The effects of xenobiotics on steroidogenesis in human: "in vitro" and "in vivo" investigations

Steroid hormones have a pivotal role in many physiological processes. For example, the glucocorticoids are crucial in the regulation and maintenance of sugar balance, immunity, stress response, and mood, whereas the mineralocorticoids are involved in electrolyte- and water balance thus regulating blood pressure. Androgens are crucial for muscle function, cardiovascular system and the development and maintenance of male characteristics. Therefore, the disruption of the steroidogenesis is associated with severe diseases such as cancer, metabolic syndrome, cardiovascular diseases, immune disorders, impaired brain function, and developmental dysfunctions.
In the first part of this thesis, we were interested in the in vitro investigation of xenobiotics affecting the human steroidogenesis. We focused on the adrenal steroidogenesis, which is rather neglected by many regulatory agencies, despite its pivotal role in humans. We provided a critical overview of the current available cell lines used to screen for potential endocrine disruptors and to study their effects on adrenal steroidogenesis. Moreover, we discussed their advantages/disadvantages, and the need for improvements of the well-established human carcinoma cell line H295R and the associated validated OECD test guideline 456, namely the “H295R steroidogenesis assay”. This resulted in a refined version of the H295R steroidogenesis assay, which is distinguished from the currently used OECD protocols by analyzing multiple adrenal steroids simultaneously with exclusive separation techniques combined with mass spectrometry, as well as including additional controls, such as medium composition at the starting time and reference compounds with known mechanism. The obtained results of the steroid changes can then be further combined with the observed effects on gene expression, providing first mechanistic hints on steroidogenesis disruption. By using the newly established refined version of the H295R steroidogenesis assay, we demonstrated that exposure of H295R cells to the UV-filter octyl methoxycinnamate and the plasticizer acetyl tributylcitrate resulted in increased corticosteroid levels, as well as enhanced CYP11B2 expression, similar to the corticosteroid inducer torcetrapib (positive control). To summarize, the refined H295R steroidogenesis assay is a valuable in vitro tool to screen and study chemicals potentially disrupting the production of adrenal steroids and provides initial mechanistic evidence in combination with gene expression data.
Many psychoactive drugs can lead to immense increases in cortisol by stimulating the hypothalamic-pituitary-adrenal (HPA) axis. However, a comprehensive analysis of drug induced changes of several steroids, such as glucocorticoids, mineralocorticoids and adrenal androgens along with their full time courses is missing. In the second part of this thesis, we studied the effects of lysergic acid diethylamide (LSD), which has sparked a renewed interest in psychiatric research, lisdexamfetamine, a new drug for the treatment of attention deficit hyperactivity disorder (ADHD), and D-amphetamine on the circulating steroids in vivo. Plasma samples were obtained from two individual clinical trials, where healthy volunteers were administered a single dose of either LSD (200 µg), lisdexamfetamine dimesylate (100 mg) or immediate-release D-amphetamine sulfate (40.3 mg) at equimolar doses. Both studies were conducted using a randomized, double-blind, placebo-controlled, cross-over design and plasma steroids for the concentration–time profiles were quantified by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). We could demonstrate, that LSD produces significant acute effects on circulating steroids compared to placebo in 16 healthy volunteers. The glucocorticoids cortisol, cortisone, corticosterone and 11-dehydrocorticosterone were significantly increased following LSD administration, indicating HPA axis stimulation. Cortisol and corticosterone reached the maximum concentration (cmax) after 2.5 h and 1.9 h of LSD administration, respectively. Evaluation of the relationship between the LSD concentration in plasma and the glucocorticoid response to LSD indicated no acute pharmacological tolerance. Furthermore, the androgens dehydroepiandrosterone (cmax and the area under the concentration-time curve from time 0 to 10 h (AUC10)) and androstenedione (AUC10) were significantly increased by LSD, but not the other androgens, mineralocorticoids or progestogens compared to placebo.
We showed, that the administration of equivalent doses of lisdexamfetamine and D-amphetamine exhibit an identical pharmacokinetic profile for plasma D-amphetamine. However, lisdexamfetamine administration showed a significantly longer onset time (1.4 vs. 0.8 h) and tmax (4.4 vs. 3.2 h) for plasma D-amphetamine compared to D-amphetamine administration, due to the rate-limiting hydrolysis of lisdexamfetamine. Furthermore, lisdexamfetamine and D-amphetamine showed a similar enhancement of glucocorticoid production (cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, and 11-deoxycortisol), increases in androgen precursors (dehydroepiandrosterone, its sulphated metabolite, and androstenedione) and adrenocorticotropic hormone (ACTH) in plasma in 24 healthy volunteers. This suggests a HPA axis stimulation. Moreover, an acute pharmacological tolerance of the drug-induced change in active glucocorticoids was demonstrated. The other circulating steroids, such as the mineralocorticoids (aldosterone and 11-deoxycorticosteone), androgens (testosterone and androsterone) and progestins (17α-hydroxyprogesterone and progesterone (but not the male progesterone levels)), were not affected by lisdexamfetamine or D-amphetamine.
In conclusion, LSD, lisdexamfetamine and D-amphetamine had an acute and profound effect on the circulating steroids, especially on the glucocorticoids, suggesting HPA stimulation. This emphasizes the need for further research to understand drug induced changes in steroid homeostasis during chronic administration of amphetamine based ADHD treatments, notably in the pediatric population. Obtained results, should then support an appropriate benefit-risk assessment of these drugs.