Potency of Hexaconazole to Disrupt Endocrine Function with Sex Hormone-Binding Globulin.

Hexaconazole is widely used as a fungicide for agricultural purposes. However, the endocrine-disrupting potential of hexaconazole is still under investigation. In addition, an experimental study found that hexaconazole may disrupt the normal synthesis of steroidal hormones. The potency of hexaconazole to bind with sex hormone-binding globulin (SHBG), a plasma carrier protein that binds androgens and oestrogens, is unknown. In this study, we evaluated the efficacy of hexaconazole to bind with SHBG by molecular interaction, a molecular dynamics method. In addition, principal component analysis was performed to understand the dynamical behaviour of hexaconazole with SHBG in comparison with dihydrotestosterone and aminoglutethimide. The binding scores of hexaconazole, dihydrotestosterone, and aminoglutethimide with SHBG were found to be -7.12 kcal/mol, -11.41 kcal/mol, and -6.84 kcal/mol, respectively. With respect to stable molecular interaction, hexaconazole showed similar molecular dynamics patterns of root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and hydrogen bonding. The solvent surface area (SASA) and principal component analysis (PCA) of hexaconazole exhibit similar patterns in comparison with dihydrotestosterone and aminoglutethimide. These results show that hexaconazole has a stable molecular interaction with SHBG, which may acquire the active site of the native ligand, resulting in significant endocrine disruption during agricultural work.

Tamsulosin facilitates depressive-like behaviors in mice: Involvement of endogenous glucocorticoids.

The benign prostatic hyperplasia (BPH) is the main source of lower urinary tract symptoms. The BPH is a common age-dependent disease and tamsulosin is an α1-adrenoceptor blocker widely prescribed for BPH. Beyond the common adverse effects of tamsulosin, increased diagnosis of dementia after prescription was observed. Importantly, a clinical study suggested that tamsulosin may exert antidepressant effects in BPH patients. Considering the expression of α1-adrenoceptors in the brain, this study aimed to investigate the effects of tamsulosin in the forced swimming and open field tests in mice. For this, tamsulosin (0.001-1 mg/kg) was orally administered subacutely (1, 5 and 23 hr) and acutely (60 min) before tests. Mifepristone (10 mg/kg), a glucocorticoid receptor antagonist, and aminoglutethimide (10 mg/kg), a streoidogenesis inhibitor, were intraperitoneally injected before tamsulosin to investigate the role of the hypothalamic-pituitary-adrenal axis in the mediation of tamsulosin-induced effects. Subacute and acute administrations of tamsulosin increased the immobility time in the first exposition to an inescapable stressful situation. In the re-exposition to the swim task, controls displayed a natural increase in the immobility time, and the treatment with tamsulosin further increased this behavioral parameter. Tamsuslosin did not affect spontaneous locomotion neither in naïve nor in stressed mice. Our findings also showed that mifepristone and aminoglutethimide prevented the tamsulosin-induced increase in the immobility time in the first and second swimming sessions, respectively. In conclusion, tamsulosin may contribute to increased susceptibility to depressive-like behaviors, by facilitating the acquisition of a passive stress-copying strategy. These effects seem to be dependent on endogenous glucocorticoids.

Expression and activation of the steroidogenic enzyme CYP11A1 is associated with IL-13 production in T cells from peanut allergic children.

