RESUMEN
Mutations of the androgen receptor (AR) gene are the most frequent cause of 46,XY disorders of sex development. They are associated with a variety of phenotypes, ranging from phenotypic women (complete androgen insensitivity syndrome, CAIS) to milder degrees of undervirilization (partial forms) or men with only infertility (mild form). We identified a new W752G AR mutation responsible for a familial case of CAIS and performed an in vitro study and structural analysis of this mutation and the only other reported substitution affecting the same amino acid (W752R). Although sex assignment is not discussed in cases of CAIS, we show how the phenotype-genotype correlation can be refined by in vitro and structural studies according to the nature of the amino acid substitution, which in turn may have interesting impacts on the follow-up of these patients.
RESUMEN
PURPOSE: While familial forms of complex disorders/differences of sex development have been widely reported, data regarding isolated hypospadias are sparse and a family history is thought to be less frequent. We aimed to determine the frequency of hypospadias in families of boys with hypospadias, to establish whether these familial forms exhibit a particular phenotype and to evaluate the prevalence of genetic defects of the main candidate genes. MATERIALS AND METHODS: A total of 395 boys with hypospadias were prospectively screened for a family history with a standardized questionnaire, extensive clinical description, family tree and sequencing of AR, SF1, SRD5A2 and MAMLD1. RESULTS: Family history of hypospadias was more frequent than expected (88 patients, 22.3%). In 17 instances (19.3%) familial hypospadias cases were multiple. Familial hypospadias was related to the paternal side in 59.1% of cases, consisting of the father himself (30.7%) as well as paternal uncles and cousins. Premature birth, assisted reproductive techniques, other congenital abnormalities and growth retardation were not more frequent in familial hypospadias than in sporadic cases. The severity of phenotype was similar in both groups. The results of genetic analysis combined with previous data on androgen receptor sequencing revealed that familial cases more frequently tend to demonstrate genetic defects than sporadic cases (5.68% vs 1.63%, p = 0.048). CONCLUSIONS: Familial forms of hypospadias are far more frequent than previously reported. Even minor and isolated forms justify a full clinical investigation of the family history. Detecting these hereditary forms may help to determine the underlying genetic defects, and may improve followup and counseling of these patients.
Asunto(s)
Predisposición Genética a la Enfermedad/epidemiología , Hipospadias/epidemiología , Hipospadias/genética , Linaje , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Preescolar , Estudios de Seguimiento , Humanos , Incidencia , Lactante , Masculino , Tamizaje Masivo/métodos , Estudios Prospectivos , Receptores Androgénicos/genéticaRESUMEN
OBJECTIVE: Disorders of sex development (DSD) are a heterogeneous group of conditions affecting the differentiation and development of the internal and external genitalia. Here, we aimed at identifying the genetic cause of DSD in two 46,XY sisters from a consanguineous family. DESIGN: We performed a whole-exome sequencing of two 46,XY female individuals. Sanger sequencing was used to validate the most likely candidate variant, affecting the desert hedgehog (DHH) gene. Molecular dynamics simulations were performed to get insights into the impact of the variant on protein structure and on its interaction with the protein partner BOC (brother of CDO/cell adhesion molecule, downregulated by oncogenes). PATIENTS: The index patient presented with a female phenotype, primary amenorrhoea (low oestradiol and testosterone and high FSH and LH). She also had an apparent absence of intra-abdominal gonads and uterus, facial dysmorphy, psychomotor retardation and neuropathy. Her sister displayed a similar gonadal and endocrinological picture, without dysmorphy or psychomotor retardation. RESULTS: Whole-exome sequencing revealed a homozygous variant in DHH leading to the p.Trp173Cys substitution. The relevant Trp residue is conserved, and its alteration was predicted to be deleterious. Molecular dynamics simulations showed that the mutation increases the conformational flexibility of the protein and potentially alters its interaction with BOC, a positive regulator of Hedgehog signalling. We do not exclude an interference of the mutation with DHH-intein-mediated auto-processing. CONCLUSIONS: This report increases the number of described homozygous DHH variants and highlights the importance of advanced bioinformatic tools to better understand the pathogenicity of human variants.
