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1.
Acta Histochem ; 121(8): 151441, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31522738

RESUMO

PURPOSE: This study aimed to evaluate the effects of estrogen reduction on amyloid deposition, some lipid metabolism and oxidative stress markers, PSA-like production and p63 expression in the prostate of the adult rat. METHODS: Aromatase inhibitor: Formestane (4-OHA), was administrated to male rats, at a dose of 0.1 mg/kg b.w./day, for 10 days. The control group (CONT) received the same volume of placebo injection (NaCl 0.9%). RESULTS: 4-OHA treatment induced a significant accumulation of intraprostatic cholesterol (138.90 ±â€¯17.64 vs 85.12 ±â€¯2.87, p = 0.01); against an insignificant diminution of malondialdehyde (412.6 ±â€¯54.35 vs 842.70 ±â€¯336.50, p > 0.05) and glutathione (2.40 ±â€¯0.23 vs 3.65 ±â€¯0.88, p > 0.05). This was associated with a significant decrease of nitric oxide (31.76 ±â€¯7.07 vs 179.40 ±â€¯58.35, p = 0.024). Additionally, 4-OHA significantly increased the intraprostatic production of PSA-like (11.12 ±â€¯2.78 vs 3.91 ±â€¯0.43, p = 0.043). The prostatic histology revealed an amyloid deposition, in all prostatic lobes and a smooth muscle layer growth (p < 0.05); especially significant in the dorsal and lateral lobes. Theses lobes manifested a basal cells proliferation, with a 3-fold increase of p63 expression (p < 0.001). The ventral lobe presented epithelial atrophy (37.80 ±â€¯16.20 vs 167.60 ±â€¯5.16, p < 0.05); with occasional and significant proliferative foci (247.00 ±â€¯9.573 vs 167.60 ±â€¯5.16 p < 0.05). DISCUSSION AND CONCLUSION: Aromatase inhibition, in the adult male rat, alters the prostatic function by reducing nitric oxide availability and inducing amyloid deposition along with limiting the differentiation of basal cells, through a lobe-specific p63-overexpression.


Assuntos
Amiloide/metabolismo , Androstenodiona/análogos & derivados , Inibidores da Aromatase/efeitos adversos , Aromatase/metabolismo , Próstata/enzimologia , Androstenodiona/efeitos adversos , Androstenodiona/farmacologia , Animais , Inibidores da Aromatase/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Próstata/patologia , Ratos , Ratos Wistar , Fatores de Tempo , Proteínas Supressoras de Tumor/biossíntese
2.
J Steroid Biochem Mol Biol ; 191: 105369, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31039398

RESUMO

11α-Hydroxyprogesterone (11αOHP4) and 11ß-hydroxyprogesterone (11ßOHP4) have been reported to be inhibitors of 11ß-hydroxysteroid dehydrogenase (11ßHSD) type 2, together with 11ß-hydroxytestosterone and 11ß-hydroxyandrostenedione, and their C11-keto derivatives being inhibitors of 11ßHSD1. Our in vitro assays in transiently transfected HEK293 cells, however, show that 11αOHP4 is a potent inhibitor of 11ßHSD2 and while this steroid does not serve as a substrate for the enzyme, the aforementioned C11-oxy steroids are indeed substrates for both 11ßHSD isozymes. 11ßOHP4 is metabolised by 11ßHSD2 yielding 11-ketoprogesterone with 11ßHSD1 catalysing the reverse reaction, similar to the reduction of the other C11-oxy steroids. In the same model system, novel 11αOHP4 metabolites were detected in its conversion by steroid-5α-reductase (SRD5A) types 1 and 2 yielding 11α-hydroxydihydroprogesterone and its conversion by cytochrome P450 17A1 (CYP17A1) yielding the hydroxylase product, 11α,17α-dihydroxyprogesterone, and the 17,20 lyase product, 11α-hydroxyandrostenedione. We also detected both 11αOHP4 and 11ßOHP4 in prostate cancer tissue- ∼23 and ∼32 ng/g respectively with 11KP4 levels >300 ng/g. In vitro assays in PC3 and LNCaP prostate cancer cell models, showed that the metabolism of 11αOHP4 and 11ßOHP4 was comparable. In LNCaP cells expressing CYP17A1, 11αOHP4 and 11ßOHP4 were metabolised with negligible substrate, 4%, remaining after 48 h, while the steroid substrate 11ß,17α-dihydroxyprogesterone (21dF) was metabolised to C11-keto C19 steroids yielding 11-ketotestosterone. Despite the fact that 11αOHP4 is not metabolised by 11ßHSD2, it is a substrate for SRD5A and CYP17A1, yielding C11α-hydroxy C19 steroids as well as the C11α-hydroxy derivative of 21dF-the latter associated with clinical conditions characterised by androgen excess. With our data showing that 11αOHP4 is present at high levels in prostate cancer tissue, the steroid may serve as a precursor to unique C11α-hydroxy C19 steroids. The potential impact of 11αOHP4 and its metabolites on human pathophysiology can however only be fully assessed once C11α-hydroxyl metabolite levels are comprehensively analysed.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Androstenodiona/análogos & derivados , Hidroxiprogesteronas/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Idoso , Androstenodiona/metabolismo , Linhagem Celular Tumoral , Cortodoxona/metabolismo , Células HEK293 , Humanos , Masculino , Neoplasias da Próstata/metabolismo
3.
Environ Toxicol Pharmacol ; 68: 133-140, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30889543

