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1.
Development ; 147(4)2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32001440

RESUMO

Sex determination and differentiation are complex processes controlled by many different factors; however, the relationships among these factors are poorly understood. Zebrafish gonadal differentiation exhibits high plasticity involving multiple factors and pathways, which provides an excellent model for investigating the interactions between them. Ovarian aromatase (cyp19a1a) and dmrt1 are key factors in directing vertebrate ovary and testis differentiation, respectively. Knockout of zebrafish cyp19a1a leads to all-male offspring, whereas the loss of dmrt1 results in a female-biased sex ratio. In the present study, we established dmrt1-/- ;cyp19a1a-/- double mutant zebrafish and discovered that the introduction of the dmrt1 mutation into the cyp19a1a mutant could rescue the all-male phenotype of the latter. Interestingly, despite the lack of aromatase/estrogens, the follicles in the ovary of the rescued cyp19a1a mutant could develop normally up to the previtellogenic stage. Further evidence suggested the ovarian aromatase directed ovarian differentiation by suppressing dmrt1 expression via nuclear estrogen receptors (nERs). Our results provide solid evidence for an interaction between cyp19a1a and dmrt1 in zebrafish gonadal differentiation, and for the dispensability of estrogens in controlling early folliculogenesis.


Assuntos
Aromatase/genética , Aromatase/fisiologia , Folículo Ovariano/embriologia , Testículo/embriologia , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Alelos , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Estrogênios/fisiologia , Feminino , Técnicas de Inativação de Genes , Genótipo , Heterozigoto , Masculino , Mutação , Fenótipo , Receptores Estrogênicos/fisiologia , Processos de Determinação Sexual , Diferenciação Sexual , Peixe-Zebra
2.
Ecotoxicol Environ Saf ; 193: 110324, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32088548

RESUMO

This study assessed the transcription levels of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL, ChgH, and ChgHm), cytochrome P450 aromatase (CYP19a1b), and ER subtypes (ERα, ERß1, and ERß2), in 7 days-post-fertilization (dpf) embryos and 9 and 12 dpf larvae of medaka (Oryzias latipes) exposed to estrogenic endocrine-disrupting chemicals (EDCs). The <5 h-post-fertilization embryos were exposed to EDCs such as 17ß-estradiol (E2), p-n-nonylphenol (NP), and bisphenol A (BPA). In E2 (0.10-222 nM)-treated 7 dpf embryos and 9 or 12 dpf larvae, ChgL, ChgH, and ChgHm expression was up-regulated in a concentration-dependent manner. By contrast, interestingly, Vtg1 and Vtg2 expression was not induced in E2-treated 7 dpf embryos but was significantly induced in 9 and 12 dpf larvae, suggesting a developmental-stage-specific regulatory mechanism underlying Vtg expression. The maximum concentrations of NP (0.09-1.5 µM) and BPA (1.8-30 µM) up-regulated Chg expression in 9 or 12 dpf larvae, and the relative estrogenic potencies (REPs) of E2, NP, and BPA were 1, 2.1 × 10-4, and 1.0 × 10-5, respectively. Chg messenger RNA (mRNA) in medaka embryos and larvae can be used as a sensitive biomarker for screening potential estrogenic EDCs. Our assay system using embryos and larvae can be used as an in vivo alternative model because independent feeding stages (e.g., embryonic and early larval stages) are suitable alternatives.


Assuntos
Proteínas do Ovo/biossíntese , Disruptores Endócrinos/toxicidade , Estrogênios/toxicidade , Oryzias/embriologia , Oryzias/crescimento & desenvolvimento , Animais , Aromatase/biossíntese , Aromatase/genética , Compostos Benzidrílicos/toxicidade , Proteínas do Ovo/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Estradiol/toxicidade , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/biossíntese , Receptor beta de Estrogênio/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Masculino , Modelos Animais , Oryzias/genética , Fenóis/toxicidade , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Vitelogeninas/biossíntese , Vitelogeninas/genética
3.
Artigo em Inglês | MEDLINE | ID: mdl-32033145

RESUMO

Benzo[a]pyrene (BaP) is a common environmental disrupting chemical that can cause endocrine disorders in organisms. However, the continued interference effects of BaP on multi-generation fish needs further research. In this study, we performed different periods (G1F1-3, G2F2-3, G3F3) of BaP exposure on marine medaka. We determined the embryo toxicity, and analyzed relative reproductive genes (ERα, cyp19a and vtg1) to predict the sexual differentiation of marine medaka. The results showed that high concentrations of BaP (200 µg·L-1) significantly delayed the hatching time of embryos. Moreover, medium/high concentrations of BaP (20 and 200 µg·L-1) prolonged the sexual maturity time of marine medaka. The relative gene expression of ERα, cyp19a and vtg1 were measured at 5 dpf of embryos. We found that BaP had significantly inhibited the expression of the genes related to female fish development. Consequently, there were more males in the offspring sex ratio at BaP exposure. Overall, BaP can cause embryonic toxicity and abnormal sexual differentiation, while the expression of related reproductive genes can effectively indicate the sex ratio.


