Dehydroepiandrosterone Production Research Articles

Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes.Key points• A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD• Both SwiKR and GDH are identified with ketosteroid isomerase activity.• Development of catalytic strategy to control by-product and achieve highly selective DHEA productionGraphical abstract

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system

BackgroundPregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones.ResultsBased on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses.ConclusionThe use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones—progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Effect of Essential Oil Components on the Activity of Steroidogenic Cytochrome P450.

Endocrine-disrupting chemicals (EDCs) may impact the development of prostate cancer (PCa) by altering the steroid metabolism. Although their exact mechanism of action in controlling tumor growth is not known, EDCs may inhibit steroidogenic enzymes such as CYP17A1 or CYP19A1 which are involved in the production of androgens or estrogens. High levels of circulating androgens are linked to PCa in men and Polycystic Ovary Syndrome (PCOS) in women. Essential oils or their metabolites, like lavender oil and tea tree oil, have been reported to act as potential EDCs and contribute towards sex steroid imbalance in cases of prepubertal gynecomastia in boys and premature thelarche in girls due to the exposure to lavender-based fragrances. We screened a range of EO components to determine their effects on CYP17A1 and CYP19A1. Computational docking was performed to predict the binding of essential oils with CYP17A1 and CYP19A1. Functional assays were performed using the radiolabeled substrates or Liquid Chromatography-High-Resolution Mass Spectrometry and cell viability assays were carried out in LNCaP cells. Many of the tested compounds bind close to the active site of CYP17A1, and (+)-Cedrol had the best binding with CYP17A1 and CYP19A1. Eucalyptol, Dihydro-β-Ionone, and (-)-α-pinene showed 20% to 40% inhibition of dehydroepiandrosterone production; and some compounds also effected CYP19A1. Extensive use of these essential oils in various beauty and hygiene products is common, but only limited knowledge about their potential detrimental side effects exists. Our results suggest that prolonged exposure to some of these essential oils may result in steroid imbalances. On the other hand, due to their effect on lowering androgen output and ability to bind at the active site of steroidogenic cytochrome P450s, these compounds may provide design ideas for novel compounds against hyperandrogenic disorders such as PCa and PCOS.

Ultrasonication-assisted synthesis of fluorescent borophene quantum dots for sensing of dehydroepiandrosterone biomarker

A novel fluorescence approach is introduced for sensing of dehydroepiandrosterone (DHEA). The primary characteristic of pediatric adrenocortical carcinoma disease causes due to the excessive production of DHEA, a hormone whose levels in the bloodstream can potentially facilitate the early diagnosis of cancer. In this paper, a water-dispersed borophene quantum dots (BQDs) based sensor was developed for the detection of DHEA, marking the first instance of such an application. The as-synthesized BQDs display blue fluorescence under 365 nm of UV light and exhibit λEx/Em at 370/428 nm with a good quantum yield of 24.12%. The as-prepared BQDs acted as a fluorescent “turn-on” sensor for the detection of DHEA with a dynamic range from 0.1 to 125 μM with the detection limit of 18.72 nM. The BQDs-based paper strip was designed for visual detection of DHEA. In addition, BQDs were utilized as imaging probes to visualize Saccharomyces cerevisiae cells. The developed method was extended for DHEA assay in biofluids (plasma and urine).

The relationship between host defense peptides and adrenal steroids. An account of reciprocal influences