Activation of the steroidogenic enzyme CYP11A1 was shown to be necessary for the development of peanut-induced intestinal anaphylaxis and IL-13 production in allergic mice. We determined if levels of CYP11A1 in peripheral blood T cells from peanut-allergic (PA) children compared to non-allergic controls were increased and if levels correlated to IL-13 production and oral challenge outcomes to peanut. CYP11A1 mRNA and protein levels were significantly increased in activated CD4+ T cells from PA patients. In parallel, IL-13 production was significantly increased; IFNγ levels were not different between groups. There were significant correlations between expression levels of CYP11A1 mRNA and levels of IL13 mRNA and protein, levels of serum IgE anti-Ara h 2 and to outcomes of peanut challenge. The importance of CYP11A1 on cytokine production was tested using a CYP11A1 CRISPR/Cas9 KO plasmid or an inhibitor of enzymatic CYP11A1 activity. Inhibition of CYP11A1 activation in patient cells treated with the inhibitor, aminoglutethimide, or CD4+ T cell line transfected with the CYP11A1 KO plasmid resulted in reduced IL-13 production. These data suggest that the CYP11A1-CD4+Tcell-IL-13 axis in activated CD4+ T cells from PA children is associated with development of PA reactions. CYP11A1 may represent a novel target for therapeutic intervention in PA children.

Pharmacological suppression of endogenous glucocorticoid synthesis attenuated blood pressure and heart rate response to acute restraint in Wistar rats.

Glucocorticoids (GCS) are known to modulate cardiovascular response during stress conditions. The present study was aimed to test the hypothesis that permissive and/or stimulating effect of GCs is essential for the maintenance of peripheral vascular resistance and for the adequate response of cardiovascular system to stressor exposure. The effects of acute pharmacological adrenalectomy (PhADX) on humoral and cardiovascular parameters were studied in adult Wistar rats under the basal conditions and during the acute restraint stress. Acute PhADX was performed by the administration of metyrapone and aminoglutethimide (100 mg/kg s.c. of each drug) resulting in a suppression of endogenous glucocorticoid synthesis. Blood pressure (BP), heart rate (HR) and core body temperature were measured using radiotelemetry. BP responses to administration of vasoactive agents were determined in pentobarbital-anesthetized animals. PhADX considerably attenuated stress-induced increase of BP, HR and core body temperature. PhADX did not abolish BP and HR lowering effects of ganglionic blocker pentolinium indicating preserved sympathetic function in PhADX rats. BP response to exogenous norepinephrine administration was attenuated in PhADX rats, suggesting reduced sensitivity of cardiovascular system. Suppression of corticosterone synthesis by PhADX increased basal plasma levels of ACTH, aldosterone and plasma renin activity in unstressed animals but there was no further increase of these hormones following stressor exposure. In conclusion, PhADX attenuated stress-induced rise of blood pressure, heart rate and core body temperature indicating an important permissive and/or stimulating role of glucocorticoids in the maintenance of the adequate response of cardiovascular system and thermoregulation to several stimuli including acute exposure to stressor.

Microglial Pro-Inflammatory and Anti-Inflammatory Phenotypes Are Modulated by Translocator Protein Activation.

A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated pathways underlying the modulation of reactive microglia. In the present work, the TSPO activation was explored in an in vitro human primary microglia model (immortalized C20 cells) under inflammatory stimulus. Two different approaches were used with the aim to (i) pharmacologically amplify or (ii) silence, by the lentiviral short hairpin RNA, the TSPO physiological function. In the TSPO pharmacological stimulation model, the synthetic steroidogenic selective ligand XBD-173 attenuated the activation of microglia. Indeed, it reduces and increases the release of pro-inflammatory and anti-inflammatory cytokines, respectively. Such ligand-induced effects were abolished when C20 cells were treated with the steroidogenesis inhibitor aminoglutethimide. This suggests a role for neurosteroids in modulating the interleukin production. The highly steroidogenic ligand XBD-173 attenuated the neuroinflammatory response more effectively than the poorly steroidogenic ones, which suggests that the observed modulation on the cytokine release may be influenced by the levels of produced neurosteroids. In the TSPO silencing model, the reduction of TSPO caused a more inflamed phenotype with respect to scrambled cells. Similarly, during the inflammatory response, the TSPO silencing increased and reduced the release of pro-inflammatory and anti-inflammatory cytokines, respectively. In conclusion, the obtained results are in favor of a homeostatic role for TSPO in the context of dynamic balance between anti-inflammatory and pro-inflammatory mediators in the human microglia-mediated inflammatory response. Interestingly, our preliminary results propose that the TSPO expression could be stimulated by NF-κB during activation of the inflammatory response.

