Glomerella fusarioides-catalyzed structural transformation of steroidal drugs mesterolone and methasterone, and anti-inflammatory activity of resulting derivatives.

Transformation of steroidal drug mesterolone (1) with Glomerella fusarioides yielded two new (17α-hydroxy-1α-methyl-5α-androstan-3-one-11α-yl acetate (2) and 15α-hydroxy-1-methyl-5α-androstan-1-en-3,17-dione (3)), and four known derivatives (15α,17ß-dihydroxy-1α-methyl-5α-androstan-3-one (4), 15α-hydroxy-1α-methyl-5α-androstan-3,17-dione (5), 1α-methyl-androsta-4-en-3,17-dione (6) and 15α,17ß-dihydroxy-1-methyl-5α-androstan-1-en-3-one (7). Similarly, G. fusarioides-catalyzed transformation of steroidal drug methasterone (8) afforded four new metabolites, 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (9), 3a,11α,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (10), 1ß,3ß,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (11), and 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (12). Structures of new derivatives were determined by using 1D-, and 2D-NMR, HREI-MS, and IR spectroscopic data. New derivative 3 was identified as a potent inhibitor of NÈ® production with the IC50 value of 29.9 ± 1.8 µM, in comparison to the standard l-NMMA (IC50 = 128.2 ± 0.8 µM) in vitro. In addition, methasterone (8) (IC50 = 83.6 ± 0.22 µM) also showed a significant activity comparable to new derivative 12 (IC50 = 89.8 ± 1.2 µM). New derivatives 2 (IC50 = 102.7 ± 0.5 µM), 9 (IC50 = 99.6 ± 5.7 µM), 10 (IC50 = 123.5 ± 5.7 µM), and 11 (IC50 = 170.5 ± 5.0 µM) showed a moderate activity. NG-MonomethylL-arginine acetate (IC50 = 128.2 ± 0.8 µM) was used as standared NO⋅- free radicals have an important role in the regulation of immune responses and cellular events. Their overproduction is associated with the pathogenesis of numerous ailments, such as Alzheimer's cardiac disorders, cancer, diabetes, and degenerative diseases. Therefore, inhibition of NÈ® production can help in the treatment of chronic inflammation and associated disorders. All derivatives were found to be non-cytotoxic to human fibroblast (BJ) cell line. The results presented here form the basis of further research for the development of new anti-inflammatory agents with improved efficacy through biotransformation approaches.

Syce1 and Syce3 regulate testosterone and dihydrotestosterone synthesis via steroidogenic pathways in mouse Sertoli and Leydig cells.

Testosterone (T) and dihydrotestosterone (DHT) are the main hormones regulating reproduction and development of male animals. Although their synthesis and secretion are regulated by the endocrine system [hypothalamic-pituitary-gonadal (adrenal) axis], it is also possible to synthesize T and DHT from the induction of two proteins: Syce1 and Syce3. As central elements of the synaptonemal complex (SC), Syce1 and Syce3 play a key role in the association of homologous chromosomes during meiosis. However, Syce1 and Syce3 also promote the synthesis of T and DHT, although potential mechanisms have yet to be revealed. In this study, Leydig and Sertoli cells, which are responsible for the production and regulation of steroid hormones in testis, were transfected with recombinant Syce1/Syce3 and silence sequence. Our results revealed the highest expression of Syce1 and Syce3 in spermatogenic cells of the testis. Moreover, overexpression or knockdown of Syce1 and Syce3 in Sertoli and Leydig cells resulted in activation or suppression of steroidogenic genes Star and Hsd3b, which are involved in a steroidogenic pathway that upregulates T synthesis. Upregulated expression of Syce1 resulted in a significant increase in Srd5a1, which can promote DHT secretion. Interestingly, Syce1 and Syce3 overexpression synergistically promoted each other's abundance. Our results define a previously unknown mechanism of Syce1 and Syce3 dependent activation of steroidogenic signaling in Sertoli and Leydig cells.

Valproate selectively suppresses adolescent anabolic/androgenic steroid-induced aggressive behavior: implications for a role of hypothalamic γ-aminobutyric acid neural signaling.