Aimβ-defensins 2 and −3 (HBD-2 and HBD-3) and cathelicidin LL-37 are host defense peptides (HDPs) that play a crucial role in the immune response against mycobacteria. Given our former studies in tuberculosis patients wherein their plasma levels of such peptides correlated with steroid hormone concentrations, we now studied the reciprocal influence of cortisol and/or dehydroepiandrosterone (DHEA) on HDPs biosynthesis and LL-37 on adrenal steroidogenesis. Main methodsCultures of macrophages derived from the THP-1 line were treated with cortisol (10-6M) and/or DHEA (10-6M and 10-7M) and stimulated with irradiated M. tuberculosis (Mi) or infected M. tuberculosis strain H37Rv to assess cytokine production, HDPs, reactive oxygen species (ROS) and colony forming units. Cultures of NCI-H295-R adrenal line were treated with LL37 (5, 10, and 15 µg/ml) for 24 h to further measure cortisol and DHEA levels together with steroidogenic enzyme transcripts. Key findingsIn macrophages, M. tuberculosis produced an increase of IL-1β, TNFα, IL-6, IL-10, LL-37, HBD-2, and HBD-3 levels, irrespective of DHEA treatment. Adding cortisol to M. tuberculosis-stimulated cultures (with or without DHEA) decreased the amounts of these mediators, compared to only stimulated cultures. Although M. tuberculosis reduced ROS levels, DHEA increased these values in addition to diminishing intracellular mycobacterial growth (no matter cortisol treatment).In turn, studies on adrenal cells showed that LL-37 reduced the production of cortisol and DHEA besides modifying transcripts for some steroidogenic enzymes. Significancewhile adrenal steroids seem to influence the production of HDPs, the former compounds are also likely to modulate adrenal biogenesis.

DHEAS and nutritional status among Sidama, Ngandu, and Aka children: Effects of cortisol and implications for adrenarche.

Adrenarche, the biological event marked by rising production of dehydroepiandrosterone and its sulfate (DHEAS), may represent a sensitive period in child development, with important implications for adolescence and beyond. Nutritional status, particularly BMI and/or adiposity, has long been hypothesized as a factor in DHEAS production but findings are inconsistent, and few studies have examined this among non-industrialized societies. In addition, cortisol has not been included in these models. We here evaluate effects of height- (HAZ), weight- (WAZ), and BMI- (BMIZ) for-age on DHEAS concentrations among Sidama agropastoralist, Ngandu horticulturalist, and Aka hunter-gatherer children. Heights and weights were collected from 206 children aged 2-18 years old. HAZ, WAZ, and BMIZ were calculated using CDC standards. DHEAS and cortisol assays were used to determine biomarker concentrations in hair. Generalized linear modeling was used to examine effects of nutritional status on DHEAS concentrations, as well as cortisol, controlling for age, sex, and population. Despite the prevalence of low HAZ and WAZ scores, the majority (77%) of children had BMI z-scores >-2.0 SD. Nutritional status has no significant effect on DHEAS concentrations, controlling for age, sex, and population. Cortisol, however, is a significant predictor of DHEAS concentrations. Our findings do not support a relationship between nutritional status and DHEAS. Instead, results suggest an important role for stress and ecology in DHEAS concentrations across childhood. Specifically, effects of environment via cortisol may be influential to patterning of DHEAS. Future work should investigate local ecological stressors and their relationship to adrenarche.

Exploring the Chemical Space of CYP17A1 Inhibitors Using Cheminformatics and Machine Learning

Cytochrome P450 17A1 (CYP17A1) is one of the key enzymes in steroidogenesis that produces dehydroepiandrosterone (DHEA) from cholesterol. Abnormal DHEA production may lead to the progression of severe diseases, such as prostatic and breast cancers. Thus, CYP17A1 is a druggable target for anti-cancer molecule development. In this study, cheminformatic analyses and quantitative structure-activity relationship (QSAR) modeling were applied on a set of 962 CYP17A1 inhibitors (i.e., consisting of 279 steroidal and 683 nonsteroidal inhibitors) compiled from the ChEMBL database. For steroidal inhibitors, a QSAR classification model built using the PubChem fingerprint along with the extra trees algorithm achieved the best performance, reflected by the accuracy values of 0.933, 0.818, and 0.833 for the training, cross-validation, and test sets, respectively. For nonsteroidal inhibitors, a systematic cheminformatic analysis was applied for exploring the chemical space, Murcko scaffolds, and structure-activity relationships (SARs) for visualizing distributions, patterns, and representative scaffolds for drug discoveries. Furthermore, seven total QSAR classification models were established based on the nonsteroidal scaffolds, and two activity cliff (AC) generators were identified. The best performing model out of these seven was model VIII, which is built upon the PubChem fingerprint along with the random forest algorithm. It achieved a robust accuracy across the training set, the cross-validation set, and the test set, i.e., 0.96, 0.92, and 0.913, respectively. It is anticipated that the results presented herein would be instrumental for further CYP17A1 inhibitor drug discovery efforts.