Assuntos
Benzo(a)pireno/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Diferenciação Sexual/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Aromatase/genética , Receptor alfa de Estrogênio/genética , Feminino , Proteínas de Peixes/genética , Expressão Gênica/efeitos dos fármacos , Masculino , Oryzias/genética , Oryzias/crescimento & desenvolvimento , Reprodução/efeitos dos fármacos , Reprodução/genética , Razão de Masculinidade , Vitelogeninas/genética
4.
J Steroid Biochem Mol Biol ; 199: 105608, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31996328

RESUMO

Ovarian granulosa cells, known to be endocrine cells, have well active TLR4-/NFKB signalling mediated innate immune capabilities. We have previously shown that endotoxin not only transiently regulates proinflammatory cytokines but cells become tolerant on repeated exposure to endotoxin and impaired granulosa cells functions, which includes downregulation of CYP19A1 gene. To understand further endotoxin tolerance and impaired granulosa cells function, genome-wide transcriptomic profiling in endotoxin tolerant buffalo granulosa cells (bGCs) identified miR-326 as upregulated amongst top 5 DE miRNAs [unpublished data] and qPCR validation confirmed its upregulation during endotoxin tolerance. In silico analyses showed that miR-326 targets CYP19A1 gene. Therefore, in the present study, we elucidated the role of miR-326 in buffalo granulosa cells (bGCs). We first validated its expression vis-à-vis CYP19A1 gene expression in bGCs, both in vivo and in vitro. Results showed an inverse relationship between miR-326 and CYP19A1 expression. Similarly, transcription factors, known to be involved in CYP19A1 gene regulation, CREB and C/EBP-ß expression was also found to be decreased in granulosa cells mimicking pre-ovulatory follicular stage. Further, miR-326 mimic was transfected to bGCs in culture and expression of CYP19A1 and CREB & C/EBP-ß and genes encoding other enzymes of steroidogenesis pathway were also analyzed. The present study results showed that miR-326 significantly inhibits the expression of CYP19A1 gene while expression of transcription factors CREB and C/EBP-ß was found to be upregulated. The expression of STAR and CYP11A1 was found to be unaffected. To elucidate the molecular mechanism of miR-326 mediated downregulation of CYP19A1, binding analyses of RNA polymerase II and CEBP-ß to CYP19A1 gene promoter II was analyzed. The result also showed decreased binding of RNA polymerase II with increased binding of CEBP-ß to CYP19A1 gene promoter II in bGCs, transfected with miR-326 as compared to control. In summary, our results suggest that miR-326 upregulate CREB and CREB may activate C/EBP-ß and later inhibited the transcription of CYP19A1 and decreased estradiol-17b production. The miR-326 mediated down-regulation of the CYP19A1 gene involving CREB-C/EBP-ß can be exploited in developing strategies to attenuate endotoxin-mediated tolerance induced impaired granulosa cells function to ensure proper fertility in females.


Assuntos
Aromatase/genética , Estradiol/genética , Estrogênios/genética , MicroRNAs/genética , Animais , Búfalos/genética , Búfalos/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Bovinos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Estradiol/biossíntese , Estrogênios/biossíntese , Feminino , Regulação da Expressão Gênica/genética , Células da Granulosa/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais
5.
PLoS One ; 15(1): e0227830, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31971970