Pubertal male Syrian hamsters (Mesocricetus auratus) treated with anabolic/androgenic steroids (AASs) during adolescence (P27-P56) display a highly intense aggressive phenotype that shares many behavioral similarities with pathological aggression in youth. Anticonvulsant drugs like valproate that enhance the activity of the γ-aminobutyric acid (GABA) neural system in the brain have recently gained acceptance as a primary treatment for pathological aggression. This study examined whether valproate would selectively suppress adolescent AAS-induced aggressive behavior and whether GABA neural signaling through GABAA subtype receptors in the latero-anterior hypothalamus (LAH; an area of convergence for developmental and neuroplastic changes that underlie aggression in hamsters) modulate the aggression-suppressing effect of this anticonvulsant medication. Valproate (1.0-10.0 mg/kg, intraperitoneal) selectively suppressed the aggressive phenotype in a dose-dependent fashion, with the effective anti-aggressive effects beginning at 5 mg/kg, intraperitoneally. Microinfusion of the GABAA receptor antagonist bicuculline (7.0-700 ng) into the LAH reversed valproate's suppression of AAS-induced aggression in a dose-dependent fashion. At the 70 ng dose of bicuculline, animals expressed the highly aggressive baseline phenotype normally observed in AAS-treated animals. These studies provide preclinical evidence that the anticonvulsant valproate selectively suppresses adolescent, AAS-induced aggression and that this suppression is modulated, in part, by GABA neural signaling within the LAH.

Enzymatic Synthesis of Anabolic Steroid Glycosides by Glucosyltransferase from Terribacillus sp. PAMC 23288.

The application of steroids has steadily increased thanks to their therapeutic effects. However, alternatives are required due their severe side effects; thus, studies on the activities of steroid derivatives are underway. Sugar derivatives of nandrolone, which is used to treat breast cancer, as well as cortisone and prednisone, which reduce inflammation, pain, and edema, are unknown. We linked O-glucose to nandrolone and testosterone using UDP-glucosyltransferase (UGT-1) and, then, tested their bioactivities in vitro. Analysis by NMR showed that the derivatives were 17ß-nandrolone ß-D-glucose and 17ß-testosterone ß-D-glucose, respectively. The viability was higher and cytotoxicity was evident in PC12 cells incubated with rotenone and, testosterone derivatives, compared to the controls. SH-SY5Y cells incubated with H2O2 and nandrolone derivatives remained viable and cytotoxicity was attenuated. Both derivatives enhanced neuronal protective effects and increased the amounts of cellular ATP.

In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse.

Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17ß-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17ß-ol), reduced and dihydroxylated madol (17α-methyl-5α-androstane-2ß,3α,17ß-triol), and the isomeric enone metabolites 17ß-hydroxy-17α-methyl-5α-androst-3-en-2-one and 17ß-hydroxy-17α-methyl-5α-androst-2-en-4-one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell-based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC-MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.

Dietary soybean oil, canola oil and partially-hydrogenated soybean oil affect testicular tissue and steroid hormone levels differently in the miniature pig.

The present study was conducted to examine the influence of dietary canola oil (CAN) and partially-hydrogenated soybean oil (HSO) compared to soybean oil (SOY, control) on the morphology and function of testes using miniature pigs as the test subject. Male miniature pigs were fed a diet containing 10%SOY, 9%CAN+1%SOY, or 9%HSO+1%SOY for 18 months. The scheduled autopsies revealed no abnormalities in histopathological examination of the major organs, except the testes. Atrophy of the seminiferous tubules and hyperplasia in the Leydig cells were found in the SOY and CAN groups. DNA microarray analysis indicated downregulation in the CAN and the HSO groups of genes encoding for gonadotropins in the pituitary gland and of enzymes and proteins involved in steroid hormone metabolism in the testes, compared to the SOY group. Plasma levels of sex hormones in the CAN and HSO groups tended to be higher and testosterone and dihydrotestosteorne in the HSO group were significantly higher than in the SOY group. These results demonstrate that testes are morphologically and functionally affected by the dietary oils, while the plasma steroid hormone levels do not necessarily reflect the gene expression, probably owing to feedback regulation via the gonadal hormones in the hypothalamus-pituitary-gonadal axis.

Trans-fatty acids aggravate anabolic steroid-induced metabolic disturbances and differential gene expression in muscle, pancreas and adipose tissue.