Increased 27-hydroxycholesterol production during luteolysis may mediate the progressive decline in progesterone secretion.

STUDY QUESTION: Does 27-hydroxycholesterol (27OH) actively facilitate the progression of luteolysis? SUMMARY ANSWER: There is increased mRNA expression of the enzyme that produces 27OH during luteolysis in vivo in rhesus macaques and sheep, and 27OH reduces progesterone secretion from human luteinized granulosa cells. WHAT IS KNOWN ALREADY: There is an increase in mRNA expression of liver x receptor (LXR) and a decrease in sterol regulatory element binding protein 2 (SREBP2) target genes during spontaneous luteolysis in primates, which could result in reduced cholesterol availability for steroidogenesis. Concentrations of 27OH are also increased in primate corpora lutea (CL) during luteolysis, and 27OH is a dual LXR agonist and SREBP2 inhibitor. STUDY DESIGN SIZE, DURATION: This was an in vitro study using primary human luteinized granulosa cells in a control versus treatment(s) design. Analyses of CL from sheep undergoing induced or spontaneous luteolysis were also performed, along with database mining of microarray data from rhesus macaque CL. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary luteinizing granulosa cells were obtained from 37 women aged 24-44 who were undergoing oocyte donation or IVF for male factor or idiopathic infertility, and cells were further luteinized in vitro using human chorionic gonadotropin. Three approaches to test the effect of 27OH produced via CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1) on luteinized granulosa cells were used: (i) direct 27OH supplementation, (ii) induction of endogenous CYP27A1 activity via pharmacologic inhibition of steroidogenesis, and (iii) siRNA-mediated knockdown to directly inhibit CYP27A1 as well as cholesterol transport into the mitochondria via the steroidogenic acute regulatory protein (STAR). Endpoints included: progesterone (P4) secretion into culture media determined by enzyme immunoassay, cholesterol efflux and uptake assays using fluorescent lipid analogs, and mRNA expression determined via semi-quantitative real-time PCR (QPCR). An additional experiment involved QPCR analysis of 40 CL collected from ewes undergoing induced or spontaneous luteolysis, as well as database mining of microarray data generated from 16 rhesus macaque CL collected during spontaneous luteolysis and 13 macaque CL collected during a luteinizing hormone ablation and replacement protocol. MAIN RESULTS AND THE ROLE OF CHANCE: The mRNA expression of CYP27A1 was significantly increased during luteolysis in rhesus macaques and sheep in vivo, and CYP27A1 transcription was suppressed by luteinizing hormone and hCG. There was a significant decrease in hCG-stimulated P4 secretion from human luteinized granulosa cells caused by 27OH treatment, and a significant increase in basal and hCG-stimulated P4 synthesis when endogenous 27OH production was inhibited via CYP27A1 knockdown, indicating that 27OH inhibits steroidogenesis. Pharmacologic inhibition of steroidogenesis by aminoglutethimide significantly induced LXR and inhibited SREBP2 target gene mRNA expression, indicating that increased oxysterol production occurs when steroidogenesis is suppressed. Inhibiting cholesterol delivery into the mitochondria via knockdown of STAR resulted in reduced SREBP2 target gene mRNA expression, indicating that STAR function is necessary to maintain SREBP2-mediated transcription. The effects of 27OH treatment on markers of LXR and SREBP2 activity were moderate, and knockdown of CYP27A1 did not prevent aminoglutethimide-induced changes in LXR and SREBP2 target gene mRNA expression. These observations indicate that 27OH inhibits P4 secretion partially via mechanisms separate from its role as an LXR agonist and SREBP2 inhibitor, and also demonstrate that other oxysterols are involved in modulating LXR and SREBP2-mediated transcription when steroidogenesis is suppressed. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: Luteinized granulosa cells may differ from luteal cells, and the effect on luteal function in vivo was not directly tested. The mechanisms that cause the initial rise in CYP27A1 mRNA expression during luteolysis are also not clear. WIDER IMPLICATIONS OF THE FINDINGS: The factors causing luteolysis in primates have not yet been determined. This study provides functional evidence of a novel mechanism via increased 27OH synthesis during luteolysis, which subsequently represses progesterone secretion. Increased 27OH may also facilitate the progression of luteolysis in domestic animal species. STUDY FUNDING AND COMPETING INTEREST(S): The authors have nothing to disclose. Support was provided by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH), award number R00HD067678 to R.L.B.

