Mecanismos alternos de expresión de la actividad estrogénica del gestodeno, una progestina sintética de tercera generación
Gestodene (GSD; 13p-ethyl-I 7a-ethynyl-l7p-hydroxy-4,15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observat...
| Autor: | |
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2002 |
| País: | México |
| Institución: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:p8418n76f |
| Acceso en línea: | https://doi.org/10.24275/uami.p8418n76f |
| Access Level: | acceso abierto |
| Palabra clave: | info:eu-repo/classification/LEM/Progesterona info:eu-repo/classification/LEM/Progesterone info:eu-repo/classification/LEM/Estrógeno info:eu-repo/classification/LEM/Estrogen info:eu-repo/classification/cti/3 |
| Sumario: | Gestodene (GSD; 13p-ethyl-I 7a-ethynyl-l7p-hydroxy-4,15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observations that this contraceptive synthetic progestin exerts estrogen-like effects besides that of its progestational activity, prompted the conduction of the present investigation to understand the underlying mechanisms through which GSD administration induces estrogenic effects, even though this synthetic steroid molecule neither interacts with intracellular estrogen receptors (ER) nor undergoes in vivo aromatization. The research hypothesis behind the rationale and experimental studies of this work establishes that the slight estrogen-like effects observed after GSD administration are exerted by its enzyme-formed A-ring reduced derivatives, at peripheral organs, which specifically bind with a high relative affinity to the active site of the ER, and are able to transactivate estrogen-regulated genes at hormone sensitive tissues. To assess whether GSD, like other 19-nor synthetic progestins, could be biotransformed to non-phenolic metabolites with intrinsic estrogenic potency, at the target organ level, homogenates of castrated female rat anterior pituitaries and hypothalami, as well as castrated male rat ventral prostates were incubated in vitro with different concentrations of [3H]- and [14C]-labeled GSD at different pH, in the presence or absence of the cofactor NADPH. The isolation, purification and identification of GSD metabolites were done by a reverse isotope dilution technique which included thin layer chromatography in, at least, two different systems and repeated crystallizations to obtain constant specific activity. Incubations of rat anterior pituitaries, hypothalami and ventral prostates homogenates with [3H]-testosterone, under identical experimental conditions, were used as positive controls, while tissuefree incubations served as negative controls. The results provided evidence that GSD was extensively metabolized to A-ring reduced derivatives in the steroid hormone-sensitive tissues studied. The formation of radioactive 5a-dihydroGSD (5aGSD), 3a,5a-tetrahydroGSD (3a15aGSD) and 3P15a-tetrahydroGSD (3P15aGSD) was demonstrated, thus indicating that GSD is an adequate substrate for 5a-steroid reductases and 3a- and 3P-hydroxysteroid dehydrogenases. The bioconversion of GSD to 5aGSD in the rat pituitary and hypothalamus occurred only at pH 7.4, whereas a lack of biotransformation of GSD was observed when incubations were performed at pH 4.8, thus indicating the activity of 5a reductase type 1 only. In contrast, 5aGSD was the major product obtained in rat ventral prostate incubations at both pH 7.4 and 4.8, indicating the presence of Sa-steroid reductase type1 and 5asteroid reductase type 2, respectively. In all tissue incubations the activity of 5asteroid reductase was NADPH dependent. The most important finding in these studies was the isolation and identification of 3a15aGSD and 3P15aGSD in the three target organs, which is in line with previous reports indicating that other synthetic 19-nor contraceptive progestins, exhibit a similar metabolic conversion pattern. To investigate whether the estrogen-like effects of GSD are mediated by some of its A-ring reduced metabolites, a series of experimental studies were undertaken using different biochemical and molecular approaches. First of all, the stereospecificity of the binding of GSD and its 5a-reduced metabolites to the intracellular ER was assessed by competition analysis. The relative binding affinity and the inhibition constant of GSD and its derivatives to ER were determined in aliquots of immature rat uterine cytosol, incubated with [3H]-estradiol as radioligand, in the presence of increasing concentrations of radioinert GSD, SaGSD, 3a,5aGSD, 3p,5aGSD and estradiol (Ez). The results confirmed that indeed GSD does not bind to the ER and demonstrated that its tetrahydro 3p,5a derivative and, to a lesser extent, its 3a,5a epimer interacted with a high relative affinity with the ER, though with a significantly lower binding affinity than naturally occurring E*. The ability of increasing concentrations of GSD and its A-ring reduced derivatives to induce estrogenic activities were evaluated in HeLa cells transiently transfected with an expression vector, for either human ERa (hERa) or human ERP (hERp) genes and estrogen responsive elements fused to the chloramphenicol acetyltransferase (CAT) reporter gene. The results demonstrated that 3PGSD and 3aGSD were able to activate, in a dose dependent manner, the hERa-mediated but no the hERP-mediated transcription of the CAT reporter gene in the HeLa cells expression system. The 3p derivative of GSD exhibited a significantly higher estrogenic effect than its 3a isomer, while unchanged GSD and SaGSD were completely ineffective. However, It must be stressed that the potency of the GSD reduced metabolites to transactivate the CAT reporter gene was significantly lower than that of naturally occurring E2 The in vivo estrogenic potency of 3PGSD was also assessed by its capability induce estrogen-dependent progestin receptors (PR) in the anterior pituitary castrated female rats, after administration of three different doses (0.5, 1.0 and 1 to of .5 mg/day) for 6 consecutive days. The characteristics of PR induced by the highest doses of 3P,5aGSD were determined using a sucrose gradient labeling technique. The sedimentation coefficient (7-9 S) of the complexes formed after incubation of aliquots of anterior pituitaries cytosol with I3H]-ORG 2058 were identical to that induced by estradiol benzoate (E2B) used as experimental control. The results showed a significant dose-dependent increase of PR in the anterior pituitaries of castrated rats, as compared to vehicle treated animals, though with significantly lower potency than that observed after administration of E2B. The overall results confirmed the proposed hypothesis that peripheral metabolism of Gestodene modulates its hormone-like effects and pointed out the relevance of the biotransformation of GSD to 3pGSD and its 301 isomeric alcohol, which specifically bind to the ER and possess a weak intrinsic estrogenic activity. The data also contribute to a better understanding of the GSD mechanisms of action and allow to conclude that the slight estrogen-like effects attributable to GSD are indeed mediated by its non phenolic, tetrahydro reduced metabolites. |
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