eprintid: 24215
rev_number: 12
eprint_status: archive
userid: 3662
dir: disk0/00/02/42/15
datestamp: 2018-03-29 07:10:53
lastmod: 2018-04-03 11:59:27
status_changed: 2018-03-29 07:10:53
type: doctoralThesis
metadata_visibility: show
creators_name: Macherey, Melanie
title: Inhibition of glucocorticoid signaling and synthesis detected by an in vivo zebrafish larva screening system:
A novel tool for endocrine disruptor risk evaluation
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
abstract: Pharmaceutical residues in aquatic systems constitute a major environmental risk as pharmaceuticals are designed to interact with specific biological processes. These processes are mostly conserved among all vertebrate groups (e.g. signal transduction, metabolism), thus, drugs may have similar effects in fish as they have in mammals. In particular, endocrine disruptive chemicals (EDCs) are of high ecotoxicological concern as they may interfere with hormonal signaling via multiple pathways and can affect health and reproduction of organisms. EDCs which directly interfere with the reproductive system via sexual steroid hormones such as estrogens and androgens have been in the focus of ecotoxicological risk assessment for decades, but potential interactions with other hormonal groups are so far poorly investigated. One of these neglected hormonal groups are Glucocorticoids (GCs), a subclass of steroid hormones, which regulate metabolism and immune function. Drug interference with the GC pathway may thus hamper an organism’s survival, but research efforts examining the complex interactions of these compounds with this pathway are so far limited.
In this thesis, I developed an in vivo testing approach, which enables to detect EDCs of the GC system and to investigate their mechanisms of action. I have applied a bioluminescence-based test system with transgenic zebrafish larvae, the GRIZLY assay, to screen an FDA-approved drug library for compounds that may affect the GC pathway. By means of conducting three assay modes I aimed to identify inhibitors of GC signalingin vitro and in vivo as well as disruptors of GC biosynthesis in vivo.
I detected 29 compounds that showed significant inhibitory activity in at least one of the assay modes. Interestingly, I also found five superactivators of GC signaling, substances which increased and/ or extended the bioluminescence signal activity. Concentration-dependent retests validated high reliability of the screen performance. The combined evaluation of compound effects in three assay modes enabled me to pre-categorize the potential substance mechanisms of action according to either direct interference with GC signaling activity or disruption of GC biosynthesis. In order to follow up on the compound mechanisms of action, I conducted chemical and gene expression analysis experiments with selected in vivo inhibitors and superactivators. The obtained results for substance effects on larva-internal steroid levels and target gene expression, combined with the GRIZLY outcomes, allowed me to group the compounds according to their potential mechanisms of action. For example, anti-inflammatory drugs interfered with GC signaling without affecting the GC biosynthesis pathway. Estrogens and retinoids inhibited GC signaling and synthesis, while for progestins both inhibitory and stimulatory effects were observed, suggesting a complex interaction of this compound class with the GC pathway.
Overall, I validated that the GRIZLY assay is a highly suitable in vivo test system to detect pharmaceutical interference with the GC system via various effect pathways. The combined evaluation of three different GRIZLY modes furthermore allows for pre-categorization of compound effect mechanisms, which facilitates the selection of specific follow-up experiments for an in-depth risk assessment for effects of EDCs on the GC system. Moreover, my results show that several of the most-widely prescribed drugs which enter aquatic systems may interfere with GC signaling and steroid hormone biosynthesis in fish. Given the GC regulation of metabolism and the immune system, this may possibly lead, especially under chronic exposure, to a weakened ability of organisms to cope with stressors. In the long-term view, pharmaceutical residues in the environment might thus lead to a reduced biological fitness, which may constitute an additional, so far largely neglected burden for aquatic Organisms.
date: 2018
id_scheme: DOI
id_number: 10.11588/heidok.00024215
ppn_swb: 1654784303
own_urn: urn:nbn:de:bsz:16-heidok-242151
date_accepted: 2018-03-05
advisor: HASH(0x561a62812668)
language: eng
bibsort: MACHEREYMEINHIBITION2018
full_text_status: public
citation:   Macherey, Melanie  (2018) Inhibition of glucocorticoid signaling and synthesis detected by an in vivo zebrafish larva screening system: A novel tool for endocrine disruptor risk evaluation.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/24215/1/Thesis_final7_orig_formatiert%20f%C3%BCr%20PDFA.pdf