RESUMO

Estrogens are important for maintaining metabolic health in males. However, the key sources of local estrogen production for regulating energy metabolism have not been fully defined. Immune cells exhibit aromatase activity and are resident in metabolic tissues. To determine the relative contribution of immune cell-derived estrogens for metabolic health in males, C57BL6/J mice underwent bone marrow transplant with marrow from either wild-type (WT(WT)) or aromatase-deficient (WT(ArKO)) donors. Body weight, body composition, and glucose and insulin tolerance were assessed over 24 weeks with mice maintained on a regular chow diet. No differences were found in insulin sensitivity between groups, but WT(ArKO) mice were more glucose tolerant than WT(WT) mice 20 weeks after transplant, suggestive of enhanced glucose disposal (AUCglucose 6061±3349 in WT(WT) mice versus 3406±1367 in WT(ArKO) mice, p = 0.01). Consistent with this, skeletal muscle from WT(ArKO) mice showed higher expression of the mitochondrial genes Ppargc1a (p = 0.03) and Nrf1 (p = 0.01), as well as glucose transporter type 4 (GLUT4, Scl2a4; p = 0.02). Skeletal muscle from WT(ArKO) mice had a lower concentration of 17ß-estradiol (5489±2189 pg/gm in WT(WT) mice versus 3836±2160 pg/gm in WT(ArKO) mice, p = 0.08) but higher expression of estrogen receptor-α (ERα, Esr1), raising the possibility that aromatase deficiency in immune cells led to a compensatory increase in ERα signaling. No differences between groups were found with regard to body weight, adiposity, or gene expression within adipose tissue or liver. Immune cells are a key source of local 17ß-estradiol production and contribute to metabolic regulation in males, particularly within skeletal muscle. The respective intracrine and paracrine roles of immune cell-derived estrogens require further delineation, as do the pathways that regulate aromatase activity in immune cells specifically within metabolic tissues.


Assuntos
Aromatase/genética , Glucose/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Músculo Esquelético/metabolismo , Animais , Aromatase/metabolismo , Transplante de Medula Óssea , Células Cultivadas , Estrogênios/metabolismo , Deleção de Genes , Teste de Tolerância a Glucose , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
6.
Aquat Toxicol ; 220: 105403, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927064

RESUMO

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to "classical" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 µg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.


Assuntos
Animais Geneticamente Modificados , Aromatase/genética , Disruptores Endócrinos/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/metabolismo , Feminino , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Guias como Assunto , Organização para a Cooperação e Desenvolvimento Econômico , Ovário/efeitos dos fármacos , Ovário/metabolismo , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Vitelogeninas/metabolismo , Peixe-Zebra/metabolismo
7.
Nat Commun ; 11(1): 185, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924771

RESUMO

Developing insight into tissue-specific transcriptional mechanisms can help improve our understanding of how genetic variants exert their effects on complex traits and disease. In this study, we apply the principles of Mendelian randomization to systematically evaluate transcriptome-wide associations between gene expression (across 48 different tissue types) and 395 complex traits. Our findings indicate that variants which influence gene expression levels in multiple tissues are more likely to influence multiple complex traits. Moreover, detailed investigations of our results highlight tissue-specific associations, drug validation opportunities, insight into the likely causal pathways for trait-associated variants and also implicate putative associations at loci yet to be implicated in disease susceptibility. Similar evaluations can be conducted at http://mrcieu.mrsoftware.org/Tissue_MR_atlas/.


Assuntos
Análise da Randomização Mendeliana , Fenômica , Transcriptoma , Aromatase/genética , Fibrilina-2/genética , Regulação da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Herança Multifatorial , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Proteínas Ribossômicas/genética , Doenças da Glândula Tireoide/genética , Doenças da Glândula Tireoide/metabolismo
8.
Domest Anim Endocrinol ; 70: 106378, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514021

RESUMO

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.


Assuntos
Aromatase/metabolismo , Galinhas , Regulação da Expressão Gênica/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , Nitrofenóis/farmacologia , Folículo Ovariano , Progesterona Redutase/metabolismo , Esteroide Isomerases/metabolismo , Animais , Aromatase/genética , Regulação para Baixo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Complexos Multienzimáticos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona Redutase/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Esteroide Isomerases/genética , Técnicas de Cultura de Tecidos
9.
Toxicol In Vitro ; 62: 104658, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31629071

RESUMO

We developed an innovative co-culture system composed of Hs578t human mammary stromal-like cells and T47D hormone-dependent breast epithelial tumor cells as a representative in vitro model of the human hormone-dependent mammary tumor microenvironment. Hs578t cells expressed aromatase (CYP19) mainly via the healthy stromal CYP19 promoter I.4, but also to a lesser extent via the breast cancer-relevant promoters PII, I.3 and I.7, and produced estrogens from androgen precursors. These estrogens stimulated T47D cell proliferation and estrogen receptor-dependent expression of trefoil factor-1 (TFF1), which is known to stimulate mammary tumor cell proliferation and migration. Hs578t cells can also undergo a "promoter-switch" where the normally silent CYP19 promoters PII, I.3 and I.7 become activated, which mimics the in vivo situation in human breast cancer patients. This positive feedback loop is the hallmark of the hormone-dependent breast tumor microenvironment. Using the co-culture model we designed, we evaluated the promoter-specific expression of CYP19, expression of estrogen-dependent gene TFF1, and determined the effects exhibited by basil and fennel seed essential oils on steroidogenesis in the tumor microenvironment.