Impact of nonsteroidal aromatase inhibitors on steroid profile in a Chinese population.

Steroid profiling was introduced to determine the endogenous steroid misuse in sports. Thus, screening for the exogenous use of these prohibited substances can be established by monitoring a range of endogenous steroids, which constitute the steroid profile and evaluate their concentrations and ratios against reference values. The steroid profiling is currently based on population statistics. As large interindividual variations exist, athlete biological passport (ABP) analysis is ongoing. This study aimed to identify new biomarker(s) for aromatase inhibitor detection in sports using statistical analysis and adapt the model into ABP analysis.Forty-one Chinese nonathlete volunteers (21 males and 20 females) were administered 3 nonsteroidal aromatase inhibitors (aminoglutethimide, letrozole, and anastrozole) independently. Statistical analysis was performed on 16 steroid profile parameters.After administration, the concentrations of endogenous androgen biomarkers including testosterone (T), epitestosterone, androsterone (AN), etiocholanolone (ETIO), 5α-diol, 5ß-diol, and dehydroepiandrosterone were increased, while the level of estrogen was decreased. These biomarkers returned to the baselines levels within 1 month. In females, the concentrations of endogenous biomarkers were affected by nonsteroidal aromatase inhibitors, without a common trend. Three new endogenous biomarkers (AN/estrone, ETIO/estrone, and T/estrone) elevated significantly after treatment. The 3 new models were more sensitive than the World Anti-Doping Agency ratio biomarkers. They were also effective in exponentially weighted moving average chart analysis.Verification experiment demonstrated that the biomarker T/estrone was valid in judging the steroidal aromatase inhibitor abuse. The screening of these new endogenous biomarkers can provide additional parameters to support ABP monitoring and specific information regarding the administered steroids.

Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals.

In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

Evaluation for roles of neurosteroids in modulating forebrain mechanisms controlling vasopressin secretion and related phenomena in conscious rats.

Anteroventral third ventricular region (AV3V) that regulates autonomic functions through a GABAergic mechanism possesses neuroactive steroid (NS)-synthesizing ability. Although NS can exert effects by acting on a certain type of GABAA-receptor (R), it is not clear whether NS may operate to modulate AV3V GABAergic activity for controlling autonomic functions. This study aimed to investigate the issue. AV3V infusion with a GABAA antagonist bicuculline increased plasma vasopressin (AVP), glucose, blood pressure (BP), and heart rate in rats. These events were abolished by preinjecting its agonist muscimol, whereas the infusion with allopregnanolone, a NS capable of potentiating GABAA-R function, affected none of the variables in the absence or presence of such bicuculline actions. Similarly, AV3V infusion with pregnanolone sulfate, a NS capable of antagonizing GABAA-R, produced no effect on those variables. AV3V infusion with muscimol was effective in inhibiting the responses of plasma AVP or glucose, or BP to an osmotic loading or bleeding. However, AV3V infusion with aminoglutethimide, a NS synthesis inhibitor, did not affect any of the variables in the absence or presence of those stimuli. These results suggest that NS may not cause acute effects on the AV3V GABAergic mechanism involved in regulating AVP release and other autonomic function.