AIMS: Although anabolic steroids (AS) and trans-fatty acids overload exerts systemic toxicity and are independent risk factors for metabolic and cardiovascular disorders, their interaction remains poorly understood. Thus, we investigated the impact of a diet rich in trans-fatty acids (HFD) combined with AS on glycemic control, lipid profile, adipose tissue, skeletal muscle and pancreas microstructure and expression of genes involved in energy metabolism. MAIN METHODS: Forty-eight C57BL/6 mice were randomized into 6 groups treated for 12 weeks with a standard diet (SD) or a diet rich in C18:1 trans-fatty isomers (HFD), alone or combined with 10 or 20 mg/kg testosterone cypionate (AS). KEY FINDINGS: Our results indicated that AS improved glycemic control, upregulated gene expression of Glut-4 and CPT-1 in skeletal muscle, FAS, ACC and UCP-1 in adipose tissue. AS also reduced total and LDL cholesterol in mice fed a SD. When combined with the HFD, AS was unable to induce microstructural adaptations in adipose tissue, pancreatic islets and ß-cells, but potentiated GCK and Glut-2 (pancreas) and Glut-4 and CPT-1 (skeletal muscle) upregulation. HFD plus AS also downregulated FAS and ACC gene expression in adipose tissue. Combined with HFD, AS increased triacylglycerol circulating levels, improved insulin sensitivity and glycemic control in mice. SIGNIFICANCE: Our findings indicated that HFD and AS can interact to modulates glycemic control and lipid profile by a mechanism potentially related with a reprogramming of genes expression in organs such as the pancreas, adipose tissue and skeletal muscle.

Bovine bone particulates containing bone anabolic factors as a potential xenogenic bone graft substitute.

BACKGROUND: Alternative grafts are needed to improve the healing of bone non-union. Here, we assessed a bovine bone product which retains the inorganic and organic components of bone, as an alternative bone graft. METHODS: Bovine bone matrix proteins (BBMPs) were isolated from bovine bone particulates (BBPs) and tested in vitro. Primary rat osteoblast viability, differentiation, and mineralisation were assessed with alamarBlue®, real-time PCR, and von Kossa staining assays, respectively. Osteoclast formation was assessed in primary murine bone marrow cultures with TRAP staining. Human osteoblast growth and differentiation in the presence of BBPs was evaluated in 3D collagen gels in vitro using alamarBlue® and real-time PCR, respectively. The efficacy of BBPs as an alternative bone graft was tested in a rat critical-size calvarial defect model, with histology scored at 4 and 12 weeks post-surgery. RESULTS: In vitro, the highest concentration of BBMPs increased mineral deposition five-fold compared to the untreated control group (P < 0.05); enhanced the expression of key osteoblast genes encoding for RUNX2, alkaline phosphatase, and osteocalcin (P < 0.05); and decreased osteoclast formation three-fold, compared to the untreated control group (P < 0.05). However, the BBPs had no effect on primary human osteoblasts in vitro, and in vivo, no difference was found in healing between the BBP-treated group and the untreated control group. CONCLUSIONS: Overall, despite the positive effects of the BBMPs on the cells of the bone, the bovine bone product as a whole did not enhance bone healing. Finding a way to harness the positive effect of these BBMPs would provide a clear benefit for healing bone non-union.

The cell biology of systemic insulin function.

Insulin is the paramount anabolic hormone, promoting carbon energy deposition in the body. Its synthesis, quality control, delivery, and action are exquisitely regulated by highly orchestrated intracellular mechanisms in different organs or "stations" of its bodily journey. In this Beyond the Cell review, we focus on these five stages of the journey of insulin through the body and the captivating cell biology that underlies the interaction of insulin with each organ. We first analyze insulin's biosynthesis in and export from the ß-cells of the pancreas. Next, we focus on its first pass and partial clearance in the liver with its temporality and periodicity linked to secretion. Continuing the journey, we briefly describe insulin's action on the blood vasculature and its still-debated mechanisms of exit from the capillary beds. Once in the parenchymal interstitium of muscle and adipose tissue, insulin promotes glucose uptake into myofibers and adipocytes, and we elaborate on the intricate signaling and vesicle traffic mechanisms that underlie this fundamental function. Finally, we touch upon the renal degradation of insulin to end its action. Cellular discernment of insulin's availability and action should prove critical to understanding its pivotal physiological functions and how their failure leads to diabetes.

Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays.