RESUMO

Acute developmental exposure to pharmaceuticals or environmental contaminants can have deleterious, long lasting effects. Many of these compounds are endocrine disruptors (EDCs) that target estrogen signaling, with effects on reproductive and non-reproductive tissues. We recently reported that zebrafish larvae transiently exposed to the pharmaceutical EDC 4-OH-A display visual deficits as adults. Here, we examine whether these long-term effects are due to compound-induced morphological and/or cellular changes. Zebrafish aged 24 h, 48 h, 72 h, or 7 days post-fertilization (larvae) or 3-4mos (adults) were exposed to either 4-OH-A or PCB1254 for 24 h. After that time, notochord length, eye diameter, inter-eye distance, and heart rate were measured from larvae; and aromatase (estrogen synthase) activity was measured in homogenates of adult brain tissue. In general, indices of larval growth and development were not altered by 24 h exposure to either compound. 4-OH-A potently inhibited aromatase activity, while PCB1254 did not, with inhibition continuing even after removal from treatment. These results support differential function of EDCs and indicate that developmental exposure to 4-OH-A causes sustained inhibition of aromatase, which could be associated with altered adult behaviors.


Assuntos
Androstenodiona/análogos & derivados , Inibidores da Aromatase/toxicidade , Disruptores Endócrinos/toxicidade , Peixe-Zebra , Androstenodiona/toxicidade , Animais , Aromatase/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
4.
Steroids ; 146: 34-42, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30904502

RESUMO

Formestane (4-hydroxyandrost-4-ene-3,17-dione, 4OH-AED) is an aromatase inhibitor prohibited in sports. In recent years, it has been demonstrated that it can also originate endogenously by the hydroxylation in C4 position of androstenedione. Thus, the use of isotope ratio mass spectrometry (IRMS) is mandatory according to the World Antidoping Agency (WADA) to discriminate endogenous from synthetic origin. In a previous work and after oral administrations of formestane (4OH-AED), the ratio between the main formestane metabolite (4α-hydroxyepiandrosterone; 4OH-EA) and formestane parent compound could help to identify the endogenous origin, avoiding unnecessary and costly IRMS confirmations. In the present work, we investigated whether the same criteria could also be applied after transdermal applications. Six volunteers were transdermally treated once with formestane. Urine samples were collected for 120 h postadministration and analyzed by gas chromatography coupled to mass spectrometry (GC-MS and GC-MS/MS). Formestane and its major metabolites were monitored. The kinetic profile of formestane and its main metabolites was found different between oral and transdermal application. A shift on the excretion of the metabolites compared to formestane itself that can be observed after the oral administration, is absent after the transdermal one. This makes that a simple criteria cannot be applied to differentiate the endogenous from the synthetic origin based on metabolic ratios. The ratio between 4-hydroxyepiandrosterone and 4-hydroxyandrosterone (4OH-A) can be used to differentiate the route of administration. Ratios higher than one (4OH-EA/4OH-A > 1) are diagnostic of an oral administration. This allows to correctly interpret the 4OH-EA/4OH-AED ratio as proposed in our previous investigation. The results of this work demonstrate that the use of appropriate biomarkers (metabolic ratios) helps to reach correct conclusions without using complex and costly instrumentation approaches.