Synthesis and aromatase inhibitory activity of some new 16E-arylidenosteroids.

A new series of 16E-arylidene androstene derivatives has been synthesized and evaluated for aromatase inhibitory activity. The impact of various aryl substituents at 16 position of the steroid skeleton on aromatase inhibitory activity has been observed. The 16E-arylidenosteroids 6, 10 and 11 exhibited significant inhibition of the aromatase enzyme. 16-(4-Pyridylmethylene)-4-androstene-3,17-dione (6, IC(50): 5.2 µM) and 16-(benzo-[1,3]dioxol-5-ylmethylene)androsta-1,4-diene-3,17-dione (11, IC(50): 6.4 µM) were found to be approximately five times more potent in comparison to aminoglutethimide.

Continuous de novo synthesis of neurosteroids is required for normal synaptic transmission and plasticity in the dentate gyrus of the rat hippocampus.

Both in vivo and in vitro studies have shown that neurosteroids promote learning and memory by modulating synaptic functions in the hippocampus. However, we do not know to what degree endogenously synthesized neurosteroids contribute to the hippocampal synaptic functions. Cytochrome P450scc is the enzyme that converts cholesterol to pregnenolone (PREG), which is required for the biosynthesis of all other neurosteroids. To investigate the physiological roles of endogenous neurosteroids in synaptic functions, we electrophysiologically examined the effects of aminoglutethimide (AG), a selective inhibitor of P450scc, on the synaptic transmission and plasticity in the dentate gyrus of rat hippocampal slices. The application of AG (100 µM) decreased the slope of the field excitatory postsynaptic potentials (fEPSPs) in granule cells by 20-30% in 20 min through the modulation of postsynaptic AMPA receptors, while it did not affect the presynaptic properties, including the paired-pulse ratio and the probability of glutamate release from presynaptic terminals. The AG-induced depression was nearly completely rescued by exogenously applied 500 nM PREG or by 1 nM dehydroepiandrosterone sulfate (DHEAS), one of the neurosteroids synthesized from PREG, suggesting that the AG-induced depression was caused by the loss of DHEAS. AG also reduced NMDA receptor activity, and suppressed high-frequency stimulation (HFS)-induced long-term potentiation (LTP). These findings provide novel evidence that the endogenous neurosteroids locally synthesized in the brain are required to maintain the normal excitatory synaptic transmission and plasticity in the dentate gyrus of the rat hippocampus.

Pharmacological evidence that Ca²+ channels and, to a lesser extent, K+ channels mediate the relaxation of testosterone in the canine basilar artery.

Testosterone induces vasorelaxation through non-genomic mechanisms in several isolated blood vessels, but no study has reported its effects on the canine basilar artery, an important artery implicated in cerebral vasospasm. Hence, this study has investigated the mechanisms involved in testosterone-induced relaxation of the canine basilar artery. For this purpose, the vasorelaxant effects of testosterone were evaluated in KCl- and/or PGF(2α)-precontracted arterial rings in vitro in the absence or presence of several antagonists/inhibitors/blockers; the effect of testosterone on the contractile responses to CaCl2 was also determined. Testosterone (10-180 µM) produced concentration-dependent relaxations of KCl- or PGF(2α)-precontracted arterial rings which were: (i) unaffected by flutamide (10 µM), DL-aminoglutethimide (10 µM), actinomycin D (10 µM), cycloheximide (10 µM), SQ 22,536 (100 µM) or ODQ (30 µM); and (ii) significantly attenuated by the blockers 4-aminopyridine (K(V); 1 mM), BaCl2 (K(IR); 30 µM), iberiotoxin (BK(Ca²+); 20 nM), but not by glybenclamide (K(ATP); 10 µM). In addition, testosterone (31, 56 and 180 µM) and nifedipine (0.01-1 µM) produced a concentration-dependent blockade of the contraction to CaCl2 (10 µM to 10 mM) in arterial rings depolarized by 60mM KCl. These results, taken together, show that testosterone relaxes the canine basilar artery mainly by blockade of voltage-dependent Ca²+ channels and, to a lesser extent, by activation of K+ channels (K(IR), K(V) and BK(Ca²+)). This effect does not involve genomic mechanisms, production of cAMP/cGMP or the conversion of testosterone to 17ß-estradiol.