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10-8 M), 4-hydroxyandrost-4-ene-3,17-dione (10-8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

Effect of the 5α-reductase enzyme inhibitor dutasteride in the brain of intact and parkinsonian mice.

Dutasteride is a 5alpha-reductase inhibitor in clinical use to treat endocrine conditions. The present study investigated the neuroprotective mechanisms of action of dutasteride in intact and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice using a low dose of MPTP not affecting motor activity modeling early stages of Parkinson's disease (PD). We hypothesized that dutasteride neuroprotection is due to altered steroids levels. Dutasteride pre-treatment prevented loss of striatal dopamine (DA) and its metabolite DOPAC. Dutasteride decreased effects of MPTP on striatal dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2) and D2 DA receptor specific binding while D1 receptor specific binding remained unchanged. Dutasteride enhanced DAT specific binding and the glycosylated form of DAT in intact mice. MPTP-lesioned mice had plasma and brain testosterone and dihydrotestosterone levels lower than control mice whereas progesterone and its metabolites (dihydroprogesterone, isopregnanolone and tetrahydroprogesterone) pathway showed increases. Dutasteride treatment by inhibiting transformation of progesterone and testosterone to its metabolites elevated plasma and brain concentrations of testosterone compared to MPTP mice and decreased DHT levels in intact mice. Plasma and brain estradiol levels were low and remained unchanged by MPTP and/or dutasteride treatment. Dutasteride treatment did not affect striatal phosphorylation of Akt and its downstream substrate GSK3ß as well as phosphorylation of ERK1/2 in intact and MPTP lesioned MPTP mice. Striatal glial fibrillary acidic protein (GFAP) levels were markedly elevated in MPTP compared to control mice and dutasteride reduced GFAP levels in MPTP mice. Treatment with dutasteride post-lesion left unchanged striatal DA levels. These results suggest dutasteride as promising drug for PD neuroprotection.

IGF-1 Attenuates Hypoxia-Induced Atrophy but Inhibits Myoglobin Expression in C2C12 Skeletal Muscle Myotubes.

Chronic hypoxia is associated with muscle wasting and decreased oxidative capacity. By contrast, training under hypoxia may enhance hypertrophy and increase oxidative capacity as well as oxygen transport to the mitochondria, by increasing myoglobin (Mb) expression. The latter may be a feasible strategy to prevent atrophy under hypoxia and enhance an eventual hypertrophic response to anabolic stimulation. Mb expression may be further enhanced by lipid supplementation. We investigated individual and combined effects of hypoxia, insulin-like growth factor (IGF)-1 and lipids, in mouse skeletal muscle C2C12 myotubes. Differentiated C2C12 myotubes were cultured for 24 h under 20%, 5% and 2% oxygen with or without IGF-1 and/or lipid treatment. In culture under 20% oxygen, IGF-1 induced 51% hypertrophy. Hypertrophy was only 32% under 5% and abrogated under 2% oxygen. This was not explained by changes in expression of genes involved in contractile protein synthesis or degradation, suggesting a reduced rate of translation rather than of transcription. Myoglobin mRNA expression increased by 75% under 5% O2 but decreased by 50% upon IGF-1 treatment under 20% O2, compared to control. Inhibition of mammalian target of rapamycin (mTOR) activation using rapamycin restored Mb mRNA expression to control levels. Lipid supplementation had no effect on Mb gene expression. Thus, IGF-1-induced anabolic signaling can be a strategy to improve muscle size under mild hypoxia, but lowers Mb gene expression.

Applications of Isotope Ratio Mass Spectrometry in Sports Drug Testing Accounting for Isotope Fractionation in Analysis of Biological Samples.

