OBJECTIVES: The aim of this study was to assess the possible in vitro apoptotic effects of anatoxin-a on the selected immune cells isolated from the blood of carp.
DESIGN: In the experiments pure anatoxin-a was used at concentrations of 0.01, 0.05, 0.1 and 1 µg/ml RPMI-1640 medium. Apoptosis or necrosis of fish leukocytes (lymphocytes and phagocytes) induced by the toxin was determined by measurement of the activity of caspases-3/7 and the analysis of phosphatidylserine on the outer leaflet of apoptotic cell-membranes using Annexin-V-Fluorescein and Propidium Iodide. Moreover, fluorescent measurement of the release of lactate dehydrogenase from the cells with damaged membranes was done.
RESULTS: The viability of the lymphocytes exposed only to the highest concentration of anatoxin-a (1 µg/ml) was significantly decreased. The exposure to the toxin at higher concentrations (0.1 and 1 µg/ml) resulted in a significant increase of caspases 3/7 activity in phagocytes and lymphocytes. Moreover, fluorescent analysis with the use of annexin-V-fluorescein and propidium iodide staining showed more cells at the apoptotic stage than necrotic cells.
CONCLUSION: Our study showed that anatoxin-a is an inducer of apoptosis in fish immune cells....