Heat shock protein 70 levels in rainbow trout primary epidermal cultures in response to 2,4-dichloroaniline exposure: A novelin vitro aquatic toxicity marker

Abstract

The aim of this work was to investigate the use of the heat shock protein, HSP 70, as a sublethal measurement of ecotoxicity and to identify if the amount of HSP 70 synthesized is proportional to the chemical stress applied. This was achieved by quantifying the HSP 70 levels in primary cultured rainbow trout, Oncorhynchus mykiss (R.), skin epidermal cells in response to 2,4-dichloroaniline (2,4-DCA) exposure. The cellular stress response protects organisms from damage resulting from exposure to a wide variety of stressors including xenobiotics. The use of a HSP 70 polyclonal antibody on rainbow trout primary epidermal skin cultures exposed to 2,4-DCA was investigated as a possible biomarker for environmental stress using an immunocytochemical approach. The epidermis is highly susceptible, as it is the interface between the fish and its aquatic environment. In this study we have developed a simple in vitro system for aquatic-toxicity risk assessment. A method for the quantification of heat shock (stress) protein levels by immunocytochemistry is described. The antibody dilution range enabled the detection and quantification of only the inducible HSP 70 fraction. A 1:2000 dilution was decided upon. This assay was effective in detecting and quantifying the induced HSP 70. There was a direct toxicant concentration-dependent increase in the levels of the cellular stress protein in the primary epidermal cultures. Enhanced localization of HSP 70 in the nuclei of the epidermal cells was observed following exposure to 2,4-DCA. This work indicated the possibility of using heat shock protein induction and subsequent quantification as a sensitive system for aquatic toxicity risk assessment.