Zebrafish and safety pharmacology. Effects of ethanol on early brain and eye development.

Abstract

Ethanol is well studied with regard to its teratogenic effects in different animal models including humans. Exposure of a fetus to alcohol during pregnancy can lead to fetal alcohol syndrome (FAS), or ethanol mediated toxicity in animal models.

During early embryogenesis, development of the most important organs including the central nervous system (CNS) occurs. Disturbance of any function that contributes to the normal development can lead to defects and dysfunctions of the brain and other important organs. Zebrafish (Danio rerio) is a small fresh water fish increasingly being used as a model system for human disease, drug development and safety pharmacology. It is also a perfect model for studying the effects of ethanol on molecular level due to the similarities between zebrafish and humans with regards to genetic cascades and signaling pathways during early embryogenesis. Genes that are members of Pax and Atoh families of transcription factors are expressed in specific parts of the brain. Visualization of gene expression by using in situ RNA hybridisation can provide useful information about the brain development of embryos exposed to drugs compared to their normal counterparts. An important part of the project was therefore to isolate RNA and make cDNA, so that the Atoh1a and Atoh1c genes could be cloned and used as probes for in situ hybridisation. Probes for in situ hybridisation with Pax6.1 and Pax2.1 were already available. Zebrafish embryos were incubated in different concentrations of ethanol 0.01 %, 0.1 %, 1 % and 2 % for three days. After every 24 hours within the three days the embryos were observed in a dissecting microscope and development changes and mortalities were recorded. Embryos incubated in 2 % ethanol were overall deformed, while embryos incubated in 1% ethanol developed malformations including pericardial edema, yolk sac edema, axial malformations, axial blistering and truncated body axis. Some of the embryos had late development and were inactive. In situ hybridisations were done to look for changes in the brain and eye morphology. In addition, changes in Pax6.1 gene expressions were paid extra attention because it is previously reported that ethanol decreases the level of Pax6 expression. This was confirmed by in situ hybridisation of 72 hpf embryos and by Western blot of 24 hpf embryos.