Assuntos
Androstenodiona/análogos & derivados , Doping nos Esportes/prevenção & controle , Administração Oral , Adulto , Androstenodiona/administração & dosagem , Androstenodiona/metabolismo , Biomarcadores/metabolismo , Biomarcadores/urina , Humanos , Masculino
5.
Neurosci Lett ; 701: 65-70, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30742936

RESUMO

CA1 hippocampal expression of α4ßδ GABAA receptors (GABARs) increases at the onset of puberty in female mice, an effect dependent upon the decline in hippocampal levels of the neurosteroid THP (3α-OH-5α-pregnan-20-one) which occurs at this time. The present study further characterized the mechanisms underlying α4ßδ expression, assessed in vivo. Blockade of pubertal levels of 17ß-estradiol (E2) (formestane, 0.5 mg/kg, i.p. 3 d) reduced α4 and δ expression by 75-80% (P < 0.05) in CA1 hippocampus of female mice, assessed using Western blot techniques. Conversely, E2 administration increased α4 and δ expression by 50-100% in adults, an effect enhanced by more than 2-fold by concomitant administration of the 5α-reductase blocker finasteride (50 mg/kg, i.p., 3d, P < 0.05), suggesting that both declining THP levels and increasing E2 levels before puberty trigger α4ßδ expression. This effect was blocked by ICI 182,780 (20 mg/kg, s.c., 3 d), a selective blocker of E2 receptor-α (ER-α). These results suggest that both the rise in circulating levels of E2 and the decline in hippocampal THP levels at the onset of puberty trigger maximal levels of α4ßδ expression in the CA1 hippocampus.


Assuntos
Região CA1 Hipocampal/metabolismo , Estradiol/farmacologia , Pregnanolona/análogos & derivados , Receptores de GABA-A/metabolismo , Androstenodiona/análogos & derivados , Androstenodiona/farmacologia , Animais , Inibidores da Aromatase/farmacologia , Antagonistas de Estrogênios , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Pregnanolona/antagonistas & inibidores , Pregnanolona/farmacologia
6.
J Inorg Biochem ; 184: 79-87, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29684698

RESUMO

Cytochrome P450 19 (CYP19, aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. Aromatase is a phylogenetically-ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming the neural circuits controlling song and in the determination of sex in fish and reptiles. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin, af) aromatase. Using human cytochrome P450 reductase as a redox partner, afCYP19 displayed similar substrate turnover and LC/MS/MS confirmed that afCYP19 catalyzes the transformations through the intermediates 19-hydroxy- and 19-oxo-androstenedione. Androstenedione and anastrozole had the highest affinity for the enzyme and were followed closely by 19-hydroxyandrostenedione and testosterone. The affinity of 19-oxo-androstenedione for afCYP19 was ten-fold lower. The time-dependent changes in the Soret bands observed in stopped-flow mixing experiments of the steroidal ligands and the inhibitor anastrozole with afCYP19 were best described by a two-step binding mechanism. In summary, these studies describe the first biophysical characterization of an avian aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme and could serve as a convenient model system for studies of the enigmatic transformation of androgens to estrogens.


Assuntos
Aromatase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Anastrozol/metabolismo , Androstenodiona/análogos & derivados , Androstenodiona/metabolismo , Análise Espectral Raman , Testosterona/metabolismo
7.
Toxicol Lett ; 292: 39-45, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29702199

RESUMO

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.


Assuntos
Androgênios/farmacologia , Androstenodiona/análogos & derivados , Doping nos Esportes , Receptor alfa de Estrogênio/agonistas , Estrogênios/farmacologia , Hidroxitestosteronas/farmacologia , Substâncias para Melhoria do Desempenho/farmacologia , Receptores Androgênicos/efeitos dos fármacos , Detecção do Abuso de Substâncias/métodos , Congêneres da Testosterona/farmacologia , Leveduras/efeitos dos fármacos , Androgênios/química , Androstenodiona/química , Androstenodiona/metabolismo , Androstenodiona/farmacologia , Biotransformação , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/química , Estrogênios/metabolismo , Humanos , Hidroxitestosteronas/química , Hidroxitestosteronas/metabolismo , Simulação de Acoplamento Molecular , Substâncias para Melhoria do Desempenho/química , Substâncias para Melhoria do Desempenho/metabolismo , Conformação Proteica , Receptores Androgênicos/química , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Congêneres da Testosterona/química , Congêneres da Testosterona/metabolismo , Leveduras/genética , Leveduras/metabolismo
8.
Biotechnol Lett ; 40(4): 673-678, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29392454