Age-dependent role of steroids in the regulation of growth of the hen follicular wall.

BACKGROUND: The ovaries are the primary targets of senescence effects in mammalian and avian species. In the present study, relationships between reproductive aging, sex steroids and the growth pattern of the pre-ovulatory follicle wall were investigated using young hens with long clutch (YLC), old hens with long clutch (OLC), old hens with short clutch (OSC), and old hens with interrupted long clutch (OILC). METHODS: Experiment 1: Hens were sacrificed 1.5 and 14.5 h after ovulation. Experiment 2: YLC and OILC hens were sacrificed 3.5 h after treatments with LH and/or aminoglutethimide (AG), an inhibitor of steroid synthesis. Volumes of pre-ovulatory follicles (F1-F5) and plasma concentrations of ovarian steroids were determined. Experiment 3: Granulosa and theca cells from F3 follicles of OSC and/or YLC hens were exposed in vitro to estradiol-17beta (E2), testosterone (T) and LH and the proliferative activity of the cells was examined using CellTiter 96 Aqueous One Solution Assay. RESULTS: In YLC and OLC groups, the total volume of F1-F5 follicles rose between 1.5 and 14.5 h after ovulation (P < 0.01), negatively correlating with the plasma level of E2 (P < 0.01). There was no growth of pre-ovulatory follicles in the middle of the ovulatory cycle in the OSC group, with a positive correlation being present between E2 and the follicular volume (P < 0.05). In young hens, AG caused a rise in the total follicular volume. This rise was associated with a fall in E2 (r = -0.54, P < 0.05). E2 enhanced proliferation of granulosa cells from YLC and OSC groups. The proliferative activity of granulosa and theca cells of YLC hens depended on the interaction between T and LH (P < 0.01). CONCLUSIONS: These data indicate for the first time that the growth pattern of pre-ovulatory follicles during the ovulatory cycle changes in the course of reproductive aging. E2 seems to play a dual role in this adjustment; it stimulates the growth of the follicular wall in reproductive aged hens, whereas it may inhibit this process in young birds. T and LH are apparently involved in the growth regulation during the pre-ovulatory surge in young hens.

Human cytomegalovirus productively infects adrenocortical cells and induces an early cortisol response.

Following our recent findings on the presence of human cytomegalovirus (HCMV) in the normal human adrenal cortex and in adrenocortical tumors, especially in cortisol-secreting tumors, aim of the present study was to investigate the direct effects of HCMV infection on human adrenocortical cells. To this aim, both clinical isolates and laboratory strains of HCMV were used to assess the early effects of infection on human adrenocortical cell morphology, proliferation, gene expression, and steroidogenic function. Both clinical and laboratory HCMV strains could infect and replicate in primary human adrenocortical cell cultures and in adrenocortical carcinoma cell lines, leading to cytopathic changes. Most importantly, in the first hours post-infection (p.i.), adrenocortical cells showed a significant increase of cortisol and estrogen production, paralleled by up-regulation of steroidogenic acute regulatory protein and expression of steroidogenic enzymes involved in the last steps of adrenal steroidogenesis. This effect was probably due to HCMV immediate-early gene expression, since it was most evident in the early phases p.i. and UV-inactivated viral particles did not affect hormone production. Moreover, the effect on steroidogenesis was HCMV specific, since it was not observed after infection with herpes simplex virus. These data suggest that human adrenocortical cells are permissive to HCMV infection and acutely respond to infection with increased cortisol production. An acute glucocorticoid response is typically triggered by infections and is considered to be critical to host defense against pathogens, although, in the case of HCMV infection, it might also enhance viral replication and reactivation from latency.