Asunto(s)
Trastorno del Desarrollo Sexual 46,XY/genética , Proteínas Hedgehog/genética , Adulto , Sustitución de Aminoácidos , Salud de la Familia , Femenino , Variación Genética , Homocigoto , Humanos , Simulación de Dinámica Molecular , Linaje , Conformación Proteica , Hermanos , Secuenciación del ExomaRESUMEN
BACKGROUND: Numerous studies have focused on the association between endocrine-disrupting chemicals (EDCs) and hypospadias. Phenotype variability, the absence of representative comparison groups and concomitant genetic testing prevent any definitive conclusions. OBJECTIVE: To identify the role of occupational and environmental exposures to EDCs in nongenetic isolated hypospadias. DESIGN, SETTING, AND PARTICIPANTS: A total of 408 consecutive children with isolated hypospadias and 302 normal boys were prospectively included (2009-2014) in a multi-institutional study in the south of France, the area of the country with the highest prevalence of hypospadias surgery. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: In patients without AR, SRD5A2, and MAMLD1 mutations, parental occupational and professional exposures to EDCs were evaluated based on European questionnaire QLK4-1999-01422 and a validated job-exposure matrix for EDCs. Environmental exposure was estimated using the zip code, the type of surrounding hazards, and distance from these hazards. Multivariate analysis was performed. RESULTS: Fetal exposure to EDCs around the window of genital differentiation was more frequent in the case of hypospadias (40.00% vs 17.55%, odds ratio 3.13, 95% confidence interval 2.11-4.65). The substances were paints/solvents/adhesives (16.0%), detergents (11.0%), pesticides (9.0%), cosmetics (5.6%), and industrial chemicals (4.0%). Jobs with exposure were more frequent in mothers of hypospadiac boys (19.73% vs 10.26%, p=0.0019), especially cleaners, hairdressers, beauticians, and laboratory workers. Paternal job exposure was more frequent in the cases of hypospadias (40.13% vs 27.48%, p=0.02). Industrial areas, incinerators, and waste areas were more frequent within a 3-km radius for mothers of hypospadiac boys (13.29% vs. 6.64%, p<0.00005). Association of occupational and environmental exposures increases this risk. CONCLUSIONS: This multicenter prospective controlled study with a homogeneous cohort of hypospadiac boys without genetic defects strongly suggests that EDCs are a risk factor for hypospadias through occupational and environmental exposure during fetal life. The association of various types of exposures may increase this risk. PATIENT SUMMARY: Our multi-institutional study showed that parental professional, occupational, and environmental exposures to chemical products increase the risk of hypospadias in children.
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Disruptores Endocrinos/efectos adversos , Exposición a Riesgos Ambientales/efectos adversos , Hipospadias/inducido químicamente , Exposición Profesional/efectos adversos , Efectos Tardíos de la Exposición Prenatal , Niño , Preescolar , Femenino , Francia , Humanos , Lactante , Recién Nacido , Masculino , Embarazo , Estudios ProspectivosRESUMEN
The use of complementary and alternative medicine and herbal products, especially traditional Chinese medicines, is progressively rising for both adults and children. This increased use is based on the popular belief that these medicines are safe and harmless. In this report, we describe the results of a bedside-to-bench study that involved a short-statured 4-year-old boy with deficiencies in growth hormone, thyroid stimulating hormone, and adrenocorticotropic hormone due to an ectopic posterior pituitary gland and invisible pituitary stalk. Although the boy was given replacement therapy with hydrocortisone and L-thyroxin, the parents refused to treat him with growth hormone and consulted a naturopath who prescribed a traditional Chinese medicine (TCM) to stimulate the boy's growth. From the age of 20 months, the child's growth was regularly monitored while he was being treated with hydrocortisone, thyroxin, and the TCM. Over a 36-month period, the child's growth velocity accelerated (3 cm/year to 8 cm/year), his height increment substantially increased (-2 SD to -0.8 SD), and his bones matured. In the laboratory investigation, estrogen receptor (ER)alpha and ERbeta reporter cell lines were used to characterize the estrogenic activity of the TCM medicine and its 18 components, and the results established that the medicine and some of its components have estrogen receptor ERalpha and ERbeta selectivity and partial estrogen agonism. Partial estrogenic activity of the TCM was confirmed using whole-cell competitive binding, cell proliferation, and endogenous gene expression assays in the ERalpha-positive breast cancer cell lines. Although the presence of evidence is not always evidence of causality, we have concluded that this traditional Chinese medicine contains ingredients with estrogenic activity that can sustain bone growth and maturation without affecting other estrogen-dependent tissues.