The misuse of anabolic-androgenic steroids (AAS) in sports aiming at enhancing athletic performance has been a challenging matter for doping control laboratories for decades. While the presence of a xenobiotic AAS or its metabolite(s) in human urine immediately represents an antidoping rule violation, the detection of the misuse of endogenous steroids such as testosterone necessitates comparably complex procedures. Concentration thresholds and diagnostic analyte ratios computed from urinary steroid concentrations of, e.g., testosterone and epitestosterone have aided identifying suspicious doping control samples in the past. These ratios can however also be affected by confounding factors and are therefore not sufficient to prove illicit steroid administrations. Here, carbon and, in rare cases, hydrogen isotope ratio mass spectrometry (IRMS) has become an indispensable tool. Importantly, the isotopic signatures of pharmaceutical steroid preparations commonly differ slightly but significantly from those found with endogenously produced steroids. By comparing the isotope ratios of endogenous reference compounds like pregnanediol to that of testosterone and its metabolites, the unambiguous identification of the urinary steroids' origin is accomplished. Due to the complex urinary matrix, several steps in sample preparation are inevitable as pure analyte peaks are a prerequisite for valid IRMS determinations. The sample cleanup encompasses steps such as solid phase or liquid-liquid extraction that are presumably not accompanied by isotopic fractionation processes, as well as more critical steps like enzymatic hydrolysis, high-performance liquid chromatography fractionation, and derivatization of analytes. In order to exclude any bias of the analytical results, each step of the analytical procedure is optimized and validated to exclude, or at least result in constant, isotopic fractionation. These efforts are explained in detail.

Rapid Analysis of Anabolic Steroid Metabolites in Urine by Combining Field Asymmetric Waveform Ion Mobility Spectrometry with Liquid Chromatography and Mass Spectrometry.

The combination of field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry (LC-FAIMS-MS) has been developed for the analysis of glucuronide and sulfate metabolites of seven anabolic-androgenic steroids in urine. Separation by FAIMS-MS was investigated in positive ion mode for selected cationic adducts (H+, NH4+, Na+, K+, and Cs+). LC-FAIMS-MS analysis of the doubly sodiated adducts ([M + 2Na - H]+) of isobaric and coeluting steroid metabolites allowed their rapid (8 min) qualitative and quantitative determination in spiked urine using hydrophilic interaction liquid chromatography prior to FAIMS-MS separation, with discrimination >95% achieved between the steroids investigated. A quantitative evaluation of the LC-FAIMS-MS method was performed giving limits of detection in the range 1-6 ng mL-1, limits of quantification in the range 3-20 ng mL-1, with reproducibility (%RSD < 10%; n = 6) and linearity (R2 > 0.99). The LC-FAIMS-MS method demonstrates increases in signal-to-noise ratios for the doubly sodiated steroid metabolites in unspiked urine (>250%) by the reduction of isobaric interferences from the matrix. An alternative or additional tool for identification of the steroid metabolites is based on the observations of different patterns of sodium acetate clusters that are characteristic for each metabolite.

Biotransformation of anabolic compound methasterone with Macrophomina phaseolina, Cunninghamella blakesleeana, and Fusarium lini, and TNF-α inhibitory effect of transformed products.

Microbial transformation of methasterone (1) was investigated with Macrophomina phaseolina, Cunninghamella blakesleeana, and Fusarium lini. Biotransformation of 1 with M. phaseolina yielded metabolite 2, while metabolites 3-7 were obtained from the incubation of 1 with C. blakesleeana. Metabolites 8-13 were obtained through biotransformation with F. lini. All metabolites, except 13, were found to be new. Methasterone (1) and its metabolites 2-6, 9, 10, and 13 were then evaluated for their immunomodulatory effects against TNF-α, NO, and ROS production. Among all tested compounds, metabolite 6 showed a potent inhibition of proinflammatory cytokine TNF-α (IC50=8.1±0.9µg/mL), as compared to pentoxifylline used as a standard (IC50=94.8±2.1µg/mL). All metabolites were also evaluated for the inhibition of NO production at concentration of 25µg/mL. Metabolites 6 (86.7±2.3%) and 13 (62.5±1.5%) were found to be the most potent inhibitors of NO as compared to the standard NG-monomethyl-l-arginine acetate (65.6±1.1%). All metabolites were found to be non-toxic against PC3, HeLa, and 3T3 cell lines. Observed inhibitory potential of metabolites 6 and 13 against pro-inflammatory cytokine TNF-α, as well as NO production makes them interesting leads for further studies.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Relationship between the climbing up and climbing down stairs domain scores on the FES-DMD, the score on the Vignos Scale, age and timed performance of functional activities in boys with Duchenne muscular dystrophy / Relação entre escore FES-DMD-subir e descer escada com escore Escala Vignos, idade e tempo de realização das atividades em meninos com Distrofia Muscular de Duchenne

