Advancing understanding of glucocorticoid effects in fish

Synthetic glucocorticoid drugs are used by millions of people worldwide on a daily basis to treat a wide variety of conditions that involve inflammation. However, a fundamental understanding of the mechanisms of glucocorticoid action in non-mammalian vertebrates is lacking and this project aims to help fill that knowledge gap for fish. Recently members of this team have published a quantitative adverse outcome pathway (qAOP) for one glucocorticoid (Margiotta-Casaluci et al 2016) that explains effects observed in extensive and multiple in vivo studies and goes some way to addressing the lack of fundamental understanding as to how this class of compounds acts in fish. However, some high-quality studies used in regulatory submissions have recorded unexpected effects that led to poor survival in F1 generation of zebrafish. These findings cannot be explained from our current understanding of the mechanisms of fish physiology, and there is a need to identify predictive biomarkers of key events (KEs) in the physiological cascade.
The aim of this PhD studentship is to understand the fundamental mechanism(s) of glucocorticoid action using the zebrafish as the experimental model. We hypothesise that the adverse (lethal) effects reported in the offspring (F1) derived from F0 corticosteroid exposed zebrafish may be explained by a hypersensitisation in the F1 generation to glucocorticoid drug action. The most likely effect pathways for this are via alterations to insulin/insulin like growth factor signalling, blood glucose control, immune function, and/or disruption to the development of the cardio vascular system. The studentship will focus on these pathways but flexibility will be maintained to allow the project results to dictate the direction of study. Following an initial training in some of the molecular, physiology and imaging techniques required for the project and the undertaking of comprehensive literature review, the student will generate juvenile zebrafish from adults exposed to the glucocorticoids at concentrations already known to cause detectable effects. (S)he will then expose the F1 to glucocorticoid drugs to establish when (in which life stages) the adverse effects occur to refine the study life stage window. Assessments of the heart development and function (blood flow, valve function) will be undertaken in the F1 via a combination of morphometry and imaging. Effects on IGF signalling will be assessed via comparisons of the hepatic transciptomes (RNA Seq) in exposed and non exposed fish. Comparison between drug treated and non treated fish will also be made on blood glucose, cortisol levels (which can be measured non-invasively via the water), appetite and growth. To investigate for possible effects on immune function we will generate the required glucocorticoid drug exposed F1 for transgenic fish in which we are able to quantify responses of fluorescently labelled macrophages and neutrophils via imaging where they have been subject to an immunological challenge. Once identified which systems are impacted, and as time allows, models can be generated using the principles of inducing response either by direct inflammation or knock-in approaches (using CRISPR-Cas) to provide zebrafish with a phenotype that can be rescued with the glucocorticoid. The results will shape the current understanding of how drugs designed for human use can act in fish with known similar targets but unknown pharmacological mechanism. Characterising this response will require multiple glucocorticoid drugs that will provide a range of different efficacies and so allow further questions as to the effects of simple mixtures and poly-pharmacology to be addressed in non-mammal models.