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Medicamentos Herbarios Chinos/uso terapéutico , Receptor alfa de Estrógeno/agonistas , Receptor beta de Estrógeno/agonistas , Trastornos del Crecimiento/tratamiento farmacológico , Fitoestrógenos/uso terapéutico , Preescolar , Agonismo Parcial de Drogas , Medicamentos Herbarios Chinos/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Genes Reporteros/efectos de los fármacos , Trastornos del Crecimiento/metabolismo , Trastornos del Crecimiento/patología , Humanos , Células MCF-7 , Masculino , Osteogénesis/efectos de los fármacos , Fitoestrógenos/farmacología , Resultado del TratamientoRESUMEN
The aim of the work was to investigate the pathophysiology of isolated premature thelarche (IPT) by determining the impact of pre/postnatal exposure to endocrine disrupting chemicals (EDCs) through evaluation of total serum estrogenic bioactivity (EBA). The pathophysiology remains elusive, although recent investigations suggested the role of EDCs in premature female breast development. We investigated 15 girls with IPT. Plasma estradiol, follicle-stimulating hormone, and luteinizing hormone were measured in basal state and after gonadotropin-releasing hormone testing; bone age and uterine length were also assessed for all patients. Total EBA of patient serum was analyzed with an ultrasensitive bioassay that we previously developed and compared with that of 18 age-matched control girls. Parents were interviewed about their environmental/occupational exposure to EDCs during the patient's prenatal/postnatal life. Nine families reported parental occupational/environmental EDCs exposure during prenatal/postnatal patient life; the mean total EBA found in these 9 IPT girls was significantly elevated (12.31 ± 6.64 pg/mL) in comparison with that of the 6 patients without exposure (2.53 ± 0.73 pg/mL) and the 18 age-matched controls (3.53 ± 2.23 pg/mL; p < 0.01). The significant increase in total EBA in these 9 girls with IPT suggests that premature female breast development may be related in some cases to higher pre/postnatal contamination by EDCs.
Asunto(s)
Disruptores Endocrinos/toxicidad , Exposición a Riesgos Ambientales , Estrógenos/sangre , Pubertad Precoz/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Preescolar , Disruptores Endocrinos/sangre , Exposición a Riesgos Ambientales/análisis , Exposición a Riesgos Ambientales/estadística & datos numéricos , Estradiol/sangre , Femenino , Humanos , Lactante , Exposición Profesional/análisis , Exposición Profesional/estadística & datos numéricos , Embarazo , Efectos Tardíos de la Exposición Prenatal/sangre , Efectos Tardíos de la Exposición Prenatal/epidemiología , Pubertad Precoz/epidemiología , Pubertad Precoz/etiología , Contaminantes Químicos del Agua/sangre , Contaminantes Químicos del Agua/toxicidadRESUMEN
OBJECTIVE: Beyond the classic triad of peripheral precocious puberty, café-au-lait skin pigmentation and polyostotic fibrous dysplasia, partial presentation McCune-Albright syndrome (MAS) has been reported, including the association of isolated recurrent ovarian cysts in early infancy. The aims of this study were to determine whether isolated voluminous fetal unilateral ovarian cysts (diameter > 4 cm) may be associated with a Gsα activating mutation, suggestive of MAS. DESIGN: We followed five female fetuses presenting with voluminous unilateral ovarian cysts by ultrasonography until delivery. At birth, all patients underwent percutaneous cyst aspiration and two patients later underwent ovariectomy. A sensitive PCR-based method was used to analyze the Gsα activating mutation in DNA obtained from ovarian cystic fluids or tissue. RESULTS: Among the five cases, one Gsα mutation (R201C) was identified in the ovarian tissue. CONCLUSIONS: We demonstrate for the first time that voluminous fetal unilateral ovarian cysts may be suggestive of MAS. Systematic search for the Gsα mutation should be performed in all newborns with voluminous fetal unilateral ovarian cysts requiring percutaneous cyst aspiration, because early diagnosis of MAS prevents unnecessary oophorectomy to eliminate questions of malignancy and imposes long-term clinical, biological, and imaging follow-up to detect other early manifestations of MAS.
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Enfermedades Fetales/diagnóstico por imagen , Displasia Fibrosa Poliostótica/diagnóstico , Quistes Ováricos/diagnóstico por imagen , Niño , Preescolar , Cromograninas , Análisis Mutacional de ADN , Diagnóstico Diferencial , Diagnóstico Precoz , Femenino , Enfermedades Fetales/diagnóstico , Enfermedades Fetales/etiología , Enfermedades Fetales/genética , Displasia Fibrosa Poliostótica/complicaciones , Displasia Fibrosa Poliostótica/diagnóstico por imagen , Displasia Fibrosa Poliostótica/genética , Estudios de Seguimiento , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Humanos , Lactante , Recién Nacido , Quistes Ováricos/diagnóstico , Quistes Ováricos/etiología , Quistes Ováricos/genética , Embarazo , Ultrasonografía PrenatalRESUMEN
OBJECTIVE: 46,XY disorders of sex differentiation (46,XY DSD) can be due to a testis determination defect, an androgen biosynthesis defect, or androgen resistance (complete or partial androgen insensitivity syndrome (PAIS), or 5α reductase deficiency). We aimed to evaluate the impact of a prenatal contamination by environmental xenoestrogens in 'idiopathic' PAIS-like phenotype. SUBJECTS: We investigated 28 newborn/infant males with 46,XY DSD, normal androgen production, and no androgen receptor or steroid-5αR type II enzyme (SRD5A2) gene mutations. METHODS: To exclude other genetic defects, we sequenced the steroidogenic factor 1 (SF1) and mastermind-like domain-containing 1 (MAMLD1) genes, which were recently found to be associated with the PAIS-like phenotype. Parents were interviewed about their environmental/occupational exposure to endocrine disrupting chemicals (EDCs) before/during the patients' fetal life. Total estrogenic bioactivity of patient serum was analyzed by ultrasensitive bioassay. RESULTS: All the patients had normal SF1 sequence and one patient showed a double polymorphism of MAMLD1. Eleven (39.3%) of the 28 patients had reported parental fetal exposure to EDCs. The mean estrogenic bioactivity in these 11 patients with fetal EDC exposure (6.65 ± 8.07 pg/ml) versus 17 cases without contamination (1.27 ± 0.34 pg/ml) and controls (1.06 ± 0.44 pg/ml; P<0.05) was elevated. CONCLUSIONS: Our results indicate that the 'idiopathic' PAIS-like phenotype may in some cases be related to EDC contamination during fetal life.
