The Anabolic Androgenic Steroid Fluoxymesterone Inhibits 11β-Hydroxysteroid Dehydrogenase 2-Dependent Glucocorticoid Inactivation

Anabolic androgenic steroids (AAS) are testosterone derivatives used either clinically, in elite sports, or for body shaping with the goal to increase muscle size and strength. Clinically developed compounds and nonclinically tested designer steroids often marketed as food supplements are widely used. Despite the considerable evidence for various adverse effects of AAS use, the underlying molecular mechanisms are insufficiently understood. Here, we investigated whether some AAS, as a result of a lack of target selectivity, might inhibit 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2)-dependent inactivation of glucocorticoids. Using recombinant human 11β-HSD2, we observed inhibitory effects for several AAS. Whereas oxymetholone, oxymesterone, danazol, and testosterone showed medium inhibitory potential, fluoxymesterone was a potent inhibitor of human 11β-HSD2 (half-maximal inhibitory concentration [IC50] of 60-100nM in cell lysates; IC50 of 160nM in intact SW-620, and 530nM in MCF-7 cells). Measurements with rat kidney microsomes and lysates of cells expressing recombinant mouse 11β-HSD2 revealed much weaker inhibition by the AAS tested, indicating that the adverse effects of AAS-dependent 11β-HSD2 inhibition cannot be investigated in rats and mice. Furthermore, we provide evidence that fluoxymesterone is metabolized to 11-oxofluoxymesterone by human 11β-HSD2. Structural modeling revealed similar binding modes for fluoxymesterone and cortisol, supporting a competitive mode of inhibition of 11β-HSD2-dependent cortisol oxidation by this AAS. No direct modulation of mineralocorticoid receptor (MR) function was observed. Thus, 11β-HSD2 inhibition by fluoxymesterone may cause cortisol-induced MR activation, thereby leading to electrolyte disturbances and contributing to the development of hypertension and cardiovascular diseas

Currently available murine Leydig cell lines can be applied to study early steps of steroidogenesis but not testosterone synthesis

Androgen biosynthesis in males occurs to a large extent in testicular Leydig cells. This study focused on the evaluation of three murine Leydig cell lines as potential screening tool to test xenobiotics interfering with gonadal androgen synthesis. The final step of testosterone (T) production in Leydig cells is catalyzed by the enzyme 17β-hydroxysteroid dehydrogenase 3 (17β-hsd3). The endogenous 17β-hsd3 mRNA expression and Δ4-androstene-3,17-dione (AD) to T conversion were determined in the murine cell lines MA-10, BLTK1 and TM3. Additionally, effects of 8-Br-cAMP and forskolin stimulation on steroidogenesis and T production were analyzed. Steroids were quantified in supernatants of cells using liquid chromatography-tandem mass spectrometry. Unstimulated cells incubated with AD produced only very low T but substantial amounts of the inactive androsterone. Stimulated cells produced low amounts of T, moderate amounts of AD, but high amounts of progesterone. Gene expression analyses revealed barely detectable 17β-hsd3 levels, absence of 17β-hsd5 (Akr1c6), but substantial 17β-hsd1 expression in all three cell lines. Thus, MA-10, BLTK1 and TM3 cells are not suitable to study the expression and activity of the gonadal T synthesizing enzyme 17β-hsd3. The low T production reported in stimulated MA-10 cells are likely a result of the expression of 17β-hsd1. This study substantiates that the investigated Leydig cell lines MA-10, BLTK1, and TM3 are not suitable to study gonadal androgen biosynthesis due to altered steroidogenic pathways. Furthermore, this study emphasizes the necessity of mass spectrometry-based steroid quantification in experiments using steroidogenic cells such as Leydig cells

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

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

Steroidomic Footprinting Based on Ultra-High Performance Liquid Chromatography Coupled with Qualitative and Quantitative High-Resolution Mass Spectrometry for the Evaluation of Endocrine Disrupting Chemicals in H295R Cells

The screening of endocrine disrupting chemicals (EDCs) that may alter steroidogenesis represents a highly important field mainly due to the numerous pathologies, such as cancer, diabetes, obesity, osteoporosis, and infertility that have been related to impaired steroid-mediated regulation. The adrenal H295R cell model has been validated to study steroidogenesis by the Organization for Economic Co-operation and Development (OECD) guideline. However, this guideline focuses solely on testosterone and estradiol monitoring, hormones not typically produced by the adrenals, hence limiting possible in-depth mechanistic investigations. The present work proposes an untargeted steroidomic footprinting workflow based on ultra-high pressure liquid chromatography (UHPLC) coupled to high-resolution MS for the screening and mechanistic investigations of EDCs in H295R cell supernatants. A suspected EDC, triclocarban (TCC), used in detergents, cosmetics, and personal care products, was selected to demonstrate the efficiency of the reported methodology, allowing the simultaneous assessment of a steroidomic footprint and quantification of a selected subset of steroids in a single analysis. The effects of exposure to increasing TCC concentrations were assessed, and the selection of features with database matching followed by multivariate analysis has led to the selection of the most salient affected steroids. Using correlation analysis, 11 steroids were associated with a high, 18 with a medium, and 8 with a relatively low sensitivity behavior to TCC. Among the candidates, 13 identified steroids were simultaneously quantified, leading to the evaluation and localization of the disruption of steroidogenesis caused by TCC upstream of the formation of pregnenolone. The remaining candidates could be associated with a specific steroid class (progestogens and corticosteroids, or androgens) and represent a specific footprint of steroidogenesis disruption by TCC. This strategy was devised to be compatible with medium/high-throughput screening and could be useful for the mechanistic elucidation of EDCs

A Novel Aspartic Proteinase-Like Gene Expressed in Stratified Epithelia and Squamous Cell Carcinoma of the Skin

Homeostasis of stratified epithelia, such as the epidermis of the skin, is a sophisticated process that represents a tightly controlled balance between proliferation and differentiation. Alterations of this balance are associated with common human diseases including cancer. Here, we report the cloning of a novel cDNA sequence, from mouse back skin, that is induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and codes for a hitherto unknown aspartic proteinase-like protein (Taps). Taps represents a potential AP-1 target gene because TPA-induced expression in epidermal keratinocytes critically depends on c-Fos, and co-treatment with dexamethasone, a potent inhibitor of AP-1-mediated gene regulation, resulted in impaired activation of Taps expression. Taps mRNA and protein are restricted to stratified epithelia in mouse embryos and adult tissues, implicating a crucial role for this aspartic proteinase-like gene in differentiation and homeostasis of multilayered epithelia. During chemically induced carcinogenesis, transient elevation of Taps mRNA and protein levels was detected in benign skin tumors. However, its expression is negatively associated with dedifferentiation and malignant progression in squamous cell carcinomas of the skin. Similar expression was observed in squamous skin tumors of patients, suggesting that detection of Taps levels represents a novel strategy to discriminate the progression state of squamous skin cancers