The oestrogenic effects of gestodene, a potent contraceptive progestin, are mediated by its A-ring reduced metabolites

Abstract

Gestodene (17 -ethynyl-13 -ethyl-17 -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 observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether gestodene (GSD) administration may induce oestrogenic effects, even though the GSD molecule does not interact with intracellular oestrogen receptors (ER).To assess whether GSD may exert oestrogenic effects through some of its neutral metabolites, a series of experimental studies were undertaken using GSD and three of its A-ring reduced metabolites. Receptor binding studies by displacement analysis confirmed that indeed GSD does not bind to the ER, whereas its 3 ,5 -tetrahydro reduced derivative (3 GSD) interacts with a relative high affinity with the ER. The 3 ,5 GSD isomer (3 GSD) also binds to the ER, though to a lesser extent. The ability of the A-ring reduced GSD derivatives to induce oestrogenic actions was evaluated by the use of two different molecular bioassays: (a) transactivation of a yeast system cotransfected with the human ER (hER ) gene and oestrogen responsive elements fused to the -galactosidase reporter vector and (b) transactivation of the hERmediated transcription of the chloramphenicol acetyl transferase (CAT) reporter gene in a HeLa cells expression system. The oestrogenic potency of 3 GSD was also assessed by its capability to induce oestrogen-dependent progestin receptors (PR) in the anterior pituitary of castrated female rats.The results demonstrated that 3 GSD and 3 GSD were able to activate, in a dose-dependent manner, the hERmediated transcription of both the -galactosidase and the CAT reporter genes in the yeast and HeLa cells expression systems respectively. In both assays the 3 derivative of GSD exhibited a significantly greater oestrogenic effect than its 3 isomer, while unchanged GSD and 5 GSD were completely ineffective. Neither 3 GSD nor 3 GSD exhibited oestrogen synergistic actions. Interestingly, the pure steroidal anti-oestrogen ICI-182,780 diminished the transactivation induced by 3 GSD and 3 GSD in the yeast expression system. Furthermore, administration of 3 GSD resulted in a significant increase of oestrogendependent PR in the anterior pituitaries of castrated rats in comparison with vehicle-treated animals. The characteristics of the 3 GSD-induced PR were identical to those induced by oestradiol benzoate.The overall results demonstrate that 3 GSD and its 3 isomeric alcohol specifically bind to the ER and possess a weak intrinsic oestrogenic activity, whereas unmodified GSD does not. The data contribute to a better understanding of the GSD mechanism of action and allow the hypothesis to be advanced that the slight oestrogenlike effects attributable to GSD are mediated by its non-phenolic, tetrahydro reduced metabolites.

Enhanced formation of non-phenolic androgen metabolites with intrinsic oestrogen-like gene transactivation potency in human breast cancer cells: a distinctive metabolic pattern

“…(Maggiolini et al 1999, García-Becerra et al 2002. These data are similar, though non-identical, with our previous observations with the tetrahydro-reduced metabolites of synthetic 19-nor progestins, which are also able to transactivate oestrogen-dependent genes mediated through hERa, but not through hERb, behaving as selective hER modulators (Lemus et al 2000, Larrea et al Figure 8 Oestrogen-like transactivation effects of androstanediols. Increasing concentrations of 3a,5a-androstanediol (3a,5a-diol), 3b,5a-androstanediol (3b,5a-diol) and oestradiol (E 2 ) were incubated with HeLa cells transiently co-transfected with expression vectors for hERa or hERb and an ERE-E1b-CAT reporter gene, for 24 h, at 37 8C.…”

“…Further interest for the conduction of this study stemmed from the recent observations in our laboratory (Lemus et al 2000, Larrea et al 2001, García-Becerra et al 2002, demonstrating that the A-ring tetrahydro-reduced metabolites of norethisterone, levonorgestrel and gestodene, possess oestrogen-agonistic activities, suggesting that they could be involved in the activation of breast cancer cell proliferation induced by high doses of synthetic contraceptive progestins derived from 19-nor testosterone, as it has been previously reported (Catherino et al 1993, Schoonen et al 1995a,1995b. …”

“…Incubations were carried out in the absence or presence of 1!10 K7 M ICI-182 780 (Zeneca Farma, Mexico City, Mexico), a potent steroidal antioestrogen, in a 95% air:5% CO 2 atmosphere, for 24 h at 37 8C. At the end of the incubation period, cells were harvested and submitted to the liquid CAT assay as previously described (Lemus et al 2000, García-Becerra et al 2002. Results of the transactivation studies are expressed as the effective concentration values (EC 50 ) of 3a,5a-diol, 3b,5a-diol and oestradiol, obtained by a non-linear regression analysis, using a scientific graphic software (Origin 6$1; OriginLab, Northampton, MA, USA).…”

Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells

“…Competition studies Stereospecificity of the binding of NET, 5a-NET, 3a,5a-NET, and 3b,5a-NET to ER, was assessed by competition analysis (Lemus et al 2000), using dexamethasone as control. Cytosol aliquots (400 mg protein/ml) were incubated with 0 .…”

“…Previous studies from our laboratory have demonstrated that synthetic 19-nor progestins are extensively metabolized in target organs to A-ring reduced tetrahydro derivatives (Larrea et al 1987, Lemus et al 1992), which exert estrogen-like effects (Vilchis et al 1986, Moralí et al 1990, Lemus et al 2000.…”