Assuntos
Aromatase/genética , Disruptores Endócrinos/toxicidade , Estrogênios/toxicidade , Foeniculum , Ocimum basilicum , Óleos Voláteis/toxicidade , Fator Trefoil-1/genética , Aromatase/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Regiões Promotoras Genéticas , Sementes
10.
Reprod Biol Endocrinol ; 17(1): 111, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31878927

RESUMO

BACKGROUND: Previous studies of expression profiles of major endometrial effectors of steroid physiology in endometriosis have yielded markedly conflicting conclusions, presumably because the relative effects of type of endometriosis, fertility history and menstrual cycle phases on the measured variables were not considered. In the present study, endometrial mRNA and protein levels of several effectors of steroid biosynthesis and action in patients with stage III-IV ovarian endometriosis (OE) with known fertility and menstrual cycle histories were compared with the levels in control endometrium to test this concept. METHODS: Endometrial samples were collected from patients without endometriosis (n = 32) or OE stages III-IV (n = 52) with known fertility and cycle histories. qRT-PCR and immunoblotting experiments were performed to measure levels of NR5A1, STAR, CYP19A1, HSD17Bs, ESRs and PGR transcripts and proteins, respectively. Tissue concentrations of steroids (P4, T, E1 and E2) were measured using ELISAs. RESULTS: The levels of expression of aromatase and ERß were lower (P < 0.0001) and 17ß-HSD1 (P < 0.0001) and PRA (P < 0.01) were higher in OE endometrium. Lower aromatase levels and higher 17ß-HSD1 levels were detected in fertile (aromatase: P < 0.05; 17ß-HSD1: P < 0.0001) and infertile (aromatase: P < 0.0001; 17ß-HSD1: P < 0.0001) OE endometrium than in the matched control tissues. Both proliferative (PP) and secretory (SP) phase OE samples expressed aromatase (P < 0.0001) and ERß (PP: P < 0.001; SP: P < 0.01) at lower levels and 17ß-HSD1 (P < 0.0001) and PRA (PP: P < 0.01; SP: P < 0.0001) at higher levels than matched controls. Higher 17ß-HSD1 (P < 0.01) and E2 (P < 0.05) levels and a lower (P < 0.01) PRB/PRA ratio was observed in infertile secretory phase OE endometrium than in control. CONCLUSIONS: We report that dysregulated expression of 17ß-HSD1 and PGR resulting in hyperestrogenism and progesterone resistance during the secretory phase of the menstrual cycle, rather than an anomaly in aromatase expression, was the hallmark of eutopic endometrium from infertile OE patients. Furthermore, the results provide proof of concept that the fertility and menstrual cycle histories exerted relatively different effects on steroid physiology in the endometrium from OE patients compared with the control subjects.


Assuntos
Endometriose/metabolismo , Endométrio/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Doenças Ovarianas/metabolismo , Receptores de Esteroides/metabolismo , 17-Hidroxiesteroide Desidrogenases/análise , 17-Hidroxiesteroide Desidrogenases/genética , Adolescente , Adulto , Aromatase/análise , Aromatase/genética , Endométrio/química , Estradiol/análise , Feminino , Expressão Gênica , Humanos , Infertilidade Feminina/metabolismo , Ciclo Menstrual , Progesterona/análise , Receptores Estrogênicos/análise , Receptores de Progesterona/análise , Receptores de Esteroides/genética , Adulto Jovem
11.
Anticancer Res ; 39(11): 6107-6114, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704838

RESUMO

AIM: The present investigation aimed to examine the therapeutic potential of the new coumarin derivative bis(4-hydroxy-2H-chromen-2-one) coumarin (4HC) against breast cancer. MATERIALS AND METHODS: For this purpose, the effects of 4HC treatment on the proliferation of MCF-7 breast cancer cells and on MCF-10a non-cancerous cells were evaluated using a fluorescent assay. Cell cycle distribution and apoptosis were measured by image cytometry. The expression level of aromatase (CYP19A1) and apoptosis-related genes were determined by real-time PCR. RESULTS: MCF-7 mammary cancer cell proliferation was significantly decreased within 24 h after treatment with 4HC at 50 µM, while no effect was observed on the viability of MCF-10a non-cancerous mammary cells. 4HC also increased the percentage of the cells in the G2/M phase, inducing apoptosis. Real-time PCR revealed that 4HC induced MCF-7 mortality through an up-regulation of Bax and a down-regulation of Bcl-2, resulting in an increase in caspase-3 gene expression. The increased expression of apoptosis-related genes was accompanied by a decrease in CYP19A1 gene expression. CONCLUSION: 4HC selectively inhibits proliferation of MCF-7cells in vitro. Moreover, 4HC has inhibitory effects on aromatase gene expression and promoting effects on apoptosis, in MCF-7 cells.