BACKGROUND: Knowing the potential for and limitations of information generated using different evaluation instruments favors the development of more accurate functional diagnoses and therapeutic decision-making. OBJECTIVE: To investigate the relationship between the number of compensatory movements when climbing up and going down stairs, age, functional classification and time taken to perform a tested activity (TA) of going up and down stairs in boys with Duchenne muscular dystrophy (DMD). METHOD: A bank of movies featuring 30 boys with DMD performing functional activities was evaluated. Compensatory movements were assessed using the climbing up and going down stairs domain of the Functional Evaluation Scale for Duchenne Muscular Dystrophy (FES-DMD); age in years; functional classification using the Vignos Scale (VS), and TA using a timer. Statistical analyses were performed using the Spearman correlation test. RESULTS: There is a moderate relationship between the climbing up stairs domain of the FES-DMD and age (r=0.53, p=0.004) and strong relationships with VS (r=0.72, p=0.001) and TA for this task (r=0.83, p<0.001). There were weak relationships between the going down stairs domain of the FES-DMD-going down stairs with age (r=0.40, p=0.032), VS (r=0.65, p=0.002) and TA for this task (r=0.40, p=0.034). CONCLUSION: These findings indicate that the evaluation of compensatory movements used when climbing up stairs can provide more relevant information about the evolution of the disease, although the activity of going down stairs should be investigated, with the aim of enriching guidance and strengthening accident prevention. Data from the FES-DMD, age, VS and TA can be used in a complementary way to formulate functional diagnoses. Longitudinal studies and with broader age groups may supplement this information. .

CONTEXTUALIZAÇÃO: Conhecer as potencialidades e limitações das informações geradas por diferentes instrumentos de avaliação favorece o desenvolvimento mais preciso do diagnóstico funcional e da tomada de decisão terapêutica. OBJETIVO : Investigar a relação entre o número de movimentos compensatórios ao subir e descer escadas, idade, classificação funcional e tempo de realização de atividade (TA) em meninos com Distrofia Muscular de Duchenne (DMD). MÉTODO : Foi utilizado banco de filmes de 30 meninos com DMD realizando atividades funcionais. Os movimentos compensatórios foram avaliados pela Escala de Avaliação Funcional para Distrofia Muscular de Duchenne (FES-DMD), domínio subir e descer escada; a idade, mensurada em anos; a classificação funcional foi pesquisada pela Escala de Vignos (EV), e o TA foi cronometrado. Foi utilizado o teste de correlação de Spearman. RESULTADOS : Existe moderada relação entre a FES-DMD-subir escada e a idade (r=0,53, p=0,004) e forte relação com a EV (r=0,72, p=0,001) e TA dessa tarefa (r=0,83, p<0,001). Houve fraca relação entre a FES-DMD-descer escada e a idade (r=0,40, p=0,032), EV (r=0,65, p=0,002) e o TA dessa tarefa (r=0,40, p=0,034). CONCLUSÃO : Esses achados indicam que a avaliação da tarefa de subir escada pode trazer informações mais relevantes sobre a evolução da doença, embora a atividade de descer escada deva ser pesquisada visando à orientação e prevenção de acidentes. A utilização conjunta de dados provenientes da FES-DMD, da idade e do TA pode se complementar para formulação do diagnóstico funcional. Estudos longitudinais e com outras faixas etárias mais amplas podem complementar tal informação. .

Youngest reported patient presenting with an androgen producing sclerosing stromal ovarian tumor.

BACKGROUND: Sclerosing stromal tumors are extremely rare sex cord stromal tumors of the ovary, with approximately 100 cases reported since first described in 1973. These tumors present predominantly in the 2nd and 3rd decades of life, typically present with pelvic/abdominal pain and tenderness, mass, and/or abnormal menses, and with hormonal activity reported predominantly in postmenarchal females. Only 5 cases of these tumors have been reported in premenarchal girls, with age ranging from 7 months to 12 years. Only 2 demonstrated hormonal manifestations, with vaginal bleeding due to hyperestrogenism in the 7 month old, and virilization in an 11-year-old female. CASE: We report a 9-year-old female who was diagnosed with this ovarian tumor, and who presented with virilization. SUMMARY AND CONCLUSION: This report is remarkable as our patient not only was diagnosed with an ovarian tumor that is extremely rare in this age group but is the youngest reported patient with this tumor who presented with virilization.

Sertoli cell androgen receptor expression regulates temporal fetal and adult Leydig cell differentiation, function, and population size.

