Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Abstract

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanolinduced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

Utility of the Zebrafish Model for Studying Neuronal and Behavioral Disturbances Induced by Embryonic Exposure to Alcohol, Nicotine, and Cannabis

“…Studies of alcohol exposure in zebrafish show that the internal concentration within developing eggs after 2 h of exposure, which ranges from 4-10% of the alcohol concentration in the water as measured using an AM1 Alcohol Analyser (Analox Instruments, London, UK), correlates linearly with the alcohol bath concentration [108,109]. Also, while one cannot measure blood alcohol concentrations in zebrafish embryos, due to their lack of a developed circulatory system [110], alcohol levels in embryonic tissue can be precisely measured using headspace gas chromatography; they show the internal embryo concentrations at 24 h to be 25-35% of the bath concentration, independent of genetic background [111,112], producing a range generally equivalent to the blood alcohol concentration reported in mammalian studies. Thus, with exposure to the relatively low alcohol concentration (0.5% v/v) commonly used in the literature, which produces no change in gross morphology [113,114], blood alcohol concentration is found to be ~0.12 g/dL, which falls within the range (0.005-0.212 g/dL) of that observed in humans at birth who are prenatally exposed to alcohol [115].…”