Evaluation of a transgenic zebrafish model for assessing arsenic toxicity

Abstract

The objective of this thesis was to evaluate hsp70 expression as an indicator of arsenic exposure in zebrafish larvae and to assess the accuracy of the hsp70-eGFP reporter gene construct as a reliable indicator of endogenous hsp70 expression. The relative toxicity of arsenite and arsenate was also examined and gross developmental effects were recorded following an acute 96 hour range finding exposure. Gross effects observed included edema, trunk abnormalities, immobility, and mortality for both arsenite and arsenate, with arsenite more toxic than arsenate. The median lethal concentrations (LC50) for arsenite and arsenate were calculated from the data obtained in the 96 hour exposure. They were determined to be 771.98 μM and 1347 μM respectively. The effective concentrations (EC50) were determined to be 570 μM for arsenite and 1172 μM for arsenate. Results from the 96 hour exposures were also used to determine concentrations used in subsequent exposures. Induction of hsp70 was examined in wild-type larvae following a three hour exposure to arsenic and subsequent in situ hybridization. It was determined that both trivalent and pentavelant arsenic induced expression in the olfactory rosette, gills and skin, GIT, liver, and pericardial muscle. Expression was found to be dose-dependent and tissue-specific for both. Induction of hsp70 was evident in the skin, liver, and gastrointestinal tract of zebrafish larvae exposed to 700 μM arsenite and in the skin, gills, liver, pericardial muscle, and gastrointestinal tract in those exposed to 1000 μM or 2000 μM arsenite. Exposure to 1500 μM arsenate resulted in expression in skin, liver, and gastrointestinal tract, while induction was observed in skin, gills, liver, pericardial muscle and gastrointestinal tract of larvae exposed to 2500 μM or 7500 μM arsenate. Overall expression patterns of hsp70-eGFP in transgenic zebrafish larvae exposed to arsenic were found to closely mimic that of endogenous hsp70 expression patterns in wild-type larvae suggesting that it is an accurate indicator of endogenous hsp70 expression.