RESUMO

OBJECTIVES: To enhance the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from phytosterols, a phytosterol transport system was constructed in Mycobacterium sp. strain MS136. RESULTS: 9-OHAD can be produced via the controlled degradation of phytosterols by mycobacteria. This involves an active transport process that requires trans-membrane proteins and ATP. A phytosterol transport system from Mycobacterium tuberculosis H37Rv was constructed in Mycobacterium sp. strain MS136 by co-expression of an energy-related gene, mceG, and two integrated membrane protein genes, yrbE4A and yrbE4B. The resultant of the Mycobacterium sp. strain MS136-GAB gave 5.7 g 9-OHAD l-1, which was a 20% increase over 4.7 g l-1 by the wild-type strain. The yield of 9-OHAD was increased to 6.0 g l-1 by optimization of fermentation conditions, when 13 g phytosterols l-1 were fermented for 84 h in 30 ml biotransformation medium in shake flasks. CONCLUSIONS: Phytosterol transport system plays an active role in the uptake and transport of sterols, cloning of the system improved the mass transfer of phytosterols and increased the production of 9-OHAD.


Assuntos
Androstenodiona/biossíntese , Transporte Biológico/genética , Engenharia Metabólica , Mycobacterium tuberculosis/genética , Androstenodiona/análogos & derivados , Androstenodiona/química , Fermentação , Mycobacterium tuberculosis/enzimologia , Fitosteróis/química , Fitosteróis/metabolismo
9.
Mol Cell Endocrinol ; 461: 265-276, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-28939401

RESUMO

Although the adrenal C19 steroids, androstenedione and testosterone, contribute to prostate cancer (PCa) progression the full complement of adrenal androgens, including the C11-oxy C19 steroids, 11ß-hydroxyandrostenedione (11OHA4) and 11ß-hydroxytestosterone (11OHT) and their androgenic metabolites, 11keto-testosterone (11KT) and 11keto-dihydrotestosterone (11KDHT) have, to date, not been considered. This study investigated the contribution of 11OHA4 and 11OHT to the pool of active androgens in the prostate. Steroid profiles were determined in LNCaP, C4-2B and VCaP cell models, in PCa tissue, and in plasma focussing on the inactivation, reactivation and glucuronidation of 11OHA4, 11OHT and their downstream products using ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). The C11-oxy C19 steroids were the predominant steroids with the production of 11KT and 11KDHT in prostate cell models identifying 11ß-hydroxysteroid dehydrogenase type 2 activity. Active:inactive steroid ratios indicated efficient inactivation of dihydrotestosterone (DHT) and 11KDHT by 3α-hydroxysteroid dehydrogenases, while the reactivation of DHT by retinol-like dehydrogenases was greater than the reactivation of 11KDHT. In PCa tissue, inactive C11-oxy C19 steroids ranged from 27 to 30 ng/g, whereas inactive C19 steroids were below 1 ng/g. Steroid glucuronidation was impeded: in VCaP cells, the C11-oxy C19 steroids were unconjugated and the C19 steroids fully conjugated; in C4-2B cells, all steroids were unconjugated, except for DHT of which 50% was conjugated; in LNCaP cells only androsterone, 11KT and 11ß-hydroxyandrosterone were unconjugated. In PCa patients' plasma 11KDHT was present only in the unconjugated form, with 11KT also predominantly unconjugated (90-95%). Even though plasma and tissue sample numbers were limited, this study serves to demonstrate the abundance of C11-oxy C19 steroids, with notable differences in their metabolism, dictated by steroidogenic enzymes and hampered conjugation, affecting active androgen levels. Larger cohorts are required to analyse profiles in modulated metabolic pathways, in order to shed light on treatment outcomes. The C11-oxy C19 steroids are involved in PCa, with impeded glucuronidation in PCa ascribing a dominant role to these steroids in disease progression.


