Zebrafish as a model system for safety pharmacology, using a glucocorticoid and an antimicrobial peptide as test substances

Abstract

The zebrafish is an exciting model organism with a lot to offer and many possible areas of application. Toxicity assays using zebrafish embryos are becoming increasingly popular with the pharmaceutical industry as a tool to bridge the gap between conventional cell based screens and animal testing. The main aim of this project was to explore the zebrafish as a model system for use in safety pharmacology, and to look into some of the different methods that can be used to do so. Dexamethasone and a novel antimicrobial drug lead were tested for toxicity in the zebrafish embryo by visual assessment of mortality and malformations, and by performance of apoptosis assay and western blotting to look at apoptosis and proliferation. Treatment with relative low concentrations of dexamethasone caused a significant increase in embryo malformation rate, while staining with acridine orange revealed altered apoptotic activity in brain tissues and in the urogenital tract of treated embryos. The antimicrobial peptide, β2,2-amino acid derivative 161, showed generally low toxicity to zebrafish embryos, as measured by the LC50 estimated in this project, compared to previously reported MIC and IC50 values for the drug against multi-resistant bacterial strains and cancer cells. However, truncation of tail and damage to tail tissue were seen after treatment with the drug. Staining with acridine orange showed increased apoptotic activity in brain tissues and the urogenital tract of treated embryos. Western blots of whole embryo extracts of embryos treated with either drug gave conflicting result, and a refinement of the method is suggested for future experiments. In parallel with the zebrafish experiments, studies using mammalian cell cultures were carried out. Both excess glucocorticoids and decreased levels of the transcription factor PAX6 have been associated with development of cataract and glaucoma. Glucocorticoids and PAX6 also influences glioblastoma cancer patient prognosis. We wanted to explore if treatment with dexamethasone would influence expression levels of PAX6 in human lens epithelial cells and human glioblastoma cells. We were also interested in looking at proliferation and apoptosis in these cell lines following dexamethasone treatment. To investigate this, western blots were done. However, results were inconclusive, and further studies are needed to answer this.