Fish Primary Cultures for Toxicity and Pathological Studies

In this study, epithelial primary cultures from the rainbow trout (Oncorhynchus mykiss) were developed as a model for environmental effects assessment. Cadmium was used as representative of heavy metals. The possible use of this system for pathological studies was also evaluated. The differential cytotoxic effects of cadmium on fish and mammalian epithelial cells in established and primary culture wee assessed by looking at the reduction of colony-forming ability and the reduction in the extent of growth. The influence of medium composition on the toxicity of cadmium was also studied using serum-free and serum-containing media. The experiments using immortalised cell lines showed that mammalian cells were more sensitive than fish to cadmium. Both cell types were grown at the same serum concentration. However, using the normal priamy system, human epithelial cells displayed less sensitivity to cadmium than did similar cultures from rainbow trout. It is likely that cellular mechanisms of cadmium resistance in the different cells are responsible for these effects. Cadmium proved to be more toxic when tested in serum-free medium. With fish primary cultures, reduction of cell numbers was observed at concentrations as loa as 5 umol/L in serum free compared to 100 umol/L in serum-containing medium. This was determined to be caused by the reduction of free cadmium ions, due to the presence of serum in the medium. Cultured epidermal cells from explants of skin of rainbow trout were employed to assess the cytological and functional changes following sublethal exposure to cadmium stress. The aim was to develop diagnostic markers for ecotoxicology. Acute and sub-chronic exposures were performed. Cell structural and cytological changes were established by ligh and electron microscopy. Functional alternations of glucose metabolism and melanin procution were detected by hisochemistry. The relationship between intiation of cellular alterations and cadmium concentrations was explored in cultures exposed in commercially available serum-free and serum-containing medium. The expression of stress proteins (metallothionein and heat shock protein) was also examined. Rainbow trout epithelial cells exposed to cadmium exhibited typical morphological changes indicative of cell death by apoptosis. Sublethal exposure also resulted in cellular metabolic disturbances with increased deposits of glycogen. Enhanced melanisation was also observed. These changes appeared at lower concentrations of cadmium when cells were exposed in serum-free media than serum-containing media. Cadmium induces express of heat shock proteins but not of metallothioneins. Primary and established fish cells were analysed for detection of the pathogen Renibacterium salmoninarum. The results of this thesis have contributed to the development of a technique for the culture of fish epithelial cells that is applicable in toxicological and pathological studies