Hair and Claw Dehydroepiandrosterone Concentrations in Newborn Puppies Spontaneously Dead within 30 Days of Age.

The latest intrauterine fetal developmental stage and the neonatal period represent the most challenging phases for mammalian offspring. Toward the term of pregnancy, during parturition, and after birth, the hypothalamic-pituitary-adrenal axis (HPA) is a key system regulating several physiologic processes, through the production of cortisol and dehydroepiandrosterone (DHEA). This study was aimed to assess DHEA concentrations in hair and claws of 126 spontaneously dead newborn puppies, classified as premature puppies (PRE-P), stillborn puppies (STILL-P) and puppies that died from the 1st to the 30th day of life (NEON-P). The possible influence of newborn sex, breed body size, and timing of death on DHEA concentrations in both matrices was evaluated. Claw DHEA concentrations were higher in the PRE-P group when compared to STILL-P and NEON-P puppies (p < 0.05), whilst no significant differences were found in hair for all the studied factors. The results confirm the hypothesis that higher amounts of DHEA are produced during the intrauterine life in dogs, also in puppies that will die soon after birth.

Effect of maternal body mass index on the steroid profile in women with gestational diabetes mellitus.

To explore the effect of maternal body mass index (BMI) on steroid hormone profiles in women with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT). We enrolled 79 women with NGT and 80 women with GDM who had a gestational age of 24-28 weeks. The participants were grouped according to their BMI. We quantified 11 steroid hormones profiles by liquid chromatography-tandem mass spectrometry and calculated the product-to-precursor ratios in the steroidogenic pathway. Women with GDM and BMI<25kg/m2 showed higher concentrations of dehydroepiandrosterone (DHEA) (p<0.001), testosterone (T) (p=0.020), estrone (E1) (p=0.010) and estradiol (E2) (p=0.040) and lower Matsuda index and HOMA-β than women with NGT and BMI<25kg/m2. In women with GDM, concentrations of E1 (p=0.006) and E2 (p=0.009) declined, accompanied by reduced E2/T (p=0.008) and E1/androstenedione (A4) (p=0.010) in the BMI>25 kg/m2 group, when compared to that in the BMI<25 kg/m2 group. The values of E2/T and E1/A4 were used to evaluate the cytochrome P450 aromatase enzyme activity in the steroidogenic pathway. Both aromatase activities negatively correlated with the maternal BMI and positively correlated with the Matsuda index in women with GDM. NGT women and GDM women with normal weight presented with different steroid hormone profiles. Steroidogenic pathway profiling of sex hormones synthesis showed a significant increase in the production of DHEA, T, E1, and E2 in GDM women with normal weight. Additionally, the alteration of steroid hormone metabolism was related to maternal BMI in women with GDM, and GDM women with overweight showed reduced estrogen production and decreased insulin sensitivity compared with GDM women with normal weight.

OR13-5 Bone Morphogenetic Protein-4 (BMP4) Inhibits Adrenal Cell Cortisol Synthesis by Blocking CYP17A1 and CYP11B1 Expression.

