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

Fürstenberger, Cornelia. Disruption of androgen metabolism, regulation and effects : involvement of steroidogenic enzymes. 2014, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10854

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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.
Advisors:Odermatt, Alex
Committee Members:Eggen, Rik
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molecular and Systems Toxicology (Odermatt)
Item Type:Thesis
Thesis no:10854
Bibsysno:Link to catalogue
Number of Pages:97 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:56
Deposited On:29 Jul 2014 15:15

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