In vitro kinetic properties of the Thr201Met variant of human aromatase gene CYP19A1: functional responses to substrate and product inhibition and enzyme inhibitors.

CONTEXT: The T(201)M variant (rs28757184) within exon 5 of the human aromatase gene CYP19A1, present in up to 20% of some populations, has been reported to reduce prostate cancer progression. OBJECTIVE: We hypothesized that the T(201)M variant would alter the structure of the enzyme and thus would also affect function compared to wild-type human aromatase. DESIGN: HEK293 cells were transiently transfected with CYP19A1 wild-type or T(201)M variant gene transcripts made by site-directed mutagenesis and enzyme activity measured using tritiated androstenedione as the substrate. The effects of differing concentrations of substrate and product (E1 and E2) and four aromatase inhibitors were assessed. RESULTS: At all substrate concentrations tested, the T(201)M variant showed substantially increased activity compared to the wild-type (Vmax: variant, 738 +/- 36 pmol/h . mg; wild-type, 189 +/- 17 pmol/h . mg, P < 0.0001; Km: variant, 64.4 +/- 19.3 nm; wild-type, 46.6 +/- 9.1 nm, P = 0.04). Kinetic analysis showed evidence of substrate inhibition for the wild-type, but no product inhibition was demonstrated for either transcript. Formestane, chrysin, and letrozole had no differential inhibitory effect on the two transcripts, but aminoglutethimide inhibition was substantially reduced in the variant compared to wild-type (IC(50): wild-type, 1.3 +/- 0.2 nm; variant, 45 +/- 14.2 nm, P = 0.002; and Ki: wild-type, 0.7 +/- 0.2 nm; variant, 29.6 +/- 9.7 nm, P = 0.0001). CONCLUSIONS: In addition to loss of function mutations previously described, a new naturally occurring relatively common alteration of enzyme structure at T(201)M increases enzyme activity and reduces the inhibitory effect of aminoglutethimide. These findings identify the T(201)M site, distant from the substrate-binding site and not previously considered to play a role in enzyme activity, as a functionally important area of the enzyme that may play a role in the propensity to disease. Common to other cytochrome P450 enzymes, wild-type aromatase demonstrates substrate but not product inhibition.

Regulation of inhibin/activin expression in rat early antral follicles.

The aim of the present study was to determine the endocrine activity of cultured early antral follicles (EAF) isolated from prepubertal diethylstilbestrol-treated rats. The effect of steroidogenic substrates and FSH on steroid, inhibin A and B, Pro-alphaC and activin A production was evaluated. Androsterone was the predominant steroid produced by EAF. The addition of androstenedione, androstenedione+FSH and progesterone stimulated oestradiol production, whereas 25-hydroxycholesterol (25-OH-Chol) increased progesterone production. Inhibin A, B, Pro-alphaC, and activin A were produced under basal conditions. The predominance of inhibin B over inhibin A was not affected by the addition of androstenedione or progesterone. Inhibin A and activin A production was stimulated by FSH. 25-OH-Chol increased Inha, Inhba and Inhbb mRNA expression and the production of the three molecular forms of inhibins but decreased activin A production. These results show that FSH and the steroid follicular microenvironment differentially modulate the gene expression of inhibin/activin subunits, their assembly and secretion.

Dehydroepiandrosterone and neurotrophins favor axonal growth in a sensory neuron-keratinocyte coculture model.

