Inhibition of glucocorticoid signaling and synthesis detected by an in vivo zebrafish larva screening system: A novel tool for endocrine disruptor risk evaluation

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

Glucocorticoids

As one class of the most important steroid hormones, glucocorticoids have long been recognised and their therapeutic benefits have been widely used in clinical treatment, especially in anti-inflammation cases. Glucocorticoids regulate various processes in the body including the mobilization of energy stores, immune functions, gene expression, and maintenance of the homeostasis as well as the stress response, this is not surprising that the concept of "glucocorticoids" is mentioned in almost all medical text books that focus on specific organs or systems such as the cardiovascular system, the immune system, and the neuroendocrine system. The book of Glucocorticoids - New Recognition of Our Familiar Friend aims to introduce the latest findings relating to glucocorticoids, either freshly from the laboratory or from clinical case studies, and to open up a new angle of looking at the issue of balancing the therapeutic benefits and side effects brought up by glucocorticoids

Role of macrophage 11β-HSD1 in inflammation mediated angiogenesis, arthritis and obesity

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1) is an enzyme that predominantly converts inactive glucocorticoids (cortisone in human and most mammals, 11dehydro-corticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively). Thus 11β-HSD1 amplifies intracellular levels of glucocorticoids. Studies in globally 11β-HSD1 deficient mice have revealed changes in glucocorticoid-regulated physiological and pathological processes, including metabolism, aging, arthritis and angiogenesis. The function of macrophages, which play an important role in inflammation, is also altered. For example, 11β-HSD1 deficiency in macrophages causes a delay in their acquisition of phagocytic capacity. To dissect the role of macrophage 11β-HSD1 in angiogenesis, arthritis and obesity, both in vitro macrophage stimulation and in vivo functional assays in macrophage-specific 11β-HSD1 knockout mice, were conducted. Thioglycollate-elicited peritoneal macrophages from globally 11β-HSD1 deficient and control C57BL/6 mice were used for in vitro studies. In M1/M2 macrophage polarisation experiments, 11β-HSD1 deficient macrophages showed increased expression of mRNAs encoding pro-inflammatory factors upon lipopolysaccharide and interferon-ϒ treatment and decreased expression of pro-resolution genes with interleukin-4 stimulation. However, at cytokine or protein levels, there was little difference between the genotypes except for decrease IL12 p40 levels in 11β-HSD1 deficient macrophages. Hypoxic stress failed to show differences between genotypes in hypoxia-regulated gene expression. These data do not support a strong role for macrophage 11β-HSD1 in inflammation regulation, nor in response to hypoxia, at least when measured in vitro. The discrepancy between transcriptional and translational responses is currently unexplained, but may reflect altered posttranscriptional activity. To investigate the role of macrophage 11β-HSD1 in vivo, macrophage-specific Hsd11b1 knockout mice, LysM-Cre Hsd11b1 flox/flox (MKO) mice and Hsd11b1flox/flox littermate controls were generated. In MKO mice, 11β-HSD1 protein levels and enzyme activity were reduced by >80% in resident peritoneal macrophages. However, 11β-HSD1 protein and enzyme activity levels were unchanged or only modestly reduced in thioglycocollate-elicited peritoneal neutrophils, monocytes/macrophages, or in bone marrow-derived macrophages, despite >80% decrease in Hsd11b1 mRNA levels in these cells. A relatively long half-life of 11β-HSD1 protein compared to that of circulating myeloid cells may underlie this mismatch between transcriptional and translational expression. Furthermore, following 12 days of inflammatory arthritis induced by K/BxN serum transfer, the reduction in 11β-HSD1 protein levels in circulating neutrophils of MKO mice is consistently around 50%, which corroborates the above explanation. MKO mice and littermate controls were subjected to inflammatory models which may involve resident macrophages. First, to address the role of 11β-HSD1 in macrophages in angiogenesis, sponge implants were inserted subcutaneously into the flanks of adult male mice and harvested after 21 days. Chalkley counting on hematoxylin and eosin stained sponge sections showed significantly increased angiogenesis in MKO mice (scores: 5.2±1.0 versus 4.3±0.7; p<0.05, n=9-11). Cdh5 expression (encoding VE-cadherin, a marker of endothelial cells) was higher in sponges from MKO mice (relative expression: 1.5±0.9 versus 0.8±0.6; p<0.05), as was Il1b (encoding IL-1 beta, a marker of inflammation, relative expression: 6.5±6.4 versus 1.5±0.9; p<0.05). Vegfa mRNA (encoding vascular endothelial growth factor alpha) was unchanged, with a trend for higher Angpt1 (encoding angiopoietin 1, p=0.09) expression levels in the MKO group. These results suggest that lack of 11β- HSD1 in resident macrophages increases their pro-angiogenic activity, independently of VEGF-. The K/BxN serum transfer model of arthritis was used to investigate the role of macrophage 11β-HSD1 in arthritis. Adult male MKO and control mice received a single i.p. injection of 125μl K/BxN serum per mouse, followed by 21 days of clinical scoring to assess joint inflammation. The onset of inflammation (d1-8) was similar between MKO and control mice, but MKO mice exhibited greater clinical inflammation scores in the resolution phase of arthritis (d13-21; area-under-the-curve: 86.6±14.7 versus 60.1±13.4; p<0.005), indistinguishable from globally 11β-HSD1- deficient mice. Hematoxylin and eosin staining revealed pronounced fibroplasia predominantly in the supporting mesenchyme associated with the tenosynovium, with new bone and blood vessel formation. These results suggest that macrophage 11β-HSD1 deficiency is fully accountable for the worse arthritis resolution phenotype in the globally 11β-HSD1 deficient mice, but not the earlier onset of inflammation with global 11β-HSD1 deficiency. Macrophage activation states are closely linked with adipose insulin sensitivity. Globally 11β-HSD1 deficient mice are protected from high fat diet induced insulin resistance and adipose tissue hypoxia and fibrosis. To study the effect of macrophage 11β-HSD1 deficiency on insulin sensitivity, adult male MKO and control mice were given a 14 week high fat diet, which typically causes insulin resistance in control but not globally 11β-HSD1 KO mice. The level of fibrosis in subcutaneous adipose tissues was reduced as indicated by quantification of picrosirius red staining of collagen, though GTT data so far does not support protection from insulin resistance in MKO mice. In summary, in vitro macrophage polarisation experiments do not support a strong role of 11β-HSD in M1/M2 macrophage polarisations or response to hypoxia. However, MKO mice reveal, for the first time, an important in vivo role of macrophage 11β-HSD1 to promote angiogenesis and facilitate resolution of K/BxN serum transfer induced arthritis. Modulation of fibrosis is context dependent. Reduced adipose fibrosis may be one of the mechanisms that improve insulin sensitivity. Meanwhile, these findings suggest caution regarding the potential side effects of 11β-HSD1 inhibitors in treating metabolic disease in patients with inflammation-related co-morbidities, such as rheumatoid arthritis