| The warm-water fish Colossoma Brachypomum cannot survive when water temperature is below 10.Using tissue culture ,we have successfully established a fin tail cell line named CBT. Results showed that the CBT cell line was epithelioid-like cells and the model number of its chromosome was 54.The aim of this study was to evaluate the effect of cold stress based on CBT cell line. Hoechst33258 dying was used to observe cold induced morphological changes. The cytoskeleton of CBT cells was observed by light microscope. Cell viability was measured by trypan blue exclusion assay and thiazolyl blue (MTT) assay. Ultrastructure examination was performed by using transmission electron microscope. Externalization of phosphatidylserine(PS) and mitochondrial membrane potential (MMP) were monitored with confocal laser scanning microscope.Flow cytometry was used to determine the apoptotic indexes of cells and agarose gel electropheresis to monitor the internucleosomal DNA fragmentation. Results showed that cell viability and fluorescence intensity of MMP decreased obviously in a time dependent manner when exposed to 10(P<0.01). The early events of apoptosis such as chromatin condensation, margination and PS externalization occurred during cold incubation. The apoptotic percent of CBT cells treated at 10 for 4 days was 24.03%.However, the appearance of DNA ladder, which is a biochemical hallmark of apoptosis, did notoccur during cold incubation until rewarming the cells to 32. It could be concluded that low temperature (10) could induce apoptosis in Colossoma Brachypomum CBT cells.Compared with the control cells, the intracellular reactive oxygen species (ROS) was significantly elevated when cells were incubated at 10. Furthermore, cold injury could be inhibited to a great extent by antioxidant a -Tocopherol. These results suggest that the formation of reactive oxygen species (ROS) may be a key mediator of cold-induced apoptosis in Colossoma Brachypomum CBT cell line...
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