Asunto(s)
Síndrome de Resistencia Androgénica/inducido químicamente , Disruptores Endocrinos/efectos adversos , Contaminantes Ambientales/efectos adversos , Efectos Tardíos de la Exposición Prenatal , Bioensayo , Criptorquidismo/inducido químicamente , ADN/genética , Trastorno del Desarrollo Sexual 46,XY/inducido químicamente , Trastorno del Desarrollo Sexual 46,XY/epidemiología , Exposición a Riesgos Ambientales , Estrógenos no Esteroides/sangre , Europa (Continente) , Femenino , Células HeLa , Humanos , Hipospadias/inducido químicamente , Lactante , Recién Nacido , Masculino , Exposición Profesional , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Testosterona/sangreRESUMEN
CONTEXT: In 46,XY disorders of sex development, 5α-reductase deficiency is rare and is not usually the first-intention diagnosis in newborn ambiguous genitalia, contrary to partial androgen insensitivity syndrome. Yet the cause of ambiguous genitalia may guide sex assignment, and rapid, precise diagnosis of 5α-reductase deficiency is essential. OBJECTIVE: The aim of the study was to describe relevant data for clinical diagnosis, biological investigation, and molecular determination from 55 patients with srd5A2 mutations identified in our laboratory over 20 yr to improve early diagnosis. SETTING: The study was performed at Montpellier University Hospital. PATIENTS: We studied a cohort of 55 patients with srd5A2 gene mutations. MAIN OUTCOME MEASURE(S): Genetic analysis of srd5A2 was conducted. RESULTS: Clitoromegaly (49.1%) and microphallus with various degrees of hypospadias (32.7%) were frequent phenotypes. Female external genitalia (7.3%) and isolated micropenis (3.6%) were rare. Seventy-two percent of patients were initially assigned to female gender; five of them (12.5%) switched to male sex in peripuberty. Over 72% of patients were considered for 5α-reductase deficiency diagnosis when the testosterone/dihydrotestosterone cutoff was 10. In 55 patients (with 20 having a history of consanguinity), we identified 33 different mutations. Five have never been reported: p.G32S, p.Y91H, p.G104E, p.F223S, and c.461delT. Homozygous mutations were present in 69.1% of cases, compound heterozygous mutations in 25.5%, and compound heterozygous mutations alone with the V89L polymorphism in 5.4%. Exons 1 and 4 were most affected, with 35.8 and 21.7% mutant alleles per exon, respectively. CONCLUSIONS: In the largest cohort to date, we demonstrate a wide spectrum of phenotypes and biological profiles in patients with 5α-reductase deficiency, whatever their geographical or ethnic origins.