Assuntos
Apoptose/efeitos dos fármacos , Aromatase/química , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Cromonas/química , Cumarínicos/química , Cumarínicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Aromatase/genética , Aromatase/metabolismo , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células Tumorais Cultivadas
12.
BMC Med Genet ; 20(1): 148, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477036

RESUMO

BACKGROUND: Single nucleotide polymorphisms (SNPs) in several CYP genes have been associated with altered breast cancer (BC) risk in different populations. Despite this, there is a dearth of information on the roles of these SNPs in Jordanian BC patients. Therefore, this study aims to determine if there is any single nucleotide polymorphism (SNP) within CYP19A1, CYP2C19, CYP2C9, CYP1B1, CYP3A4, and CYP1A2 genes associated with BC in the Jordanian population. In addition, this work investigates the association between selected BC prognostic factors and variants of the aforementioned CYP candidate genes. METHODS: Blood samples were withdrawn from 221 BC patients and 218 healthy volunteers recruited from the Jordanian population. Genomic DNA was withdrawn and, after quantification and quality control, was genotyped using the Sequenom MassARRAY® system (iPLEX GOLD). Statistical analysis was then carried out to assess allelic and genotypic frequencies as well as genetic association between cases and controls. RESULTS: The CYP19A1 SNP rs7176005 (p < 0.0045) and the CYP1A2 SNP rs762551 (p = 0.004) were significantly associated with BC risk. However, no such association was found for the screened SNPs of the CYP2C9, CYP1B1, CYP2C19 and CYP3A4 genes. Regarding the prognostic factors of BC, several of the screened SNPs were associated with different pathological and clinical features. CONCLUSIONS: Certain CYP genes, particularly CYP19A1 and CYP1A2, were associated with BC risk and development in the Jordanian population.


Assuntos
Neoplasias da Mama/genética , Ciclofilinas/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aromatase/genética , Estudos de Coortes , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP3A/genética , Feminino , Genótipo , Humanos , Jordânia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Risco , Adulto Jovem
13.
J Steroid Biochem Mol Biol ; 195: 105486, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31557516

RESUMO

Estrogen receptor-positive (ER+) breast cancers require estrogens for their growth. Aromatase inhibitors (AIs) are considered the first-line therapy for this type of tumours. Despite the well-established clinical benefit of this therapy, the search for novel potent AIs that present higher efficacy and fewer side effects is still demanded. Thus, taking into account the known interactions of the natural substrate, androstenedione, within the aromatase active-site, a range of new steroidal compounds have been designed, synthesized and studied by our group. In this work, it was evaluated in MCF-7aro, an ER+ breast cancer cell line that overexpress aromatase, the anti-aromatase efficacy and the biological effects of eight new AIs: 6α-methyl-5α-androst-3-en-17-one (1a), 6α-methyl-3α,4α-epoxy-5α-androstan-17-one (3a), 6α-methylandrost-4-ene-3,17-dione (9), 6α-allylandrosta-1,4-diene-3,17-dione (13), 6α-allylandrost-4-ene-3,17-dione (15), 6α-allylandrost-4-en-17-one (17), 6ß-hydroxyandrost-4-ene-3,17-dione (19) and 6α-hydroxyandrost-4-ene-3,17-dione (20). Their anti-cancer properties were elucidated, as well as, the dependence of their mechanism of action on aromatase inhibition and/or on steroid receptors modulation, such as estrogen and androgen receptors, which are key targets for this type of cancer. Results demonstrate that the studied AIs present high anti-aromatase activity, disrupt MCF-7aro cell cycle progression and induce apoptosis, through the mitochondrial pathway. Compounds 1a, 3a, 9, 13, 15 and 17 exhibited an aromatase-dependent effect on cells and, interestingly, steroids 9 and 13 displayed the ability to decrease aromatase protein levels without affecting CYP19A1 mRNA levels. Furthermore, the effects of compounds 1a, 3a and 15 were dependent on ER and on AR modulation, whereas compounds 9 and 19 were only dependent on AR modulation. From a clinical point of view, these actions can be considered as a therapeutic advantage for this type of tumours. Thus, new promising AIs that impair ER+ breast cancer cell growth, by acting on aromatase, and even, on ER and AR were discovered. Furthermore, new insights on the most favourable structural modifications in the steroidal core structure were provided, helping to a more rational drug design of new and potent AIs.


Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/metabolismo , Receptores Androgênicos/metabolismo , Aromatase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , RNA Mensageiro/metabolismo
14.
J Agric Food Chem ; 67(36): 10207-10213, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31426637

RESUMO

Forchlorfenuron (FCF) is a synthetic plant cytokine-like growth regulator that is massively used in agriculture to increase fruit size and weight. There is an insufficiency of published data on the safety profile of FCF, especially as it is involved in ovarian function. In our study, a chronic toxicity study on FCF was conducted and designed by feeding at dosage levels of 0, 0.6, and 60 mg/kg body weight in Sprague-Dawley rats for 180 days. During the 180 day FCF administration, no biologically relevant changes were observed in the body weight, clinical signs, food consumption, organ weight, hematology, and clinical biochemistry of the tested animals. However, macroscopic and microscopic evaluations revealed the presence of severe hydrometra in the uterus and pathological changes in the ovaries. In addition, it was found that FCF inhibited the proliferation of granulosa cells (GCs) and H295R cells, as well as downregulated the expression of CYP17A1 and CYP19A1 in estradiol and progesterone production, resulting in decreased steroidogenesis in GCs and H295R cells. Taken together, our findings suggest that FCF has potential adverse effects on the ovaries and on steroidogenesis.


Assuntos
Hormônios Esteroides Gonadais/metabolismo , Compostos de Fenilureia/toxicidade , Reguladores de Crescimento de Planta/toxicidade , Piridinas/toxicidade , Administração Oral , Animais , Aromatase/genética , Aromatase/metabolismo , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Ovário/patologia , Compostos de Fenilureia/administração & dosagem , Piridinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Fatores de Tempo , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimento , Útero/metabolismo , Útero/patologia
15.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(12): 158512, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31454668

RESUMO

In each menstrual cycle endometrial stromal cells (hESC) proliferate and differentiate into specialized decidual cells, a process termed decidualization, which regulates endometrial receptivity. Decidualization is mainly controlled by sex ovarian hormones, estradiol (E2) and progesterone. E2 plays an important role in the expression of the progesterone receptor and promotes the endometrial stromal cells differentiation. Our group previously reported that anandamide (AEA) impairs decidualization through cannabinoid receptor 1 (CB1). In this study, we hypothesized whether AEA inhibitory effect on cell decidualization could be mediated through interaction with aromatase and consequent interference in estradiol production/signaling. We used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) derived from human term placenta. In cells exposed to a differentiation stimulus, AEA-treatment prevents the increase of the expression of CYP19A1 gene encoding aromatase, E2 levels and of estradiol receptor expression, that are observed in differentiating cells. Regarding CYP19A1 mRNA levels, the effect was partially reverted by a CB1 receptor antagonist and by a COX2 inhibitor. In addition, we report that AEA presents anti-aromatase activity in placental microsomes, the nature of the inhibition being the uncommon mixed type as revealed by the kinetic studies. Structural analysis of the AEA-Aromatase complexes determined that AEA may bind to the active site pocket of the enzyme. In overall we report that AEA inhibits aromatase activity and may affect E2 signaling crucial for the decidualization process, indicating that a deregulation of the endocannabinoid system may be implicated in endometrial dysfunction and in fertility/infertility disorders.


Assuntos
Ácidos Araquidônicos/metabolismo , Aromatase/metabolismo , Decídua/citologia , Endocanabinoides/metabolismo , Endométrio/citologia , Alcamidas Poli-Insaturadas/metabolismo , Adulto , Aromatase/genética , Linhagem Celular , Células Cultivadas , Decídua/metabolismo , Regulação para Baixo , Endométrio/metabolismo , Feminino , Humanos , Simulação de Acoplamento Molecular , Gravidez , Células Estromais/citologia , Células Estromais/metabolismo , Adulto Jovem
16.
J Steroid Biochem Mol Biol ; 194: 105433, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31376460