Assuntos
Glândulas Suprarrenais/metabolismo , Androgênios/metabolismo , Androstenodiona/análogos & derivados , Glucuronosiltransferase/metabolismo , Metaboloma , Neoplasias da Próstata/metabolismo , Esteroides/metabolismo , Androgênios/sangue , Androstenodiona/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Modelos Biológicos , Neoplasias da Próstata/sangue
10.
J Agric Food Chem ; 65(48): 10520-10525, 2017 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-29131627

RESUMO

Modification of the sterol catabolism pathway in mycobacteria may result in the accumulation of some valuable steroid pharmaceutical intermediates, such as 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). In previous work, sigma factor D (SigD) was identified as a negative factor of the 9-OHAD production in Mycobacterium neoaurum. Here, the deficiency of rip1 putatively coding for a regulated intramembrane proteolysis metalloprotease (Rip1), which could cleave the negative regulator of SigD (anti-SigD), enhanced the transcription of some key genes (choM1, kshA, and hsd4A) in the sterol catabolic pathway. Furthermore, the deletion of rip1 increased the consumption of phytosterols by 37.8% after 96 h of growth in M. neoaurum. The production of 9-OHAD in the engineered M. neoaurumΔkstD1ΔkstD2ΔkstD3Δrip1 (MnΔk123Δrip1) strain was ultimately increased by 27.3% compared to that in its parental strain M. neoaurumΔkstD1ΔkstD2ΔkstD3 (MnΔk123). This study further confirms the important role of SigD-related factors in the catabolism of sterols.


Assuntos
Androstenodiona/análogos & derivados , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Metaloproteases/metabolismo , Mycobacterium/enzimologia , Fitosteróis/metabolismo , Androstenodiona/química , Androstenodiona/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/genética , Engenharia Genética , Metaloproteases/genética , Mycobacterium/genética , Mycobacterium/metabolismo , Fitosteróis/química , Proteólise , Soja/metabolismo , Soja/microbiologia
11.
Neurotoxicol Teratol ; 64: 45-49, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29031477

RESUMO

Estrogenic and anti-estrogenic endocrine disrupting compounds (EDCs) are recognized as critical modulators of neural development, including sensory system development. Using the zebrafish model, we tested the effect of transient developmental exposure to a known anti-estrogenic EDC on adult visually-guided behavior. In particular, we exposed zebrafish aged 24-hour post-fertilization (hpf), 72 hpf, or 7-days post-fertilization (dpf) to the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A) for 24h. After this time, the fish were removed from treatment, placed into control conditions, and reared until adulthood (3-4months) when visually-guided optomotor responses (OMR) were assessed. Our results show significant decreases in positive OMR in adults exposed to 4-OH-A at 72 hpf and 7 dpf. These deficits were not accompanied by changes in overall swimming behaviors and startle responses, suggesting 4-OH-A specifically effected the visual system. Overall, this study identified long-term, quantifiable effects in visually-guided adult behaviors resulting from transient developmental exposure to the anti-estrogenic EDC, 4-OH-A. Further, these effects were noted when 4-OH-A exposure occurred after hatching, suggesting estrogen signaling is important for visual system maturation.


Assuntos
Androstenodiona/análogos & derivados , Inibidores da Aromatase/administração & dosagem , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/administração & dosagem , Desempenho Psicomotor/efeitos dos fármacos , Visão Ocular/efeitos dos fármacos , Androstenodiona/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Estimulação Luminosa , Percepção Visual/efeitos dos fármacos , Peixe-Zebra
12.
Sheng Wu Gong Cheng Xue Bao ; 33(7): 1198-1206, 2017 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-28869739

RESUMO

In order to improve transformation efficiency of phytosterols into 9α-hydroxylation of 4-androstene-3,17-dione (9α-OH-AD) by Mycobacterium sp. LY-1, we studied the strains breeding using atmospheric and room temperature plasma (ARTP) technology and optimized their conversion process. A high production strain named C33 with a good genetic stability was selected and the product molar yield reached to 15.5%, 34.8% higher than that of original strain with 15 g/L phytosterols. Furthermore, the fermentation medium was optimized through the design of orthogonal experiment. Besides, oil-water bidirectional transformation system was set up to improve the 9α-OH-AD molar yield of mutant strain C33. With adding 12 mL soybean oil to each 1 g phytosterols, the molar yield of 9α-OH-AD reached 47.0%, which increased twice than that of control (15.5%).


