Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes

A number of algal toxins were tested for the ability to induce apoptosis (regulated cell death) in primary hepatocytes from salmon and rat. The tested toxins included the liver targeting substances microcystin-LR and nodularin, substances associated with the diarrhetic shellfish poison complex (okadaic acid, dinophysistoxin-1 and pectenotoxin-1) and calyculin A. All toxins induced apoptosis in both salmon and rat hepatocytes in less than 2 h. The apoptotic changes were evident both by electron and light microscopy and were counteracted by the caspase inhibitor ZVAD-fmk and by the Ca 2+/calmodulin dependent kinase II inhibitor KN-93. The salmon hepatocytes were 10–20-fold more sensitive to okadaic acid and dinophysistoxin-1 ( ec 50=20 nM) than rat hepatocytes and other mammalian cell lines tested. An assay was devised using hepatocyte apoptosis as parameter for detection of algal toxins. This assay was at least as sensitive as HPLC determination for okadaic acid in mussel extracts. It also detected algal toxins which do not inhibit protein phosphatases, like pectenotoxin-1. Subapoptotic concentrations of the toxins inhibited hepatocyte aggregation. Using this parameter, less than 200 pg okadaic acid could be detected. In conclusion, salmon hepatocytes in suspension culture provide a rapid and sensitive system for detection of a broad range of apoptogenic toxins.