RESUMO

The steroid hormones not only exert various endocrine functions but also act as the autocrine or paracrine factors in different tissues of mammals. In the present study, the seasonal expressions of androgen receptor (AR), estrogen receptors alpha and beta (ERα and ERß), aromatase cytochrome P450 (P450arom) and 5α-reductase 1, 2 were investigated in the epididymis of the muskrat. HE staining showed enlarged lumen and abundant sperm in the breeding season while reduced lumen with no sperm in the non-breeding season. The staining of AR was presented in nuclei of epithelial cells of the epididymis in both seasons. The immunostaining of ERα was localized in both nuclei and cytoplasm of epithelial cells of the epididymis during the breeding season, while the weak staining of ERα was only in the nuclei of epithelial cells during the non-breeding season. In contrast, ERß signal was negative in the epididymis of the muskrat in both seasons. The positive signals for P450arom and 5α-reductase 1, 2 were found in the cytoplasm of epithelial and smooth muscle cells during both seasons. Moreover, the protein and mRNA expression levels of AR, ERα, P450arom and 5α-reductase 1, 2 were significantly higher in the epididymis during the breeding season than those of the non-breeding season, and the expression level of 5α-reductase 1 was higher when compared with 5α-reductase 2. In addition, the levels of testosterone (T) and dihydrotestosterone (DHT) in the epididymis and serum were remarkably higher during the breeding season. Taken together, these findings suggested androgen and estrogen might play an important endocrine or autocrine/paracrine role to regulate the epididymal functions of the muskrat.


Assuntos
Aromatase/metabolismo , Colestenona 5 alfa-Redutase/metabolismo , Epididimo/metabolismo , Receptores Androgênicos/metabolismo , Receptores Estrogênicos/metabolismo , Animais , Aromatase/genética , Arvicolinae , Colestenona 5 alfa-Redutase/genética , Masculino , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Receptores Estrogênicos/genética , Reprodução , Estações do Ano
17.
Toxicol In Vitro ; 61: 104615, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31374317

RESUMO

Estradiol, in some way or another, plays a critically important physiologic role in the establishment and maintenance of pregnancy. This study was designed to investigate whether BPA affects the estradiol level of human placental JEG-3 cells, which may contribute to insights into the reproductive toxicity and endocrine disruption of BPA. The JEG-3 cells were treated with increasing concentrations of BPA (0.1 to 50 µM). We observed that BPA significantly reduced estradiol level of JEG-3 cells in a dose-dependent manner, which was accompanied by an increase in CYP1A1 protein level and an inhibition of CYP19A1 protein level. Additionally, by lentiviral transduction, we determined that estradiol level of JEG-3 cells over-expressing CYP1A1 gene was notably decreased and the decrease was of 84.9% compared to the control. Meanwhile, estradiol was almost undetectable in CYP19A1 knockdown group. On the contrary, the group with over-expression of CYP19A1 gene increased estradiol level by 8.6 fold while the CYP1A1 knockdown group increased by 5.6 fold. In summary, our research clearly showed that BPA alters JEG-3 estradiol synthesis and catabolism due to its action on CYP1A1 and CYP19A1 protein levels and may interfere with the normal process of placenta formation and embryonic development during early pregnancy.


Assuntos
Aromatase/genética , Compostos Benzidrílicos/toxicidade , Citocromo P-450 CYP1A1/genética , Disruptores Endócrinos/toxicidade , Estradiol/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Fenóis/toxicidade , Aromatase/metabolismo , Linhagem Celular , Citocromo P-450 CYP1A1/metabolismo , Feminino , Humanos , Placenta/citologia , Placenta/metabolismo , Gravidez
18.
Med Sci Monit ; 25: 5589-5593, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31352466

RESUMO

BACKGROUND The aim of our study was to elucidate the biological targets and pharmacological mechanisms for calycosin (CC) against colorectal cancer (CRC) through an approach of system pharmacology. MATERIAL AND METHODS Using a web-based platform, all CRC-causing genes were identified using a database of gene-disease associations (DisGeNET), and all well-known genes of CC identified using the databases of prediction of protein targets of small molecules (Swiss Target Prediction), drug classification, and target prediction (SuperPred). The carefully selected genes of CRC and CC were concurrently constructed by using a database of functional protein association networks (STRING), and use of software for visualizing complex networks (Cytoscape), characterized with production of protein-protein interaction (PPI) network of CC against CRC. The important biological targets of CC against CRC were identified through topological analysis, then the biological processes and molecular pathways of CC against CRC were further revealed for testing these important biotargets by enrichment assays. RESULTS We found that the key predictive targets of CC against CRC were estrogen receptor 2 (ESR2), ATP-binding cassette sub-family G member 2 (ABCG2), breast cancer type 1 susceptibility protein (BRCA1), estrogen receptor 1 (ESR1), cytochrome p450 19A1 (CYP19A1), and epidermal growth factor receptor (EGFR). Visual analysis revealed that the biological processes of CC against CRC were positively linked to hormonal metabolism, regulation of genes, transport, cell communication, and signal transduction. Further, the interrelated molecular pathways were chiefly related to endogenous nuclear estrogen receptor alpha network, forkhead box protein A1 (FOXA1) transcription factor network, activating transcription factor 2 (ATF2) transcription factor network, regulation of telomerase, plasma membrane estrogen receptor signaling, estrogen biosynthesis, androgen receptor, FOXA transcription factor networks, estrogen biosynthesis, and phosphorylation of repair proteins. CONCLUSIONS Use of system pharmacology revealed the biotargets, biological processes, and pharmacological pathways of CC against CRC. Intriguingly, the identifiable predictive biomolecules are likely potential targets for effectively treating CRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Isoflavonas/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Aromatase/genética , Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/fisiopatologia , Biologia Computacional/métodos , Bases de Dados Genéticas , Receptores ErbB/genética , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Isoflavonas/farmacologia , Proteínas de Neoplasias , Fenômenos Farmacológicos e Toxicológicos , Mapas de Interação de Proteínas/genética , Transdução de Sinais , Análise de Sistemas
19.
Pol J Vet Sci ; 22(2): 279-286, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31269350