Assuntos
Androstenodiona/análogos & derivados , Biotransformação , Mycobacterium/metabolismo , Fitosteróis/metabolismo , Androstenodiona/metabolismo , Fermentação , Microbiologia Industrial , Mutação
13.
Anim Reprod Sci ; 184: 187-195, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28760664

RESUMO

The goal of the present research was to evaluate the efficiency of 11ß-hydroxyandrostenedione (OHA) applied in the diet to achieve sex reversal in the European whitefish (Coregonus lavaretus). At 32day post-hatching, fish were reared in four groups: fish fed with 10ppm of OHA (10 OHA), fish fed with 20ppm of OHA (20 OHA), fish fed without OHA (C) and fish fed without OHA and reared in the water from 20 OHA group (R). The experimental groups were conducted in separate recirculation systems and the first phase of the experiment lasted 63days. For the histological analysis of the gonads, fish from all groups were reared without OHA treatment for an additional 91days (second phase). At the end of the first phase of the experiment, survival of the whitefish ranged from 34.5±11.1% to 51.5±7.3%. The final body weight and coefficient of variation in the weight ranged from 5.6±1.2g to 6.9±1.5g and from 21.5 to 22.7%, respectively. No negative effects of OHA treatment on the growth and the survival of the whitefish were found. Six histological categories of the whitefish gonads were observed. Apart from the typical ovaries and testes, two types of the intersexual gonads (ovotestis and testis-ova) and two types of the sterile-altered gonads were distinguished. No gonadal females were found among fish from any of OHA groups. Gonadal males constituted of 60% and 50% of the fish from 10 OHA and 20 OHA groups, respectively. Intersexes were observed in all groups with the highest proportion found among fish from R variant. Rate of sterile individuals in 10 OHA and 20 OHA groups was 17% and 30%, respectively. The proportion of fish with normal testes to fish with other types of gonad varied from 0.43:1 to 1.5:1 with the higher ratio observed in both OHA groups. Lack of the females among fish from OHA groups suggested OHA affected growth and development of ovaries in the whitefish. However, a high percentage of the sterile fish in both OHA treated groups indicated application of lower doses of OHA for masculinization of the whitefish in the further research.


Assuntos
Androstenodiona/análogos & derivados , Androstenodiona/farmacologia , Salmonidae , Ração Animal/análise , Animais , Dieta , Feminino , Reprodução/efeitos dos fármacos
14.
Biol Sex Differ ; 8(1): 27, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28810930

RESUMO

BACKGROUND: Sexual differentiation of the male brain, and specifically the stress circuitry in the hypothalamus, is primarily driven by estrogen exposure during the perinatal period. Surprisingly, this single hormone promotes diverse programs of sex-specific development that vary widely between different cell types and across the developing male brain. The complexity of this phenomenon suggests that additional layers of gene regulation, including microRNAs (miRNAs), must act downstream of estrogen to mediate this specificity. METHODS: To identify noncanonical mediators of estrogen-dependent sex-specific neural development, we assayed the miRNA complement of the mouse PN2 hypothalamus by microarray following an injection of vehicle or the aromatase inhibitor, formestane. Initially, multivariate analyses were used to test the influence of sex and experimental group on the miRNA environment as a whole. Then, we utilized traditional hypothesis testing to identify individual miRNA with significantly sex-biased expression. Finally, we performed a transcriptome-wide mapping of Argonaute footprints by high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (Ago HITS-CLIP) to empirically characterize targeting relationship between estrogen-responsive miRNAs and their messenger RNA (mRNA) targets. RESULTS: In this study, we demonstrated that the neonatal hypothalamic miRNA environment has robust sex differences and is dynamically responsive to estrogen. Analyses identified 162 individual miRNAs with sex-biased expression, 92 of which were estrogen-responsive. Examining the genomic distribution of these miRNAs, we found three miRNA clusters encoded within a 175-kb region of chromosome 12 that appears to be co-regulated by estrogen, likely acting broadly to alter the epigenetic programming of this locus. Ago HITS-CLIP analysis uncovered novel miRNA-target interactions within prototypical mediators of estrogen-driven sexual differentiation of the brain, including Esr1 and Cyp19a1. Finally, using Gene Ontology annotations and empirically identified miRNA-mRNA connections, we identified a gene network regulated by estrogen-responsive miRNAs that converge on biological processes relevant to sexual differentiation of the brain. CONCLUSIONS: Sexual differentiation of the perinatal brain, and that of stress circuitry in the hypothalamus specifically, seems to be particularly susceptible to environmental programming effects. Integrating miRNA into our conceptualization of factors, directing differentiation of this circuitry could be an informative next step in efforts to understand the complexities behind these processes.