Abstract Background Cytochrome P450c17 (17α-hydroxylase, encoded by CYP17A1) and cytochrome P450c11 (11β-hydroxylase, encoded by CYP11B1) are essential for production of adrenal cortisol. The expression of CYP17A1 and CYP11B1 in adrenocortical cells is stimulated by adrenocorticotropic hormone (ACTH) through the cAMP and protein kinase A signal transduction pathway. Several growth factors can act as autocrine/paracrine regulators for modulating the steroidogenesis in the adrenal cortex. Among these factors, bone morphogenetic protein-4 (BMP4), a member of the superfamily of transforming growth factor-beta (TGFβ) has been previously reported to have an inhibitory effect on adrenal cell CYP17A1 expression and dehydroepiandrosterone (DHEA) production [1]. The role of BMP4 within the adrenal and its mechanisms regulating steroidogenesis are still unclear. In the present study, we examined human adrenal expression of BMP4 and its chronic effects on the H295R adrenal cell line. Objective To define the autocrine/paracrine role of BMP4 in adrenocortical zonation and adrenal steroidogenesis. Methods BMP4 expression in normal human adrenal glands was studied using microarray analysis with laser-captured adrenal capsule and cortical zones. Adrenal BMP4 protein expression was examined following immunohistochemistry (IHC) with a rabbit polyclonal antibody against human BMP4. In vitro studies were performed using H295R and human adrenal cells treated with or without ACTH (10nM), forskolin (10 µM) and BMP4 (50 ng/mL). The experimental medium was collected every 24 h and analyzed for cortisol. Experiments were terminated at 96 h followed by isolation of RNA and qPCR analysis for PPIA (normalization transcript), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), CYP17A1 and CYP11B1. Results Microarray analysis of adrenal zone RNA demonstrated a BMP4 expression gradient with zona glomerulosa (ZG) expressing 3 and 11-fold higher levels than the zona fasciculata (ZF) and zona reticularis (ZR), respectively. IHC confirmed that BMP4 expression was highest in the ZG and the outer ZF. Administration of BMP4 to cultures of H295R cells caused a significant decrease in basal and forskolin-stimulated cortisol production at 48 h. By 96 h, BMP4 inhibited forskolin-stimulated cortisol by 60%. Cortisol inhibition occurred in conjunction with an inhibition in forskolin-stimulated CYP17A1 (↓75%) and CYP11B1 (↓65%). However, CYP11A1 expression was unaffected. In primary cultures of human adrenal cells BMP4 inhibited the ability of ACTH to increase cortisol production (↓75%), as well as CYP11B1(↓65%) and CYP17A1 (↓57%) expression. Conclusion Our findings indicate that the human adrenal has outer cortex localized expression of BMP4 that could play a paracrine/autocrine role in zone-specific production of aldosterone vs. cortisol through BMP4 selective inhibition of CYP17A1 and CYP11B1. Bibliography: 1. Rege J, Nishimoto HK, Nishimoto K, Rodgers RJ, Auchus RJ, Rainey WE. Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis. Endocrinology 2015; 156: 2530–2540. Presentation: Sunday, June 12, 2022 12:00 p.m. - 12:15 p.m.

Impacts of adrenarcheal DHEA levels on spontaneous cortical activity during development

Dehydroepiandrosterone (DHEA) production is closely associated with the first pubertal hormonal event, adrenarche. Few studies have documented the relationships between DHEA and functional brain development, with even fewer examining the associations between DHEA and spontaneous cortical activity during the resting-state. Thus, whether DHEA levels are associated with the known developmental shifts in the brain’s idling cortical rhythms remains poorly understood. Herein, we examined spontaneous cortical activity in 71 typically-developing youth (9–16 years; 32 male) using magnetoencephalography (MEG). MEG data were source imaged and the power within five canonical frequency bands (delta, theta, alpha, beta, gamma) was computed to identify spatially- and spectrally-specific effects of salivary DHEA and DHEA-by-sex interactions using vertex-wise ANCOVAs. Our results indicated robust increases in power with increasing DHEA within parieto-occipital cortices in all frequency bands except alpha, which decreased with increasing DHEA. In the delta band, DHEA and sex interacted within frontal and temporal cortices such that with increasing DHEA, males exhibited increasing power while females showed decreasing power. These data suggest that spontaneous cortical activity changes with endogenous DHEA levels during the transition from childhood to adolescence, particularly in sensory and attentional processing regions. Sexually-divergent trajectories were only observed in later-developing frontal cortical areas.

