A tale of two steroids: The importance of the androgens DHEA and DHEAS for early neurodevelopment.

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.

Design and Synthesis of Novel Dehydroepiandrosterone Analogues as Potent Antiproliferative Agents.

The aim of the present study was to determine the cytotoxic effects of a series of novel dehydroepiandrosterone derivatives containing triazole at the C16 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)-1H-1,2,3-triazole-4-yl)methylene)-10,13-dimet-hyl-1,3,4,7,8,9,10,11,12,13,15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14)-one (compound 2n), which showed considerably high antiproliferative activity in the HepG-2 cell line, with an IC50 value of 9.10 µM, and considerably high activity against the MCF-7 cell line, with an IC50 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.

Cascade biotransformation of dehydroepiandrosterone (DHEA) by Beauveria species.

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 3ß,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: 3ß-hydroxy-17a-oxa-D-homo-androst-5-en-7,17-dione and 3ß,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, 7ß and 11α positions and then reactions of oxidation and reduction leading to 3ß,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.

Determination of dehydroepiandrosterone and its biologically active oxygenated metabolites in human plasma evinces a hormonal imbalance during HIV-TB coinfection.

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.

Quantification of dehydroepiandrosterone in human serum on a routine basis: development and validation of a tandem mass spectrometry method based on a surrogate analyte.

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-3ß-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-D5, a stable isotope-labeled analogue of DHEA, to real serum from healthy subjects. DHEA-D5 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.

Changes to fetal steroidogenesis caused by maternal smoking.

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.

BNN-20, a synthetic microneurotrophin, strongly protects dopaminergic neurons in the "weaver" mouse, a genetic model of dopamine-denervation, acting through the TrkB neurotrophin receptor.

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.

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.

Steroidal[17,16-d]pyrimidines derived from dehydroepiandrosterone: A convenient synthesis, antiproliferation activity, structure-activity relationships, and role of heterocyclic moiety.

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 IC50 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.