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 apoptos is (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) an d calyculin A. All toxins induced apoptosis in both salmon and rat hep atocytes in less than 2 h, The apoptotic changes were evident both by electron and light microscopy and were counteracted by the caspase inh ibitor ZVAD-fmk and by the Ca2+/calmodulin dependent kinase II inhibit or KN-93. The salmon hepatocytes were 10-20-fold more sensitive to oka daic acid and dinophysistoxin-1 (EC50 = 20nM) than rat hepatocytes and other mammalian cell lines tested. An assay was devised using hepatoc yte apoptosis as parameter for detection of algal toxins, This assay w as at least as sensitive as HPLC determination for okadaic acid in mus sel extracts. It also detected algal toxins which do not inhibit prote in phosphatases, like pectenotoxin-1. Subapoptotic concentrations of t he toxins inhibited hepatocyte aggregation. Using this parameter, less than 200 pg okadaic acid could be detected. In conclusion, salmon hep atocytes in suspension culture provide a rapid and sensitive system fo r detection of a broad range of apoptogenic toxins. (C) 1998 Elsevier Science Ltd. All rights reserved.