Assuntos
Epigênese Genética , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Caracteres Sexuais , Androstenodiona/análogos & derivados , Androstenodiona/farmacologia , Animais , Animais Recém-Nascidos , Inibidores da Aromatase/farmacologia , Estrogênios/metabolismo , Redes Reguladoras de Genes , Hipotálamo/efeitos dos fármacos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Análise em Microsséries , Análise Multivariada , Transcriptoma/fisiologia
16.
Methods Mol Biol ; 1645: 259-269, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28710634

RESUMO

Two-step one-pot microbial transformation enables obtaining of valuable steroids that are difficult to produce chemically. Here we describe a method for obtaining 11α-hydroxyandrost-4-ene-3,17-dione (11α-HAD) from cheap and available natural sterols (phytosterols or cholesterol).11α-HAD is a primary adrenal steroid in mammals and also a key precursor in the syntheses of halogenated corticoids. Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria.Consequent bioconversions of sterols using two microbial strains in one bioreactor vessel without separation and purification of AD provides high yield of 11α-HAD. At the first fermentation step, phytosterol is converted to AD with Mycobacterium neoaurum NRRL 3805B, or relative strains, to yield about 70% (mol/mol). At the second step, AD is almost fully (98%) hydroxylated at the position 11α with Aspergillus ochraceus VKM F-830, or other suitable organisms, in the same bioreactor. At the average, 30% (w/w) of the high-purity crystalline 11α-HAD can be obtained.The method can be exploited for production of 11α-HAD for practical use.


Assuntos
Actinobacteria/metabolismo , Corticosteroides/biossíntese , Androstenodiona/análogos & derivados , Androstenodiona/biossíntese , Esteróis/biossíntese , Actinobacteria/química , Actinobacteria/genética , Corticosteroides/química , Androstenodiona/química , Biotransformação , Colesterol/biossíntese , Colesterol/química , Fermentação , Fitosteróis/biossíntese , Fitosteróis/química , Esteroides/química , Esteróis/química
17.
J Clin Endocrinol Metab ; 102(8): 2701-2710, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28472487

RESUMO

Context: Patients with 21-hydroxylase deficiency (21OHD) have long-term complications, resulting from poor disease control and/or glucocorticoid overtreatment. Lack of optimal biomarkers has made it challenging to tailor therapy and predict long-term outcomes. Objective: To identify biomarkers of disease control and long-term complications in 21OHD. Setting and Participants: Cross-sectional study of 114 patients (70 males), ages 2 to 67 years (median, 15 years), seen in a tertiary referral center. Methods: We correlated a mass-spectrometry panel of 23 steroids, obtained before first morning medication, with bone age advancement (children), adrenal volume (adults), testicular adrenal rest tumors (TART), hirsutism, menstrual disorders, and pituitary hormones. Results: Total adrenal volume correlated positively with 18 steroids, most prominently 21-deoxycortisol and four 11-oxygenated-C19 (11oxC19) steroids: 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11ketoA4), 11ß-hydroxytestosterone (11OHT), and 11-ketotestosterone (11ketoT) (r ≈ 0.7, P < 0.0001). Nine steroids were significantly higher (P ≤ 0.01) in males with TART compared with those without TART, including 11OHA4 (6.8-fold), 11OHT (4.9-fold), 11ketoT (3.6-fold), 11ketoA4 (3.3-fold), and pregnenolone sulfate (PregS; 4.8-fold). PregS (28.5-fold) and 17-hydroxypregnenolone sulfate (19-fold) levels were higher (P < 0.01) in postpubertal females with menstrual disorders. In males, testosterone levels correlated positively with all 11oxC19 steroids in Tanner stages 1 and 2 (r ≈ 0.7; P < 0.001) but negatively in Tanner stage 5 (r = -0.3 and P < 0.05 for 11ketoA4 and 11ketoT). In females, testosterone level correlated positively with all four 11oxC19 steroids across all Tanner stages (r ≈ 0.8; P < 0.0001). Conclusion: 11oxC19 steroids and PregS might serve as clinically useful biomarkers of disease control and long-term complications in 21OHD.


Assuntos
Hiperplasia Suprarrenal Congênita/metabolismo , Tumor de Resto Suprarrenal/metabolismo , Androgênios/metabolismo , Hirsutismo/metabolismo , Distúrbios Menstruais/metabolismo , Neoplasias Testiculares/metabolismo , 17-alfa-Hidroxipregnenolona/análogos & derivados , 17-alfa-Hidroxipregnenolona/metabolismo , Adolescente , Glândulas Suprarrenais/patologia , Adulto , Determinação da Idade pelo Esqueleto , Idoso , Androstenodiona/análogos & derivados , Androstenodiona/metabolismo , Androstenos/metabolismo , Criança , Pré-Escolar , Cortodoxona/metabolismo , Estudos Transversais , Feminino , Humanos , Hidroxitestosteronas/metabolismo , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Pregnenolona/metabolismo , Testosterona/análogos & derivados , Testosterona/metabolismo , Adulto Jovem
18.
Artigo em Inglês | MEDLINE | ID: mdl-28140792

RESUMO

Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.