Commentary on adrenarche and middle childhood

Adrenarche, the post-natal onset of adrenal production of DHEA (dehydroepiandrosterone) and its sulfate (DHEA-S) marks the 5-8 transition and the onset of middle childhood. However, the role of DHEA and/or DHEA-S in the cognitive, emotional and physical changes of the so-called 5-8 transition are not well-understood

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

Prevention of non‐alcoholic steatohepatitis by long‐term exercise via the induction of phenotypic changes in Kupffer cells of hyperphagic obese mice

Exercise ameliorates nonalcoholic fatty liver disease (NAFLD) by inducing phenotypic changes in Kupffer cells (KCs). p62/Sqstm1‐knockout (p62‐KO) mice develop NAFLD alongside hyperphagia‐induced obesity. We evaluated (1) the effects of long‐term exercise on the foreign‐body phagocytic capacity of KCs, their surface marker expression, and the production of steroid hormones in p62‐KO mice; and (2) whether long‐term exercise prevented the development of non‐alcoholic steatohepatitis (NASH) in p62‐KO mice fed a high‐fat diet (HFD). In experiment 1, 30‐week‐old male p62‐KO mice were allocated to resting (p62‐KO‐Rest) or exercise (p62‐KO‐Ex) groups, and the latter performed long‐term exercise over 4 weeks. Then, the phenotype of their KCs was compared to that of p62‐KO‐Rest and wild‐type (WT) mice. In experiment 2, 5‐week‐old male p62‐KO mice that were fed a HFD performed long‐term exercise over 12 weeks. In experiment 1, the phagocytic capacity of KCs and the proportion of CD68‐positive cells were lower in the p62‐KO‐Rest group than in the WT group, but they increased with long‐term exercise. The percentage of CD11b‐positive KCs was higher in the p62‐KO‐Rest group than in the WT group, but lower in the p62‐KO‐Ex group. The circulating dehydroepiandrosterone (DHEA) concentration was higher in p62‐KO‐Ex mice than in p62‐KO‐Rest mice. In experiment 2, the body mass and composition of the p62‐KO‐Rest and p62‐KO‐Ex groups were similar, but the hepatomegaly, hepatic inflammation, and fibrosis were less marked in p62‐KO‐Ex mice. The DHEA concentration was higher in p62‐KO‐Ex mice than in WT or p62‐KO‐Rest mice. Thus, long‐term exercise restores the impaired phagocytic capacity of KCs in NAFLD obese mice, potentially through greater DHEA production, and prevents the development of NASH by ameliorating hepatic inflammation and fibrogenesis. These results suggest a molecular mechanism for the beneficial effect of exercise in the management of patients with NAFLD.

Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB−. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3β-hydroxysteroid dehydrogenase (3β-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3β-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− had an opposite effect on 3β-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11β1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.

Dehydroepiandrosterone at birth: Response to stress and relation to demographic, pregnancy and delivery factors.

Enhanced production of dehydroepiandrosterone (DHEA) by the foetal hypothalamic-pituitary-adrenal (HPA) axis enables maturational events critical for labour induction and neonatal adaptation. Despite knowledge of the interconnected nature of maternal and foetal physiology and dramatic changes in DHEA production after birth, few studies have examined DHEA levels in newborns and none have examined DHEA's response to acute stress. Understanding normative patterns of early DHEA activity is needed to accurately assess functioning of the biological stress system with relevance for health and development. The present study analysed DHEA concentrations and change after stress among 93 newborns and associations with pregnancy, delivery and demographic risk factors. Three saliva samples, collected prior to and following a blood draw stressor, were used to determine baseline and stress reactive DHEA levels. Mothers self-reported on health behaviours during pregnancy. Data on obstetric factors were obtained from medical records. DHEA levels declined from pre- to post-stressor assessments. Results also showed that post-stressor DHEA change was significantly associated with administration of medications used to treat pain and accelerate labour. However, there was no significant variation in DHEA pre-stress levels or change after stress as a function of time after birth. By capturing DHEA levels after birth, the present study provides a window into prenatal health of the HPA system. The study also advances knowledge of DHEA in newborns by providing data on reference levels and important covariates. This information on basic adrenal physiology provides a foundation that can be expanded on to enhance understanding of early hypothalamic-pituitary-adrenal axis activity.