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3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/deficiencia , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Trastornos del Desarrollo Sexual/genética , Proteínas de la Membrana/genética , Adolescente , Alelos , Sustitución de Aminoácidos/genética , Síndrome de Resistencia Androgénica/diagnóstico , Síndrome de Resistencia Androgénica/genética , Niño , Preescolar , Estudios de Cohortes , ADN/genética , Dihidrotestosterona/sangre , Exones/genética , Femenino , Genitales Femeninos/anomalías , Genitales Masculinos/anomalías , Genotipo , Heterocigoto , Humanos , Lactante , Masculino , Mutación , Fenotipo , Polimorfismo Genético/genética , Testosterona/sangre , Adulto JovenRESUMEN
OBJECTIVE: To determine the genetic cause of primary amenorrhea. DESIGN: Case series. SETTING: Pediatric endocrinology, endocrinology, and gynecology departments of academic hospitals. PATIENT(S): Three adolescents and one young woman 46, XY patients with srd5A2 gene mutations. MAIN OUTCOME MEASURE(S): Genetic analysis of srd5A2. RESULT(S): We report four srd5A2 gene mutations in three adolescents and one young woman with 46,XY primary amenorrhea. All presented clitoromegaly and two presented hypospadias; all had been reared as females. Virilization of the external genitalia was noted in the pubertal period in all four patients. Three were maintained in the female sex of rearing by personal choice, and the fourth switched gender. We identified the homozygous substitutions p.L55Q (exon 1), p.Q56R (exon 1), and p.N193S (exon 4), in patients 1, 2, and 3, respectively. Patient 4 had compound heterozygous mutations, a new c.34delG (exon 1) associated with p.R246W (exon 5). All patients had high plasma T levels (ranges, 16.2-23.2 nmol/L; normal female teenage range, 0.35-2 nmol/L). CONCLUSION(S): Our data clearly demonstrate that 5α-reductase deficiency should be considered in XY adolescents with primary amenorrhea and no breast development associated with virilization at puberty and high plasma T. Positive parental consanguinity should reinforce the diagnostic orientation.
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3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Amenorrea/diagnóstico , Amenorrea/genética , Proteínas de la Membrana/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/deficiencia , Adolescente , Amenorrea/etnología , Amenorrea/etiología , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Disgenesia Gonadal 46 XY/genética , Humanos , Proteínas de la Membrana/deficiencia , Técnicas de Diagnóstico Molecular , Adulto JovenRESUMEN
BACKGROUND: Primary amenorrhea due to 46,XY disorders of sex differentiation (DSD) is a frequent reason for consultation in endocrine and gynecology clinics. Among the genetic causes of low-testosterone primary amenorrhea due to 46,XY DSD, SRY gene is reported to be frequently involved, but other genes, such as SF1 and WT1, have never been studied for their prevalence. METHODS: We directly sequenced SRY, SF1 and WT1 genes in 15 adolescent girls with primary amenorrhea, low testosterone concentration, and XY karyotype, to determine the prevalence of mutations. We also analyzed the LH receptor gene in patients with high LH and normal FSH concentrations. RESULTS: Among the 15 adolescents with primary amenorrhea and low testosterone concentration, we identified two new SRY mutations, five new SF1 mutations and one new LH receptor gene mutation. Our study confirms the 10-15% prevalence of SRY mutations and shows the high prevalence (33%) of SF1 abnormalities in primary amenorrhea due to 46,XY DSD with low plasma testosterone concentration. CONCLUSIONS: The genetic analysis of low-testosterone primary amenorrhea is complex as several factors may be involved. This work underlines the need to systematically analyze the SF1 sequence in girls with primary amenorrhea due to 46,XY DSD and low testosterone, as well as in newborns with 46,XY DSD.
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Amenorrea/genética , Disgenesia Gonadal 46 XY/genética , Mutación Missense , Factor Esteroidogénico 1/genética , Testosterona/sangre , Adolescente , Amenorrea/sangre , Amenorrea/complicaciones , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Hormona Folículo Estimulante/sangre , Disgenesia Gonadal 46 XY/sangre , Disgenesia Gonadal 46 XY/complicaciones , Humanos , Hormona Luteinizante/sangre , Modelos Biológicos , Mutación Missense/fisiología , Concentración Osmolar , Proteína de la Región Y Determinante del Sexo/genéticaRESUMEN
Cushing's syndrome (CS) may develop at any time in childhood. In the neonatal period, the major cause is the overactivation of adrenal cells within the context of McCune-Albright syndrome (MAS). The hypercorticism usually appears with other clinical signs of MAS. We report here a case of isolated neonatal CS as the initial evidence of MAS. This newborn girl was referred to our pediatric endocrine unit at the age of 3 months for hypotonia and growth retardation. Clinical examination revealed facial plethora, moon face, and swollen limbs. Laboratory data demonstrated ACTH-independent CS. Magnetic resonance imaging showed moderate bilateral adrenal hyperplasia, though more marked in the left adrenal gland, without nodules. This peripheral hypercorticism without well-defined adrenal tumor was suggestive of MAS, although no other signs like precocious puberty or café-au-lait spots were found. An activating Gsalpha gene mutation was found on DNA extracted from blood. Because MAS is a somatic disease, usually with unilateral effects, we tried to remove only the larger adrenal gland, where the mutation was demonstrated. This female newborn later developed the classical triad of MAS, reinforcing this diagnosis. Cushing's syndrome in infants, even when isolated, may suggest a diagnosis of MAS.