We have previously shown that axonal growth from a subset of sensory neurons was promoted by keratinocytes when the two cell types were co-cultured in a low calcium medium. This phenomenon involves the production of one or several diffusible factors. Here we show that the neuritogenic effect of keratinocytes was significantly reduced in the case of rat primary sensory dorsal root ganglion (DRG) neurons, or completely suppressed in the case of the sensory neuron cell line ND7-23, when the activity of neurotrophin receptors (Trk receptors) was blocked with K252a. This trophic effect apparently involved the activation of tyrosine kinase receptors A and B (TrkA and TrkB) expressed by subpopulations of small- to medium-sized DRG neurons, or only of TrkA receptors in the case of ND7-23 neurons. A residual neurite growth promoting effect of keratinocytes persisted in a fraction of DRG neurons after Trk receptor blockade. This effect was mimicked by the steroid dehydroepiandrosterone (DHEA) but not by other steroids such as pregnenolone, progesterone or 17beta-estradiol. The use of pharmacological agents which inhibit different steps of steroidogenesis indicated that DHEA was probably synthesized from cholesterol in keratinocytes. Our results strongly suggest that DHEA might act as a neurotrophic signal derived from keratinocytes to promote axonal outgrowth from a subpopulation of sensory neurons.

Glucocorticoid inhibition in the treatment of depression: can we think outside the endocrine hypothalamus?

BACKGROUND: Major depressive disorder affects a substantial percentage of the U.S. population, and can be highly debilitating. Selective serotonin reuptake inhibitors are commonly prescribed to treat depression, but may not be as effective for more severe or persistent depression. METHODS: The authors review data concerning the effects of corticosteroid synthesis inhibitors (CSIs) in the management of depressive disorders, present a hypothesis as to their possible mechanisms of action based on recent data suggesting synergistic effects of glucocorticoids on extrahypothalamic corticotropin-releasing hormone (CRH), and consider alternative hypotheses. Published reports evaluating the efficacy of CSIs in treating depression are reviewed and presented in light of recent findings regarding actions of glucocorticoids on the central CRH system. RESULTS: Results from open label and double-blind studies by several groups have indicated that CSIs may be efficacious or of adjunctive value in some patients with depression, including those refractory to other agents; however, there is a need for more controlled studies. Several lines of data suggest that the mechanism of action of these agents may not be solely a function of inhibition of adrenal cortisol production. CONCLUSIONS: The authors propose that CSIs may be efficacious in part by reducing glucocorticoid enhancement of CRH action in neurons of the central nucleus of the amygdala and other structures outside the endocrine hypothalamus. Possible effects of systemically administered CSIs on glucocorticoid receptor regulation, neuroactive steroids, and classical monoamine systems are also discussed. We conclude that available clinical data suggest a potential role for CSIs in the management of depressive disorders, especially major depression with psychotic features.

Screening of herbal constituents for aromatase inhibitory activity.

Random Forest screening of the phytochemical constituents of 240 herbs used in traditional Chinese medicine identified a number of compounds as potential inhibitors of the human aromatase enzyme (CYP19). Molecular modelling/docking studies indicated that three of these compounds (myricetin, liquiritigenin and gossypetin) would be likely to form stable complexes with the enzyme. The results of the virtual screening studies were subsequently confirmed experimentally, by in vitro (fluorimetric) assay of the compounds' inhibitory activity. The IC-50s for the flavones, myricetin and gossypetin were determined as 10 and 11 microM, respectively, whilst the flavanone, liquiritigenin, gave an IC-50 of 0.34 microM--showing about a 10-fold increase in potency, therefore, over the first generation aromatase inhibitor, aminoglutethimide.

Synthesis and hypnotic activities of N-Cbz-alpha-aminoglutarimidooxy carboxylate derivatives.

Typical hypnotic drugs, such as barbitals and glutethimide, have a cyclic imide (-CO-NH-CO-) moiety. The N-Cbz-alpha-aminoglutarimidooxy carboxylate derivatives, which we previously showed exhibit moderate anticonvulsant activities, also have a cyclic imide (-CO-N-CO-) moiety. This structural similarity prompted us to examine the hypnotic activities of the N-Cbz-alpha-aminoglutarimidooxy carboxylate derivatives, and we describe their moderate hypnotic activities here.