The timing of adrenarche in Maya girls, Merida, Mexico.

BackgroundAdrenarche involves maturation of the hypothalamic‐pituitary‐adrenal axis and increased production of dehydroepiandrosterone and its sulfate ester, dehydroepiandrosterone‐sulfate (DHEA‐S). It occurs at ages 6 to 8 in industrialized populations, marking the transition from childhood to juvenility and cognitive development at middle childhood. Studies in subsistence level populations indicate a later age (8‐9) for adrenarche, but only two such studies currently exist for comparison.AimsTo investigate adrenarcheal age among Maya girls and its association with body composition and dietary variables. We hypothesized adrenarche would occur earlier given the current dual burden of nutrition in Mexico.Materials and Methods25 Maya girls aged 7 to 9 from Merida, Mexico using ELISAs to measure salivary DHEA‐S, standard anthropometry for height, weight, and skinfolds, bioelectrical impedance for body composition variables, as well as a food frequency questionnaire for dietary information.ResultsOur hypothesis was rejected—adrenarche occurred close to 9 years. While no measures of body composition were significantly associated with adrenarcheal status, girls eating meat and dairy products more frequently had significantly higher DHEA‐S levels.DiscussionLike other populations living in ecologically challenging environments, adrenarche occurred relatively late among Maya girls. Adrenarche has been linked to measures of body composition, particularly, the adiposity or body mass index rebound, but no relevant anthropometric measures were associated, possibly because of the small sample.ConclusionFurther studies are required to illuminate how adrenarcheal variation relates to developmental plasticity, body composition, pubertal progression, and animal product consumption in other transitional populations.

Environmental enrichment modulates inflammation during development in long-evans rats (Rattus norvegicus).

It is unknown whether increased production of pro-inflammatory mediators during the life span is caused by aging per se or via the combination of a cumulative allostatic load due to life challenges. This study aimed to investigate the long-term effects of stress and environmental enrichment on the inflammatory status associated with aging in rats. Animals were assigned to the following five treatment groups: chronic stress with and without environmental enrichment; acute stress with and without environmental enrichment; control animals. Enrichment was provided as an open field containing novel objects (30min three times per week). Animals assigned to chronic stress groups were exposed to predator sound stressors (e.g., hawk sound) for 30min daily, while animals assigned to acute stress groups were exposed once a week for 30min. The interaction between the hypothalamic-pituitary-adrenal axis and the immune system was investigated by measuring metabolized dehydroepiandrosterone (DHEA) levels and peripheral levels of the cytokines IL-6 and IL-10. Results suggested that inflamm-aging was more pronounced in animals with low DHEA levels, which in turn were affected by exposure to environmental enrichment and acute stress. This study showed that environmental enrichment and acute stress can significantly attenuate disruptions in DHEA production, consequently contributing to improved neuroimmune function of aging animals.