Assuntos
Androstenodiona/análogos & derivados , Inibidores da Aromatase/urina , Cromatografia Líquida de Alta Pressão/normas , Moduladores Seletivos de Receptor Estrogênico/urina , Detecção do Abuso de Substâncias/veterinária , Espectrometria de Massas em Tandem/normas , Androstenodiona/administração & dosagem , Androstenodiona/urina , Criação de Animais Domésticos/ética , Animais , Inibidores da Aromatase/administração & dosagem , Bovinos , Controle de Medicamentos e Entorpecentes/legislação & jurisprudência , Limite de Detecção , Reprodutibilidade dos Testes , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Detecção do Abuso de Substâncias/métodos , Suínos
19.
J Steroid Biochem Mol Biol ; 167: 182-191, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28065637

RESUMO

The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(ß)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased Km and decreased kcat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites.


Assuntos
Androstenodiona/análogos & derivados , Aromatase/metabolismo , Esteroide 21-Hidroxilase/metabolismo , Androgênios/metabolismo , Androstenodiona/metabolismo , Inibidores da Aromatase/química , Catálise , Pré-Escolar , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Sistema Endócrino , Doenças do Sistema Endócrino/diagnóstico , Doenças do Sistema Endócrino/metabolismo , Escherichia coli/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Receptores Androgênicos/metabolismo , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Esteroides/metabolismo
20.
J Clin Endocrinol Metab ; 102(3): 840-848, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27901631

RESUMO

Context: Androgen excess is a defining feature of polycystic ovary syndrome (PCOS), but the exact origin of hyperandrogenemia remains a matter of debate. Recent studies have highlighted the importance of the 11-oxygenated C19 steroid pathway to androgen metabolism in humans. In this study, we analyzed the contribution of 11-oxygenated androgens to androgen excess in women with PCOS. Methods: One hundred fourteen women with PCOS and 49 healthy control subjects underwent measurement of serum androgens by liquid chromatography-tandem mass spectrometry. Twenty-four-hour urinary androgen excretion was analyzed by gas chromatography-mass spectrometry. Fasting plasma insulin and glucose were measured for homeostatic model assessment of insulin resistance. Baseline demographic data, including body mass index, were recorded. Results: As expected, serum concentrations of the classic androgens testosterone (P < 0.001), androstenedione (P < 0.001), and dehydroepiandrosterone (P < 0.01) were significantly increased in PCOS. Mirroring this, serum 11-oxygenated androgens 11ß-hydroxyandrostenedione, 11-ketoandrostenedione, 11ß-hydroxytestosterone, and 11-ketotestosterone were significantly higher in PCOS than in control subjects, as was the urinary 11-oxygenated androgen metabolite 11ß-hydroxyandrosterone. The proportionate contribution of 11-oxygenated to total serum androgens was significantly higher in patients with PCOS compared with control subjects [53.0% (interquartile range, 48.7 to 60.3) vs 44.0% (interquartile range, 32.9 to 54.9); P < 0.0001]. Obese (n = 51) and nonobese (n = 63) patients with PCOS had significantly increased 11-oxygenated androgens. Serum 11ß-hydroxyandrostenedione and 11-ketoandrostenedione correlated significantly with markers of insulin resistance. Conclusions: We show that 11-oxygenated androgens represent the majority of circulating androgens in women with PCOS, with close correlation to markers of metabolic risk.


Assuntos
Androgênios/metabolismo , Glicemia/metabolismo , Hiperandrogenismo/metabolismo , Insulina/metabolismo , Obesidade/metabolismo , Síndrome do Ovário Policístico/metabolismo , Adulto , Androstenodiona/análogos & derivados , Androstenodiona/metabolismo , Androstenos/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida , Desidroepiandrosterona/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hidroxitestosteronas/metabolismo , Hiperandrogenismo/complicações , Resistência à Insulina , Obesidade/complicações , Síndrome do Ovário Policístico/complicações , Espectrometria de Massas em Tandem , Testosterona/análogos & derivados , Testosterona/metabolismo , Adulto Jovem
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