Assessment of the oestrogenic activity of the contraceptive progestin levonorgestrel and its non-phenolic metabolites.

Levonorgestrel (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-gonen-3-one), a potent contraceptive progestin stimulates growth and proliferation of cultured breast cancer cells through a receptor-mediated mechanism, even though levonorgestrel does not bind to the oestrogen receptor (ER). To assess whether the oestrogen-like effects induced by this synthetic progestin are exerted via its metabolic conversion products, we studied the binding affinity of three A-ring levonorgestrel derivatives to the ER and their capability to transactivate an oestrogen-dependent yeast system co-transfected with the human ER gene and oestrogen responsive elements fused to a beta-galactosidase reporter vector. The results demonstrated that the 3beta,5alpha reduced levonorgestrel derivative and to a lesser extent its 3alpha isomer interact with the oestrogen receptor, with a significantly lower relative binding affinity (2.4% and 0.4%, respectively) than that of oestradiol (100%), while levonorgestrel does not. Both levonorgestrel metabolites were able to activate, in a dose-dependent manner, the beta-galactosidase reporter gene in the yeast expression system, an effect that was precluded by a steroidal antioestrogen. The oestrogenic potency of levonorgestrel metabolites was significantly lower (750-fold) than that of oestradiol. Furthermore, high doses of 3beta,5alpha levonorgestrel (2.5 mg/day/6 days) induced an increase of oestrogen-dependent progestin receptor in the anterior pituitary of castrated rats. The overall data offer a plausible explanation for the weak oestrogenic effects induced by high, non-pharmacological doses of levonorgestrel.

Binding of prostaglandins to human PPARgamma: tool assessment and new natural ligands.

The peroxisome proliferator-activated receptors (PPAR) form a family of nuclear receptors with a wide variety of biological roles from adipogenesis to carcinogenesis. More ligands (agonist and antagonist) are needed to explore the multiple functions of PPAR, particularly PPARgamma. In order to complete such ligand screening, a binding test should be assessed versus the classical transactivation reporter gene assay. In the present work, the full-length human PPARgamma protein as well as its ligand binding domain portion were expressed in Escherichia coli. Bacterial membrane preparations expressing those constructs were characterized using a classical binding competition assay [3H]rosiglitazone as the radioligand. When the receptor preparations were soluble, binding had to be measured with a new alternative method. The systems were assessed using a series of reference PPAR (alpha, beta and gamma) ligands. The full-length human PPARgamma fused to glutathione-S-transferase, expressed in E. coli and tested as a bacterial membrane-bound protein led to the most accurate results when compared to the literature. Furthermore, in an attempt to complete the panel of natural PPARgamma ligands, 29 commercially available prostaglandins were screened in the binding assay. Prostaglandins H(1) and H(2) were found to be modest ligands, however as potent as 15Delta(12-14 )prostaglandin J(2). These results were confirmed in the classical transactivation assay. The fact that these three prostaglandins were equally potent, suggests new pathways of PPARgamma-linked gene activation.