calyculin-a and dinophysistoxin-1

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 Ca2+/calmodulin dependent kinase II inhibitor KN-93. The salmon hepatocytes were 10-20-fold more sensitive to okadaic acid and dinophysistoxin-1 (EC50=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.

Topics: Animals; Apoptosis; Cell Aggregation; Cells, Cultured; Eukaryota; Liver; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Pyrans; Rats

Effects of compounds of the okadaic acid type (okadaic acid, dinophysistoxin-1, calyculin A and tautomycin) on proliferation by digestive-tract epithelial cells were investigated in mice and rats. In mice, a single oral administration of these agents caused significant enhancement of BrdU labeling indices in a dose/response manner. Exceptions showing no response were limited to the pyloric mucosa for okadaic acid, the pyloric and fundic mucosa for calyculin A and the pyloric mucosa for tautomycin. Sequential analysis of labeling indices after a single oral administration of dinophysistoxin-1 revealed two peaks of cell proliferation at 18 h and 36 h in the esophagus, ileum and colon. The labeling indices of the forestomach, fundus, pylorus and jejunum, on the other hand, continuously increased from 6 h after the administration. Elevated proliferation was also observed in the skin after 30 h or after, but no effects on the liver or kidney were evident. A single oral administration of the okadaic acid type of compounds also dose-dependently enhanced cell proliferation of the rat digestive tract. These results strongly suggest that the okadaic acid class of compounds may exert promoting potential for the gastrointestinal mucosa when administered orally.

Topics: Administration, Oral; Animals; Antifungal Agents; Bromodeoxyuridine; Carcinogens; Cell Division; Dose-Response Relationship, Drug; Ethers, Cyclic; Intestinal Mucosa; Male; Marine Toxins; Mice; Mice, Inbred ICR; Okadaic Acid; Oxazoles; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds; Time Factors

Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.

Topics: Animals; Cell Line; Cricetinae; Cytoskeleton; Ethers, Cyclic; Interphase; Kinetics; Marine Toxins; Okadaic Acid; Oxazoles; Peptide Mapping; Phosphopeptides; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinases; Pyrans; Vimentin

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.

Topics: Antibodies, Monoclonal; Autoradiography; Blotting, Northern; Breast Neoplasms; Caseins; Cell Differentiation; Electrophoresis, Polyacrylamide Gel; Ethers, Cyclic; Female; Fluorescent Antibody Technique; Gene Expression; Genes, fos; Genes, jun; Humans; Isoenzymes; Kinetics; Marine Toxins; Microcystins; Okadaic Acid; Oligonucleotide Probes; Oxazoles; Peptides, Cyclic; Phosphates; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Pyrans; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and calyculin A are the okadaic acid class of non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, which do not bind to the phorbol ester receptors in cell membranes or activate protein kinase C in vitro. They have potent tumor-promoting activities on mouse skin, as strong as TPA-type tumor promoters, such as TPA, teleocidin, and aplysiatoxin. DNA samples isolated from tumors induced by dimethylbenz[alpha]anthracene and each of the okadaic acid class tumor promoters had the same mutation at the second nucleotide of codon 61 (CAA to CTA) in the c-H-ras gene. Okadaic acid receptors, protein phosphatases 1 and 2A, are present in the particulate as well as cytosolic fractions of various mouse tissues. The apparent "activation" of protein kinases by the okadaic acid class tumor promoters, after their incubation with 32P-ATP, protein kinases, and protein phosphatases, was observed. This activation was caused by inhibition of protein phosphatases 1 and 2A by the okadaic acid class tumor promoters. Treatment of primary human fibroblasts and human keratinocytes with the okadaic acid class tumor promoters induced the hyperphosphorylation of a 60-kDa protein in nuclear and cytosolic fractions, due to the inhibition of protein phosphatases. The 60-kDa protein is a proteolytic fragment of nucleolin, a major nonhistone protein and is designated as "N-60." The mechanisms of action of the okadaic acid class tumor promoters are discussed with emphasis on the inhibition of protein phosphatase activity.

Topics: Animals; Carcinogens; DNA; DNA Damage; DNA, Neoplasm; Enzyme Activation; Ethers, Cyclic; Genes, ras; Humans; Marine Toxins; Mice; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Proteins; Pyrans; Receptors, Drug; Skin; Skin Neoplasms; Structure-Activity Relationship

Topics: Animals; Binding, Competitive; Brain; Carcinogens; Cytosol; DNA, Neoplasm; Ethers, Cyclic; Humans; Marine Toxins; Mice; Molecular Structure; Mutation; Nuclear Proteins; Nucleolin; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Protein Kinases; Proteins; Proto-Oncogene Proteins p21(ras); Pyrans; Receptors, Drug; RNA-Binding Proteins; Skin; Skin Neoplasms; Spiro Compounds; Structure-Activity Relationship