We recently created a mouse model displaying precocious Sertoli cell (SC) and spermatogenic development induced by SC-specific transgenic androgen receptor expression (TgSCAR). Here we reveal that TgSCAR regulates the development, function, and absolute number of Leydig cells (LCs). Total fetal and adult type LC numbers were reduced in postnatal and adult TgSCAR vs control testes, despite normal circulating LH levels. Normal LC to SC ratios found in TgSCAR testes indicate that SC androgen receptor (SCAR)-mediated activity confers a quorum-dependent relationship between total SC and LC numbers. TgSCAR enhanced LC differentiation, shown by elevated ratios of advanced to immature LC types, and reduced LC proliferation in postnatal TgSCAR vs control testes. Postnatal TgSCAR testes displayed up-regulated expression of coupled ligand-receptor transcripts (Amh-Amhr2, Dhh-Ptch1, Pdgfa-Pdgfra) for potential SCAR-stimulated paracrine pathways, which may coordinate LC differentiation. Neonatal TgSCAR testes displayed normal T and dihydrotestosterone levels despite differential changes to steroidogenic gene expression, with down-regulated Star, Cyp11a1, and Cyp17a1 expression contrasting with up-regulated Hsd3b1, Hsd17b3, and Srd5a1 expression. TgSCAR males also displayed elevated postnatal and normal adult serum testosterone levels, despite reduced LC numbers. Enhanced adult-type LC steroidogenic output was revealed by increased pubertal testicular T, dihydrotestosterone, 3α-diol and 3ß-diol levels per LC and up-regulated steroidogenic gene (Nr5a1, Lhr, Cyp11a1, Cyp17a1, Hsd3b6, Srd5a1) expression in pubertal or adult TgSCAR vs control males, suggesting regulatory mechanisms maintain androgen levels independently of absolute LC numbers. Our unique gain-of-function TgSCAR model has revealed that SCAR activity controls temporal LC differentiation, steroidogenic function, and population size.

The follicular hormonal profile in low-responder patients undergoing unstimulated cycles: Is it hypoandrogenic?

STUDY QUESTION: What is the final hormonal milieu of pre-ovulatory follicles of low-responder (LR) patients undergoing unstimulated cycles? SUMMARY ANSWER: Neither androgen secretion nor LH was impaired in pre-ovulatory follicles of LR women. WHAT IS KNOWN ALREADY: Therapies currently used to improve ovarian response in LR women have an impact on the final hormonal follicular milieu, and these changes are believed to be partially responsible for determining the success rate in these women. Surprisingly, as far as we know, there is no report of the final hormonal profile of LR women undergoing unstimulated cycles or evidence that follicular androgen secretion in LR women is impaired. STUDY DESIGN, SIZE AND DURATION: A prospective case-control study including 94 women, 36 normal controls and 58 LR patients (19 Young ≤ 35 years LR and 39 Aged >35 years LR) from 2009 to 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Fifty-eight LR women were divided into two groups: Young LR (age ≤ 35; n = 19) and Aged LR (ALR; age >35; n = 39). The control group (group C) comprised 36 egg donors undergoing an unstimulated cycle in our IVF unit. Serum and follicular fluid hormonal concentrations for estradiol (E2), progesterone, testosterone and androstendione were measured. The spindle parameters of metaphase II oocytes generated from these groups were also analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-ovulatory follicles from LR patients had similar androgenic and LH concentrations to those observed in the control group. However, higher intrafollicular concentrations of FSH and progesterone were observed in ALR. Moreover, no differences were found for the spindle evaluation of oocytes between groups by the Oosight technology. LIMITATIONS, REASONS FOR CAUTION: The controls were younger and had a lower BMI than the LR women. The sample size available restricted statistical power. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that the problem with LR women is not the final pre-ovulatory follicular androgen concentration since this is similar to normal responders, but in the ability to respond to controlled ovarian stimulation protocols. Therefore, efforts should be focused on long-interval androgen priming to potentially increase the recruitment of small antral follicles rather than increasing the intraovarian androgen levels within the current cycle. STUDY FUNDING/COMPETING INTEREST: The present project has been supported by the R+D programme from the Generalitat Valenciana (Regional Valencian Government) IMPIVA MIDTF/2010/95. The authors have no conflict of interest to declare.