Using hsp70 expression as an in vivo biomarker of cellular toxicity in early life stages of zebrafish following exposure to cadmium and arsenic
Blechinger, Scott Richard
The overall goal of this thesis was to determine if expression of hsp70 and a reporter construct hsp70-eGFP could be used as biomarkers of toxic exposure in zebrafish larvae. Cadmium and arsenic were chosen as the model toxic compounds to test this hypothesis because they are both strong inducers of hsp70 in other model systems, and their toxicity in fish has been well characterized. Before investigating the effects of cadmium and arsenic on hsp70 expression, it was necessary to determine the normal expression patterns under non-stressed conditions. A time course from the early blastocyst embryo to post-hatch larvae (3-96hpf) showed that hsp70 is not expressed under non-stressed conditions except for the developing lens. It was determined that this localized lens expression is constitutive rather than stress-induced, and may play a role in the development of the lens. It was also determined that the reporter hsp70-eGFP gene closely mimicked endogenous hsp70 under non-stressed conditions (i.e. constitutive lens expression) and following heat shock (i.e. strong expression throughout) and therefore is a reliable method to monitor hsp70 expression in vivo. An initial range-finding acute toxicity study showed that post-hatch zebrafish larvae were much more sensitive to cadmium than pre-hatch embryos, and therefore only larvae were used for further toxicity tests. The dose-response relationships of the acute (96hr) toxicity of cadmium and arsenic to larvae were determined and the median effective doses calculated (LC50 and EC50). The primary gross effects of edema, trunk abnormalities, immobility, and death were observed for both cadmium and arsenic, however cadmium was much more toxic than arsenic. Based on the acute toxicity tests, relevant doses of cadmium and arsenic were chosen as exposure treatments in order to measure in vivo expression of hsp70 and hsp70-eGFP. The patterns of expression for hsp70 and hsp70-eGFP were investigated in larvae following acute pulse (3hr) exposures to cadmium and arsenic (only hsp70). The patterns of expression for hsp70 and hsp70-eGFP were dose-dependent and tissue specific. Cadmium induced expression of hsp70 and hsp70-eGFP in the skin, gills, olfactory rosette, liver, and kidney, all of which are either accumulators or target organs of cadmium in fish. Arsenic also induced expression of hsp70 in the liver, gills, olfactory rosette and skin.