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Síndrome de Cushing/diagnóstico , Displasia Fibrosa Poliostótica/diagnóstico , Secuencia de Bases , Síndrome de Cushing/complicaciones , Síndrome de Cushing/genética , Análisis Mutacional de ADN , Diagnóstico Diferencial , Femenino , Displasia Fibrosa Poliostótica/complicaciones , Displasia Fibrosa Poliostótica/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Trastornos del Crecimiento/diagnóstico , Trastornos del Crecimiento/etiología , Trastornos del Crecimiento/genética , Humanos , Lactante , Recién Nacido , Tamizaje NeonatalRESUMEN
This work was undertaken (i) to study deeply the estrogen, androgen and progestative activities of tibolone and its metabolites (ii) to determine whether tibolone and its metabolites present glucocorticoid or mineralocorticoid activity. For this purpose, we used human cell lines bearing a luciferase gene with a responsive element under the control of human estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) or androgen receptor (AR) or chimeric Gal4 fusion with progesterone receptor (PR), glucocorticoid receptor (GR) or mineralocorticoid receptor (MR). The major tibolone metabolites, the two hydroxymetabolites, bind and activate ER with a preference for ERalpha. Tibolone and the Delta(4)-tibolone are agonists for AR and PR and surprisingly 3alpha- and 3beta-OH-tibolone are antagonists for them. Moreover we showed for the first time that tibolone and its primary metabolites are GR and MR antagonists with a stronger affinity for MR than for GR. In conclusion, tibolone by these actions on different receptors and by this capacity to transform in different metabolites, has more complex effects than initially supposed.
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Moduladores de los Receptores de Estrógeno/farmacología , Norpregnenos/farmacología , Receptores de Esteroides/metabolismo , Andrógenos , Línea Celular , Moduladores de los Receptores de Estrógeno/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Norpregnanos/metabolismo , Norpregnenos/metabolismo , Receptores Androgénicos/metabolismo , Receptores de Progesterona/agonistas , Receptores de Progesterona/metabolismo , Receptores de Esteroides/genéticaRESUMEN
Estrogens control transcriptional responses through binding to two different nuclear receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). Since these two ER subtypes are thought to mediate different biological effects, there is intense interest in designing subtype-selective ER ligands. In this study, we evaluated the ERalpha and ERbeta selectivity of 19 known estrogens and antiestrogens using reporter cell lines previously developed in our laboratory. The HELN-ERalpha and HELN-ERbeta cells stably express full-length ERalpha and ERbeta, respectively, and are derived from HELN cells (HeLa cells stably transfected with an ERE-driven luciferase plasmid). We report that 16alpha-LE2, PPT and 3beta,5alpha-GSD have a high ERalpha-selective agonist potency while 8beta-VE2, DPN, genistein and biochanin A show ERbeta selectivity with 8beta-VE2 being the most potent and selective ERbeta agonist. We also tested ER antagonists and we showed that raloxifene and RU486 are ERalpha and ERbeta-selective antiestrogens, respectively. In all cases, selectivity is due to differences in binding affinities as indicated by whole-cell ligand-binding assays. Very interestingly, we demonstrate that a combination of genistein and raloxifene produces a full-ERbeta specific response. Together these results demonstrate the usefulness of our stably transfected cell lines to characterize ER ligands and indicate that treatments combining agonist/antagonist ligands produce full-ERbeta selectivity.
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Diseño de Fármacos , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Células HeLa/metabolismo , Sitios de Unión , Línea Celular , Relación Dosis-Respuesta a Droga , Antagonistas de Estrógenos/clasificación , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Estrógenos/clasificación , Genes Reporteros , Células HeLa/efectos de los fármacos , Humanos , Ligandos , Luciferasas/genética , Luciferasas/metabolismo , Clorhidrato de Raloxifeno/farmacología , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
In the past 15 years, anomalies of male sexual differentiation have greatly increased in both wildlife and humans in different parts of the world. Environmental endocrine disruptors have been implicated in the dramatic rise in neonatal ambiguous genitalia with variable rates of severity, such as micropenis, cryptorchidism, and isolated or associated hypospadias. Because most environmental pollutants, such as organochlorine pesticides, polychlorinated biphenyls, dioxins and furans, alkylphenol polyetholyethoxylates, and phytoestrogens and phtalates, have estrogenic and antiandrogenic activity, they are able to interfere with normal fetal male sexual differentiation. In a neonatal screening program of ambiguous genitalia, we had the opportunity to evaluate three newborns with male pseudohermaphroditism (MPH) whose mothers were exposed to endocrine disruptors during pregnancy. All had normal testosterone production after human chorionic gonadotrophin stimulation testing, suggesting androgen resistance or so-called idiopathic MPH. Sequences of the 5alpha reductase and androgen receptor genes were normal. Since environmental pollutants are known for their estrogenic activity and can be released progressively from the adipose tissue where they accumulate, we detected their presence by measuring the estrogenic bioactivity of the newborns' serum with a recently developed ultrasensitive bioassay. We found higher estrogenic bioactivity in these newborns than in controls. In conclusion, the maternal exposure to environmental pollutants during pregnancy and high estrogenic bioactivity in the newborns' serum highly suggest that ambiguous genitalia are related to fetal exposure to endocrine disruptors.
