Studies On Cold-induced Apoptosis In Colossoma Brachypomum Cells

The warm-water fish Colossoma Brachypomum can not survive when water temperature is below 10℃. Colossoma Brachypomum tail fin (CBT) cell line has been established in our laboratory in 1997. This cell line has the same character as the fish that this cell can not survive in the 10℃ water. It has provided a optimal study model to observe the mechanism of cold injury in warm-water fish.The aim of this study is to observe why hypothermia(10℃) could lead to Colossoma Brachypomum CBT cell death and the potential mechanism involved. Cell viability was monitored by Cell Counting Kit-8. The production of reactive oxygen species(ROS) in cells was determined by H2DCFDA. Apoptosis was analyzed by hyopdiploid apex and Tunel. Agarose gel electrophoresis was used to detect DNA fragmentation. Hoechst 33258 dying was used to observe cold-induced morphological changes. The release of dehydrogenase(LDH) was determined by colorimetric assay. The results were that treatment of CBT cells at the low temperature (10℃) , caused a great decrease in cell viability, an increase of production of ROS in cells and a release of LDH simultaneously(P<0.01). When CBT cells were treated at the low temperature for 3days, flow cytometry showed the hyopdiploid apex, and the apoptotic rate was 13%. Then if the cells temperature was returned to 32℃ and lasted for 12h more, a "DNA ladder" pattern representing DNA fragmentation could be observed. Tunel detection revealed that after 3days, 4days and 5days cold incubation (10℃) followed by 12h rewarming (32℃), the apoptotic rates of CBT cells were 23. 49%, 27. 72% and 35.10% respectively. After the CBT cells were incubated at the low temperature for 5days followed by 12h rewarming, apoptotic body could be found. The results of the test by concentration gradient indicated that the α -Tocopherol could protect the cell under 10℃ and was benefit to thedecrease of the viability and the LDH release in CBT cells. This protection was available in the concentration of a -Tocopherol between 50 uM and 1000 uM. All the results showed that low temperature of 10°C could induce early apoptosis in CBT cell, additional treatment at temperature of 32°C could promote CBT cell to late apoptosis. ROS might play a key role in cold-induced apoptosis of CBT cell.