Marine phytotoxins may become a major health problem for humans because of their ability to contaminate seafood and to cause shellfish poisoning. In this report, the cytotoxic effects and the effects on intracellular caspase activities of culture supernatants from different dinoflagellate Prorocentrum clones were determined. Among the clones tested, P. tepsium BAH ME-140 and P. lima BAH ME-130 K1 and K2 clones but not P. minimum and P. micans were found to be toxic on rat pheochromocytoma PC12 cells, mouse lymphoma L5178Y cells and rat primary neurons. A significant increase in the specific activities of caspase 1 (ICE), caspase 3 (CPP32) and caspase 6 (Mch2) to 149-167% was observed after treatment of neurons with P. lima BAH ME-130 K2 supernatant for 72 h; in PC12 cells, the increase in these enzyme activities was much smaller. An even stronger and faster effect on caspase activities, compared to the K2 clone, was observed following treatment of PC12 cells and neuronal cells with P lima BAH ME-130 K1 supernatant. The maximal increase in caspase activities in PC12 cells (CPP32, 364%; and Mch2, 166%) and in neurons (CPP32, 162%; and Mch2, 111%) was observed after 24 h; no significant change of ICE activity was found during that incubation period. After 48 h the specific activities of all caspases strongly decreased. Incubation of PC12 cells with P tepsium BAH ME140 for 24 h had almost no effect on caspase activities, while a small increase in CPP32- (148%) and Mch2- (115%) but not in ICE-activity was detected after 48 h. In neurons, only an increase in CPP32 activity (to 130%) was observed with this dinoflagellate supernatant after 24 h. The P lima protein phosphatase inhibitor okadaic acid (0.5 ng/ml) caused a time-dependent increase in caspase activities in PC12 cells. A much higher effect was observed in neuronal cells; after 72 h, the specific activities of ICE, CPP32 and Mch2 increased to 295%, 146% and 235%, respectively. These results indicate that disturbances of caspase activities may contribute to the neurotoxic effects of certain dinoflagellate supernatants.
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