Xenobiotics interfering with corticosteroid action : from adrenal steroid synthesis to peripheral receptor activity

Corticosteroids are steroid hormones synthesized by the adrenal gland and regulating a variety of physiological processes to maintain whole-body homeostasis by acting through their corresponding receptors. Although the adrenal gland is considered one of the most toxin-vulnerable organs and steroid receptor regulation is recognized to have a considerable impact on tissue- and cell-specific steroid signaling, only few studies are currently exploring and characterizing the effects of xenobiotics on corticosteroid hormone action.
The first part of this thesis aimed to establish optimized steroid profile analysis in cell culture supernatants and apply it in combination with further biological assessments and molecular modeling for the identification and characterization of exogenous chemicals potentially disrupting corticosteroid hormone production.
A widely used in vitro model for studying effects of chemicals on adrenal steroid hormone synthesis constitutes the human H295R adrenocarcinoma cell line. Since the OECD test guideline No. 456 based on H295R cells has several limitations, this thesis refined the H295R steroidogenesis assay by simultaneously analyzing the most important adrenal steroid metabolites using a mass spectrometry-based method. A medium control at the beginning of the experiment as well as reference compounds with known mechanisms were introduced and, additionally, gene expression analyses were performed, in order to not only detect chemical-induced disturbances but also providing initial mechanistic insights into the mode-of-action of a given chemical. The newly established improved version of the H295R steroidogenesis assay was then further evolved by activating the cells either with torcetrapib, a potent inducer of corticosteroid synthesis, or with forskolin, a general inducer of steroidogenesis, allowing to assess the inhibitory potential of various test chemicals.
The modified torcetrapib-stimulated H295R assay was then used to evaluate three selected hits from an in silico screening of environmental chemical databases using ligand-based pharmacophore models of 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). This proof-of-concept for the application of pharmacophore-based virtual screening followed by biological assessment has proven suitable for assessing substances potentially interfering with corticosteroid synthesis.
In another study within this thesis, the adapted version of the H295R steroidogenesis assay using forskolin-stimulated cells was applied to investigate the inhibitory effects of 19 anabolic androgenic steroids (AAS) and 3 selective androgen receptor modulators (SARMs). This enabled to group the test compounds according to their individual steroid patterns. Additionally, gene expression analysis, cell-free activity assays and molecular docking calculations contributed to providing initial mechanistic information.
Besides direct effects on adrenal steroidogenesis, xenobiotic-induced alterations in circulating steroid hormone levels may arise due to altered feedback regulation or disturbed peripheral steroid metabolism. Thus, in a further part of this thesis drug-induced changes in steroid hormone levels were studied by measuring steroid profiles in human blood and urine samples.
In a clinical study, plasma levels of steroid hormones and adrenocorticotropic hormone (ACTH) were analyzed in healthy volunteers administered a single dose of slow-release lisdexamfetamine (100 mg) or immediate-release D-amphetamine (40.3 mg) at equimolar doses. Importantly, lisdexamfetamine and D-amphetamine similarly enhanced the levels of glucocorticoids, androgen precursors and ACTH, suggesting an acute stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Although lisdexamfetamine showed a delayed time of increase and peak levels of plasma D-amphetamine
concentrations compared to the D-amphetamine treatment, drug exposure and drug effects seemed to be comparable between the two formulations.
In a clinical case study, a comprehensive analysis of blood and urinary steroid profiles was conducted in samples from two patients receiving posaconazole, an antifungal agent associated with hypertension and hypokalemia due to mineralocorticoid excess. Steroid analyses indicated interindividual differences in the mechanism of mineralocorticoid-based hypertension with preferential CYP11B inhibition in one patient and predominant inhibition of 11β-HSD2 in the second patient. These results show that steroid profiling in plasma and urine samples can not only reveal disturbances of steroid homeostasis but also provide initial mechanistic information.
Together, these findings emphasize that molecular modeling combined with biological evaluation represents a valuable approach for the identification and characterization of chemicals potentially interfering with corticosteroid production and to provide initial mechanistic insights. However, in vivo investigations are unavoidable to study the impact of chemicals acting on the HPA axis.
Xenobiotics may not only affect steroid hormone production, feedback regulation or pre-receptor control of corticosteroid metabolism, but may also interfere directly with the receptor and steroid signal transduction. In order to understand potential disturbances of glucocorticoid action by xenobiotics, it is important to further clarify the signaling pathways involved in glucocorticoid receptor (GR) activation. Therefore, another part of this thesis focused on the impact of the serine/threonine-specific protein phosphatase PP1α on the activity of the GR. PP1α was found to increase GR activity, and preliminary mechanistic investigations showed that levels of phosphorylated GR-Ser211 were altered and glycogen synthase kinase 3 might be involved. Hence, PP1α appeared to modulate the cellular response to glucocorticoids, implying that impairment of its activity could lead to aberrant glucocorticoid hormone action.
In conclusion, these studies identified a novel GR regulating protein that enhances cortisol stimulation by controlling GR phosphorylation. A profound understanding of glucocorticoid signaling might provide the basis for developing cell models and conditions for the detection of chemicals disturbing glucocorticoid sensitivity and thereby contributing to diseases.