Asunto(s)
Trastornos del Desarrollo Sexual/etiología , Disruptores Endocrinos/toxicidad , Estrógenos/sangre , Exposición Materna , Agricultura , Colestenona 5 alfa-Reductasa/genética , Colestenona 5 alfa-Reductasa/metabolismo , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/metabolismo , Femenino , Genes sry/genética , Células HeLa , Humanos , Lactante , Recién Nacido , Masculino , Plaguicidas/toxicidad , Embarazo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Análisis de Secuencia de ADNRESUMEN
Liver receptor homolog-1 (LRH-1) is a nuclear receptor previously known to have distinct functions during mouse development and essential roles in cholesterol homeostasis. Recently, a new role for LRH-1 has been discovered in tumor progression, giving LRH-1 potential transforming functions. In order to identify critical factors stimulating LRH-1 expression leading to deregulated cellular proliferation, we studied its expression and its regulation in several breast cancer cell lines. We observed that LRH-1 expression was increased in estrogen receptor (ER) alpha expressing cell lines, whereas weak-to-no expression was found in nonexpressing ERalpha cell lines. In MCF7, LRH-1 expression was highly induced after treatment with 17beta-estradiol (E2). This transcriptional regulation was the result of a direct binding of the ER to the LRH-1 promoter, as demonstrated by gelshift and chromatin immunoprecipitation assays. Interestingly, siRNA-mediated inactivation of LRH-1 decreased the E2-dependent proliferation of MCF7 cells. Finally, LRH-1 protein expression was detected by immunohistochemistry in tumor cells of human mammary ductal carcinomas. Altogether, these data demonstrate that LRH-1 is transcriptionally regulated by the ER alpha and reinforce the hypothesis that LRH-1 could exert potential oncogenic effects during breast cancer formation.
Asunto(s)
Proteínas de Unión al ADN/genética , Receptor alfa de Estrógeno/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética , Sitios de Unión , Neoplasias de la Mama , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genéticaRESUMEN
Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose-response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4'-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered.
Asunto(s)
Antagonistas de Estrógenos/análisis , Estrógenos/farmacología , Genes Reporteros/genética , Mediciones Luminiscentes/métodos , Animales , Línea Celular , Estradiol/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Luciferina de Luciérnaga/farmacología , Luciferasas/análisis , Luciferasas/biosíntesis , Masculino , Ratones , Ratones Desnudos , Transfección , Trasplante HeterólogoRESUMEN
We previously demonstrated the interactions of different chemical compounds with estrogen receptors ERalpha and ERbeta and the androgen receptor (AR) using different reporter cell lines. In this study, we characterize the ERalpha, ERbeta and AR activity of different biphenyls using the same tools. We provide evidence that several phenyl derivatives present both estrogenic and antiandrogenic activity. The extent of hydroxylation and the position of the hydroxyl function were important in determining their estrogenicity and antiandrogenicity. Of the tested compounds, bisphenol-A and 4,4' biphenol had very high estrogenic activity, although it was lower than that of the strong estrogenic alkylphenol, 4-tert-octylphenol. Bisphenol-A and 4,4' biphenol were able to activate ERs at concentrations lower than 1 microM, whereas the other compounds only activated at concentrations above 1 microM. Interestingly, 4,4' biphenol was a better agonist for ERbeta than for ERalpha. No androgenic activity was detected for any of these compounds. Bisphenol-A, 3-OH phenylphenol, 4-OH phenylphenol and 4,4' biphenol exhibited antiandrogenic activity close to that of 4-tert-octylphenol (IC(50) approximately 5 microM). In whole cell binding assays, these compounds displaced [3H] R1881 with Ki = 10 microM. Although these Ki values seem high in comparison with that of hydroxyflutamide (0.4 microM), one must keep in mind that environmental chemicals can accumulate in adipose tissues for several years. In conclusion, these environmental chemicals may have a negative impact on androgen action during fetal and post-natal life.