SUN-361 Cultured Murine Osteoblasts Convert DHEA to Testosterone

Androgens have complex effects on the skeleton. Besides gonadal testosterone (T), the adrenal produces the androgen precursor dehydroepiandrosterone (DHEA), using 3β- and 17β-hydroxysteroid dehydrogenases (3βHSD & 17βHSD) to convert to T in target tissues. Microarray analyses of murine calvarial osteoblasts, RT-PCR of long bone osteoblasts, and RNA-Seq of human bone biopsies confirmed that each of the enzyme families is expressed in osteoblasts, suggesting osteoblasts can generate androgens from the adrenal-derived androgen precursor DHEA. Activation of osteoblast androgen receptor (AR) signaling by DHEA was detected using an AR reporter construct, providing evidence that active androgens are generated. To understand how DHEA is converted to T, we treated murine primary osteoblasts with 100 nM and 1 μM DHEA, or vehicle control. Conditioned media were collected 1, 2, and 3 days after DHEA treatment and assayed for intermediate and active androgens by tandem mass spectrometry with two-dimensional chromatography. As DHEA was consumed, the androgen intermediates androstenediol (A5) and androstenedione (A4) were generated and subsequently converted to T. The peak concentrations of T generated by DHEA 100 nM and 1 μM were 22 and 101 pg/ml, respectively. The equilibrium dissociation constant of the AR for T is ~0.2 nM (57.7 pg/ml), indicating sufficient T production to activate AR in androgen-sensitive osteoblasts. Cultured osteoblasts preferentially converted DHEA to A5, via 17βHSD, rather than to A4, signifying that in the conversion of DHEA to T, 3βHSD is the rate-limiting step. Of the 13 17βHSD isoforms, 7 were expressed in these samples. In contrast, only a single gene isoform of Hsd3b—Hsd3b7—was abundantly expressed in mouse osteoblasts and human bone. We investigated the effects of 3βHSD7 in osteoblasts. An Hsd3b7 shRNA knocked-down mRNA and protein expression by >85%, and caused an osteoblast growth defect compared to an shRNA control. 3βHSD7 has known functions in bile acid synthesis, converting 7α-hydroxycholesterol to 7α-hydroxycholestenone (7HC). Treatment of Hsd3b7 knockdown osteoblasts with 7HC rescued the growth defect suggesting that osteoblasts might generate 7HC or a subsequent metabolite as a trophic factor. We now report an unreported function of bone as a source of T, by conversion of the adrenal androgen precursor DHEA, using 3βHSD7 as a common enzyme for androgen and bile acid synthesis, and leading to the activation of osteoblast AR signaling. These data suggest that the skeleton has evolved protective mechanisms against hypogonadal bone loss that exploits the continued production of adrenal DHEA. This concept is especially important in men with prostate cancer bone metastasis undergoing testicular-targeted therapies whereby adrenal DHEA may continue to fuel cancer growth, and in bone maturation during adrenarche before the pubertal rise in gonadal androgens.

Dehydroepiandrosterone and Its CYP7B1 Metabolite 7α-Hydroxydehydroepiandrosterone Regulates 11β-Hydroxysteroid Dehydrogenase 1 Directions in Rat Leydig Cells.

Background: The purpose of this study was to investigate cytochrome P450-7B1 (CYP7B1) in the human and rat testes to regulate 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activity. We hypothesized that dehydroepiandrosterone (DHEA) and its product 7α-hydroxydehydroepiandrosterone (7αOHD) after catalysis of CYP7B1 played a critical role in driving the direction of 11β-HSD1, because 7αOHD is an alternative substrate for 11β-HSD1.Methods: We examined the influence of DHEA and 7αOHD on 11β-HSD1 activities in both intact Leydig cells and microsomes using radioactive substrates and identified the location of CYP7B1 in Leydig cells using immunohistochemical staining, Western blot, and qPCR.Results: We found that DHEA stimulated 11β-HSD1 oxidase activity in intact cells (EC50 = 0.97 ± 0.11 μM) and inhibited its reductase activity (IC50 = 1.04 ± 0.06 μM). In microsomes, DHEA was a competitive inhibitor of the reductase activity. The 11β-HSD1 oxidase activity in intact cells was inhibited by 7αOHD (IC50 = 1.18 ± 0.12 μM), and the reductase activity was enhanced (EC50 = 0.7 ± 0.04 μM). 7αOHD was a competitive inhibitor of 11β-HSD1 oxidase. CYP7B1 was present in rat Leydig cells, as shown by immunohistochemistry, Western blotting, and qPCR analysis.Conclusion: Our results are consistent with a conclusion that DHEA in the circulation driving 11β-HSD1 toward an oxidase in Leydig cells mainly through inhibiting the reductase of the enzyme, while 7αOHD (CYP7B1 catalytic product of DHEA) drives the enzyme toward the opposite direction.