RESUMO

In this investigation, the effects of genistein (GEN) on the expression of steroidogenic genes such as steroidogenic acute regulatory protein (StAR), side-chain cleavage enzymes (P450scc) and cytochrome P450 aromatase (CYP19) were assessed. For this study, forty young female Sprague Dawley (SD) rats at aged 2-3 months (200±20 g) and forty aged female SD rats aged 10-12 months (490±20 g) were selected. Also, based on weight they were divided into a negative control group (NC), three different GEN dose groups, which received GEN of 15, 30, 60 mg/kg, and a positive control group (PC). The experiment lasted 30 days. Concentrations of serum hormones were determined by Enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of StAR, P450scc and CYP19 were determined by Real-Time PCR and western blot techniques. It was observed that 30-60 mg/kg GEN could increase the expression of androgen generating key enzymes in the young rat ovary. GEN also significantly increased progesterone and E2 levels in the serum of aged rats and reduced the levels of LH and FSH in the serum of both young and aged rats. Compared with young rats, the effect of GEN on the ovary of aged rats was stronger and a lower dose of GEN (15 mg/kg) showed an obvious effect on these indicators. GEN influenced both estrogen level and indicators associated with estrogen and androgen transformation processes, which indicates that GEN can impair the growth and maturation of the ovary.


Assuntos
Aromatase/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Genisteína/farmacologia , Ovário/enzimologia , Fosfoproteínas/metabolismo , Androgênios , Animais , Aromatase/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Ovário/efeitos dos fármacos , Fosfoproteínas/genética , Fitoestrógenos/farmacologia , Ratos , Ratos Sprague-Dawley
20.
J Steroid Biochem Mol Biol ; 193: 105421, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31265900

RESUMO

Increasing evidence has shown that one of the major neurosteroids, estradiol, has potent neuroprotective actions. We have reported that estradiol synthesis was enhanced when retinoic acid was added into rat hippocampal slice culture. In this study, we investigated the effects of a potent retinoid X receptor (RXR) agonist, bexarotene, on estrogen synthesis and neuroprotective action in hippocampal slices. Treatment with bexarotene increased estradiol levels as well as estrogen-synthesizing enzymes and CYP19 expression in hippocampal slice cultures. Bexarotene significantly suppressed neuronal cell death induced by oxygen-glucose deprivation (OGD)/reoxygenation. RXR agonists other than bexarotene, such as CD3254, also suppressed neuronal cell death accompanied by OGD/reoxygenation. The RXR antagonists HX531 and UVI3003 and the CYP19 inhibitor letrozole abolished the neuroprotection elicited by bexarotene, indicating that estradiol produced by RXR stimulation protects neurons from ischemic insult. The human brain-specific CYP19 promoter had 6 RXR half sites, and 2 of 6 half sites were responsible for CYP19 expression induced by bexarotene. Bexarotene increased the expression of catalase and glutathione peroxidase 1 and inhibited lipid peroxidation elicited by OGD/reoxygenation, suggesting that the antioxidative property of estrogen contributes to RXR-mediated neuroprotection. Bexarotene also suppressed neuronal injury induced by lipopolysaccharide in the hippocampal slices. Taken together, RXR stimulation can protect neurons via enhanced synthesis of estradiol with antioxidative mechanisms. The RXR-estrogen axis might be a novel mechanism-based strategy to prevent or ameliorate ischemic and/or inflammatory neuronal disorders.


Assuntos
Aromatase/genética , Bexaroteno/farmacologia , Estradiol/metabolismo , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores X Retinoide/agonistas , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Hipocampo/metabolismo , Humanos , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Gravidez , Ratos Wistar , Receptores X Retinoide/genética , Regulação para Cima
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