Disruption of androgen metabolism, regulation and effects : involvement of steroidogenic enzymes

Abstract

Communication between organs and tissues is predominately controlled by hormones. Hormones regulate a vast variety of physiological and behavioural activities, including metabolism, growth and development, reproduction, sleep and mood. Steroid hormones are characterized by their sterane backbone and are regulated by distinct enzymes which control the balance between their active and their inactive forms. The present studies of this thesis focus on the enzymes which selectively control and regulate the availability of active ligand for nuclear receptor binding. The first project described in my thesis investigated the impact of anabolic androgenic steroids (ASS) on the enzyme activity of 11beta-hydroyxsteroid deydrogenase 2 (11beta-HSD2). ASS are known to induce cardiovascular complications. The underlying mechanisms remain largely unknown. Using enzyme activity assays we observed that fluoxymesterone, a widely used ASS, potently inhibited 11beta-HSD2- dependent inactivation of cortisol to cortisone. Furthermore, using LC-MS/MS we could show that fluoxymesterone is metabolized to 11-oxofluoxymesterone by human 11beta-HSD2. Structural modelling revealed that the binding modes for fluoxymesterone and cortisol are similar, suggesting that fluoxymesterone may act as a competitive inhibitor of 11beta-HSD2. No direct modulation of the mineralocorticoid receptor (MR) could be observed in transactivation assays. Since cortisol is able to potently activate the MR, we suggested that fluoxymesterone-induced inhibition of 11beta-HSD2 could contribute to cortisol-induced MR activation, leading to electrolyte dysbalance and elevated blood pressure and subsequent cardiovascular disease development. The inhibitory potential of ASS in rat kidney microsomes and in cells expressing recombinant mouse 11beta-HSD2 revealed a much weaker inhibition, revealing important species differences. This study unveiled potential pathways involved in adverse cardiac outcomes as a result of ASS misuse. It furthermore highlights the importance of species differences, especially within the field of steroidogenesis. The second study presented in my thesis investigated the pathways involved in the generation and metabolism of androgens in Leydig cells. Our investigation in two important Leydig cell lines, the well- established MA-10 cells and the more recently established BLTK-1 cells, showed that there are marked differences regarding androgen metabolism between these two cell lines. Enzyme activity assays showed that 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3) -dependent formation of testosterone from androstenedione is not the predominant pathway in BLTK-1 cells. This observation was supported be the low expression of HSD17B3 mRNA in BLTK-1 cells. We further investigated the specific pathway by which the BLTK-1 cells degrade androstenedione. LC-MS/MS measurements confirmed that BLTK-1 cells predominately reduce androstenedione to androsterone via the intermediate metabolite, 5alpha-androstanedione. This alternative pathway is part of the “back- door” pathway, which ultimately leads to the formation of 5alpha-Dihydrotestosterone and which has not been shown before in an established cell model. Under stress conditions, cells are able to switch pathways from the well-known 17beta-HSD3-mediated androstenedione reduction to testosterone to the back-door pathway. In addition to characterizing the pathways in two different Leydig cell lines, we compared and tested different methodologies to specifically quantify androgen metabolites. Our results emphasize that for complex steroid matrices, LC-MS/MS measurement is the method of choice while enzyme immunoassay need to be evaluated carefully. Tin layer chromatography should only be carried in validated two-dimensional or even in three-dimensional systems. Our study was able to demonstrate that the MA-10 and the BLTLK-1 cells both are valuable models. However, they should be used only for investigation a specific pathway. In the third study presented in my thesis, we investigated the transcriptional regulation of the HSD17B3 promoter. 17beta-HSD3 is the key enzyme for testosterone formation of the front-door pathway. With the ultimate goal to identify compounds interfering with testosterone formation we constructed a MA-10 Leydig cell line stably expressing a 2.8 kilo base sequence of the putative human HSD17B3 promoter under the control of a luciferase reporter gene. Using this tool, we carried out two projects: A) We could show using transactivation assays, that TNF-alpha strongly activates the HSD17B3 promoter via the p38 MAPK pathway. Importantly, this activation could not be reversed by the synthetic glucocorticoid dexamethasone. The results from our novel reporter assay were supported both on the mRNA-level and by enzyme activity measurements. The key conclusion from this study was the identification of a pathway which may link cancer-related inflammation with elevated testosterone levels, subsequently contributing to the growth and progression of androgen dependant tumors. B) The mechanisms of imposex induction in aquatic organisms are still disputed. Using the screening tool described above, we showed that the retinoid X receptor (RXR) ligand 9-cis retinoic acid and specific organotins are able to activate the human HSD17B3 promoter. This finding suggests that organotins exert pro-androgenic effects. We propose in a future study to address a possible link between two established yet controversial theories of imposex onset in aquatic organisms: the involvement of RXR and the elevation of testosterone levels.