Asunto(s)
Antagonistas de Receptores Androgénicos , Estrógenos no Esteroides/farmacología , Fenoles/farmacología , Receptores de Estrógenos/agonistas , Compuestos de Bencidrilo , Compuestos de Bifenilo/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/farmacología , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Genes Reporteros , Humanos , Fenoles/química , Ensayo de Unión Radioligante , Receptores Androgénicos/genética , Receptores de Estrógenos/genética , TransfecciónRESUMEN
The evaluation of estrogenic status is necessary for many physiological and pathological conditions in pediatric as well as adult endocrinology. Because current immunoassays exclusively measure E2--and with a sensitivity that is insufficient for prepubertal children--we developed a new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity. This assay is based on human uterine cervix carcinoma cells, HeLa cells, that do not naturally express E2 receptor. These cells were transfected with plasmids encoding the human ERalpha or beta, along with an estrogen-responsive promoter fused to the luciferase gene, and called HELNalpha and HELNbeta for HeLa estrogen-responsive element luciferase neomycin alpha and beta. HELNalpha and HELNbeta are able to respond to estrogens and various compounds having estrogenic activity but, because of the importance of ERalpha in the reproductive function, we chose to work with the HELNalpha cell line. The luciferase activity we obtained was compared with an E2 standard curve specific for each serum sample and established with stripped serum. The estrogenic bioactivity was expressed in picograms of E2 equivalents, and the detection limit was < 1 pg x ml(-1) E2 equivalents. The intra and interassay error was lower than 10% and 20%, respectively. We measured estrogenic bioactivity in 18 normal prepubertal boys (age = 9.7 +/- 2.4 yr), 18 normal prepubertal girls (age = 9.2 +/- 1.7 yr) and 18 normal pubertal girls (age = 13.6 +/- 1.8 yr). The estrogenic bioactivity in the prepubertal girls was significantly higher than in the boys, i.e. 3.53 +/- 2.23 pg x ml(-1) vs. 1.44 +/- 0.87 pg x ml(-1) (P < 0.01). A significant difference was found between the pre- and pubertal girls, i.e. 3.53 +/- 2.23 pg x ml(-1) vs. 26.77 +/- 18.32 pg x ml(-1) (P < 0.01). This ultrasensitive bioassay measures total estrogenic bioactivity of serum with very high sensitivity. It has numerous potential applications in pediatric and adult endocrinology. In addition, this assay may help to evaluate excess estrogenic activity related to aromatase overexpression or contamination by environmental chemicals.
Asunto(s)
Bioensayo/métodos , Estrógenos/sangre , Recombinación Genética , Niño , Receptor alfa de Estrógeno , Femenino , Células HeLa , Humanos , Luciferasas/metabolismo , Masculino , Pubertad/sangre , Receptores de Estrógenos , Sensibilidad y Especificidad , Caracteres Sexuales , TransfecciónRESUMEN
Besides the measurement of circulating conjugated metabolites of dihydrotestosterone (DHT), which reflects androgenic activity, only one assay to measure androgenic bioactivity in human serum has been proposed thus far. This recombinant bioassay is based on the androgen-dependent interaction between the LBD and NT domains of AR fused to the Gal 4 DNA-binding domain, but its construction is highly complex. We have developed a mammalian cell (CHO 515) bioassay that measures total androgen bioactivity in human serum. The AR-deficient Chinese hamster ovary (CHO) cells were stably transfected with pSG5-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, five highly inducible clones were isolated and one was selected. The expression of human androgen receptor (hAR) was confirmed by Western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after 24 h of incubation with increasing concentrations of various androgenic and non-androgenic steroid compounds in a 96-well plate. The EC50s for each tested androgenic steroid compound were 4 x 10(-11) M, 1.5 x 10(-11) M, 1 x 10(-9) M, 2 x 10(-10) M, 3 x 10(-10) M for testosterone, DHT, dehydroepiandrosterone (DHEA), delta5-androstenediol, and delta4-androstenedione, respectively. In the physiological concentrations of the non-androgenic steroids, estradiol, cortisol, aldosterone, and progesterone, no interference was noted with the AR transactivation level. Evaluation of androgenic bioactivity in human serum was performed by incubation of CHO 515 cells with 100 microl of patient serum, diluted at 1/100 = 1% in DMEM-F12 without phenol red. The sensitivity of the assay was < 0.3 ng/ml. The mean androgenic bioactivity expressed in testosterone equivalents was 0.6 +/- 0.2 ng/ml in normal prepubertal boys, and 12.4 +/- 2 and 1.7 +/- 0.1 ng/ml in normal pubertal boys and girls, respectively. In conclusion, this new recombinant cell bioassay is today the only assay that takes into account testosterone, DHT, DHEA, delta5-androstenediol, and delta4-androstenedione. It should be of particular use in male children with cryptorchidism, delayed puberty or hypogonadotrophic hypogonadism, i.e., in pediatric patients with low androgen levels.