Identification and characterization of substances interfering with steroid metabolizing enzymes and retinoic acid-related orphan receptor γt activity

Steroid hormones regulate a wide range of physiological processes by activating nuclear receptors, which then act as transcription factors to control the expression of their target genes. Toxicological safety assessments of chemicals to which humans may be exposed also include a section on endocrine assessments, which often focus on direct effects on the nuclear receptors of the sex steroid hormones, namely the androgen and the estrogen receptors. The effects of chemicals on pre receptor control, which includes the biosynthesis of the steroid hormones themselves, as well as on other nuclear receptors such as the immunomodulating retinoic acid related orphan receptor γt (RORγt) activity have been investigated insufficiently so far. This thesis aims to identify and characterize substances that interfere with steroid metabolizing enzymes and RORγt activity. We have addressed this goal in three different projects, which are focusing on different areas of endocrinology.
The steroidogenic enzyme 11β hydroxysteroid dehydrogenase type 2 (HSD11B2), is involved in the pre receptor control of the mineralocorticoid receptor (MR) by inactivating 11 hydroxylated glucocorticoids, that can bind and activate the MR. This ensures the receptors specificity for its natural ligand, aldosterone. Inhibition of HSD11B2 can lead to pseudohyperaldosteronism, an effect that has also been described in clinical case studies with patients treated with the azole antifungals itraconazole and posaconazole. In the first project, we assessed species specific susceptibility towards azole fungicide dependent HSD11B2 inhibition. The results of this investigation provided a possible explanation, why this adverse drug effect was missed during clinical trials. The human HSD11B2 homolog was strongly inhibited by both azole fungicides, while the rat enzyme was only moderately inhibited. Mouse and zebrafish homologs were only weakly inhibited. Using predictions based on homology modeling of HSD11B2 and analysis of chimeric enzyme variants, we were able to identify the C terminal region and the amino acid residues at positions 170 and 172 as relevant structural elements that are partly responsible for the species specific differences between the mouse and human homologs. This study highlights that such species specific differences in the inhibition of steroidogenic enzymes by chemicals may be relevant for toxicological investigations and should therefore be considered in future studies. As an extension of this project, we summarized the methods used for the analysis of the enzymatic activity of HSD11B2 together with further assessment possibilities for the in vitro enzyme activity determination of HSD11B2 and 11β hydroxysteroid dehydrogenase type 1 (HSD11B1) in two chapters for the book series 'Methods in Enzymology'. HSD11B1 catalyzes the reverse reaction of HSD11B2, reducing 11 keto glucocorticoids to their active 11β hydroxy forms.
Parabens and UV filters are used as additives in body care products and cosmetics to increase their shelf life. These compounds have been measured in various human matrices, including fetal samples, and are known to have antiandrogenic effects by blocking androgen receptor (AR) activity. To date, the influence of these chemicals on the pre receptor control of AR, specifically the androgen biosynthesis, is poorly understood. 3α hydroxysteroid dehydrogenases (3α HSD) are steroidogenic enzymes that can catalyze one of the last two synthesis steps in the backdoor pathway of the most potent androgen, 5α dihydrotestosterone (DHT). Androgen mediated AR activation is required for normal male genitalia formation during embryogenesis.
In the second project, we investigated the effects of parabens and UV filters on the enzymatic activity of different 3α HSDs that can produce DHT in the backdoor pathway using a novel, radiometric enzyme activity assay. We have identified several parabens and benzophenone type UV filters as the first inhibitors of the 3α HSD 17β hydroxysteroid dehydrogenase type 6 (HSD17B6) with IC50 values in the mid and high nanomolar range. Said identified inhibitors were analyzed for their structure-activity relationship using a novel HSD17B6 homology model, which highlighted the importance of the 4-hydroxylated phenyl head group present in both substance classes. The addition of a methyl group to the 4-hydroxy group resulted in a loss of inhibitory capacity in 4-methoxylated benzophenones, as it prevented the formation of a hydrogen bond with the amide group of the cofactor nicotinamide adenine dinucleotide (NAD+) in the binding pocket of the homology model.
Parabens and UV filters, like many other chemicals, have so far mainly been investigated for their direct effects on the androgen and estrogen receptors. RORγt is involved in immune response regulation and is essential for the differentiation of T helper 17 cells and their expression of pro inflammatory interleukins. Excessive activation of RORγt has been associated with inflammatory and autoimmune diseases such as psoriasis.
In the third project, we evaluated the effect of parabens and UV filters on RORγt activity using a previously established tetracycline inducible reporter gene assay in Chinese hamster ovary cells. We identified hexylparaben, benzylparaben and benzophenone 10 as potent RORγt agonists with EC50 values within the higher nanomolar and lower micromolar range. Those RORγt agonists were also able to enhance pro inflammatory cytokine expression in a mouse EL4 T lymphocyte model. Together with structurally similar chemicals which we identified by virtual screening of a cosmetics database, the chemicals identified as RORγt agonists in this study showed additive effects on the receptor activity when assessed as mixtures.
Additional experiments are required to determine whether parabens and UV filters can reach the concentrations described in the second and third projects to exert potential antiandrogenic effects in organs expressing HSD17B6, or possibly aggravate existing inflammatory and autoimmune diseases through the additional RORγt activation.
As an additional, fourth project, we examined three case studies of Tunisian patients diagnosed with 17β hydroxysteroid dehydrogenase type 3 (HSD17B3) deficiencies for their underlying molecular causes. HSD17B3 is a steroidogenic enzyme that is exclusively expressed in the testes and catalyzes the last enzymatic step of testosterone synthesis. Testosterone, a potent androgen, is essential for normal male sexual development. Pathogenic mutations in the HSD17B3 gene can lead to undervirilization of the male sexual organs due to insufficient testosterone production during embryogenesis, which is why HSD17B3 deficiencies are classified as 46,XY disorders of sexual development. Genetic analysis of the three patients revealed in one patient the first homozygous mutation in the catalytic tetrad of HSD17B3, p.K202M, for which we were able to show a complete loss of function using a radiometric enzyme activity assay. The second patient was a compound heterozygote with a paternally inherited, already characterized, inactive truncation mutation p.C206X and a maternally inherited splice site mutation (c.490 6 T > C) for which we could show by means of a splicing assay that the mutation causes skipping of exon 7 during mRNA splicing which presumably results in a truncated and inactive enzyme. The last patient turned out to have a homozygous p.C206X mutation of HSD17B3. Consanguineous marriages can promote the emergence and establishment of deleterious mutations such as the ones identified in this project. This study highlights the importance of genetic counseling and the sensitization of medical personnel towards HSD17B3 deficiencies.
The projects conducted in this thesis address relevant limitations of endocrine studies that are part of safety assessments of chemicals. By assessing species specific inhibition of HSD11B2 by azole fungicides, and identifying parabens and UV filters as modulators of HSD17B6 and RORγt activities, we have created a foundation for further research. The methods and homology models described in the respective projects of this thesis may prove to be valuable tools for future studies.