Studies on the Toxicification Mechanism of Tetrodotoxin-containing Organisms, Especially on the Dynamic State of TTX-related Substances

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In order to clarify the mechanisms of toxicification of tetrodotoxin (TTX) -containing organisms, distribution of TTX and its related substances in benthic animals and their inhabiting environments (substratums), the optimal culturing condition of TTX-producing bacteria for its production, classification of TTXproducing bacteria by the determination of base sequence of r-RNA were examined. The isolation and characterization of tetrodonic acid (TDA) -like substance, the possible precursor of TTX in living organisms, were also performed. To conduct these experiments, microanalysis methods of TTX-related substances were also developed.1) A presumed precursor of TTX, TDA-like substance, was isolated from a ribbon worm Cephalothrix linearism It is very unstable and easily converted to TTX, having a specific toxicity of 700 MU/mg, and molecular weight of 320, larger than TTX 1 by mass unit. Another assumed precursor was isolated from a toxic xanthid crab Atergatis floridus, pressumed to be a dimer of TTX. 2) To isolate and analyze these unstable precursors, a LC/FAB MS and GC/MS were developed and used. 3) From the sediments of the Pacific Ocean sounding 4000 m and fresh water lakes, some TTX producing bacteria and Ascomycetes fungi were isolated. Some of them were classified by the sequencing the bases of r-RNA. 4) Some optimal conditions of TTX-producing ability of Vibrio alginolyticus, isolated from a toxic starfish Astropecten poplyacanthus, were estimated to be 25 C for temperature and 3 % for salt concentration of culturing medium. 5) TTX and its related substances were newly detected from some benthic invertebrates inhabiting the East China Sea and the coast of South Kyushu area.