Tamao Noguchi

Tetrodotoxin--distribution and accumulation in aquatic organisms, and cases of human intoxication.

Many pufferfish of the family Tetraodontidae possess a potent neurotoxin, tetrodotoxin (TTX). In marine pufferfish species, toxicity is generally high in the liver and ovary, whereas in brackish water and freshwater species, toxicity is higher in the skin. In 1964, the toxin of the California newt was identified as TTX as well, and since then TTX has been detected in a variety of other organisms. TTX is produced primarily by marine bacteria, and pufferfish accumulate TTX via the food chain that begins with these bacteria. Consequently, pufferfish become non-toxic when they are fed TTX-free diets in an environment in which the invasion of TTX-bearing organisms is completely shut off. Although some researchers claim that the TTX of amphibians is endogenous, we believe that it also has an exogenous origin, i.e., from organisms consumed as food. TTX-bearing animals are equipped with a high tolerance to TTX, and thus retain or accumulate TTX possibly as a biologic defense substance. There have been many cases of human intoxication due to the ingestion of TTX-bearing pufferfish, mainly in Japan, China, and Taiwan, and several victims have died. Several cases of TTX intoxication due to the ingestion of small gastropods, including some lethal cases, were recently reported in China and Taiwan, revealing a serious public health issue.

DISTRIBUTION AND ORIGIN OF TETRODOTOXIN

Among the marine toxins relevant for human intoxication, tetrodo-toxin (TTX) has been known as one of the most prejudicial. Puffer fish were originally thought to be the only animal from which TTX could be isolated. Subsequent works determined that TTX also existed in a range of marine organisms of different phylogenic classes and a very few amphibians. The production of TTX is generally accepted to be associated with bacteria. This paper presents the current evidences for the distribution of TTX and its analogs in several vertebrates and invertebrates, along with the probable origin of TTX.

Ostreopsis sp., a possible origin of palytoxin (PTX) in parrotfish Scarus ovifrons

A clone of toxic dinoflagellate Ostreopsis sp. and six specimens of a parrotfish Scarus ovifrons were collected in October 1997 at Tokushima Prefecture, Japan. Ostreopsis sp. was cultured in ESM medium for 16 days, and after rearing the cell pellet (about 4.0x10(5) cells) was extracted with 50% methanol, partitioned between an aqueous layer and 1-butanol layer, and biochemically tested. Similarly, the crude toxin from S. ovifrons was extracted, and tested. The mice injected with each 1-butanol layer from Ostreopsis sp. and S. ovifrons showed the common symptoms of convulsion, drowsiness and collapse, and died within 48 h. The lethal potency of Ostreopsis sp. was calculated to be 1.0x10(-4) MU/cell. All specimens of S. ovifrons were found to be toxic, where the highest potency was determined as 2 MU/g in muscle of one specimen. After being injected with toxins, the serum creatine phosphokinase levels of mice were found to be elevated. Toxins from Ostreopsis sp. and S. ovifrons showed delayed haemolytic activity with mouse and human erythrocytes, which was inhibited by an anti-palytoxin (PTX) antibody antibody and ouabain. Toxins from Ostreopsis sp. and S. ovifrons thus resembled each other, and strongly suggested to be PTX or its akin substance. Additionally, a considerable number of adherent Ostreopsis sp. was found in the gut contents of S. ovifrons during the heavy occurrence of Ostreopsis sp. in October 1997 at Tokushima Prefecture. From the above results, it can be strongly postulated that the dinoflagellate Ostreopsis sp. is the origin of PTX which is sequestered by the parrotfish S. ovifrons through food chain.

Vibrio alginolyticus, a tetrodotoxin-producing bacterium, in the intestines of the fish Fugu vermicularis vermicularis

To clarify the mechanism of toxification in animals contaminated with tetrodotoxin, the intestinal contents of the puffer Fugu vermicularis vermicularis were examined for bacterial flora in 1985. Twenty-six out of 33 strains belonged to the genus Vibrio. These bacteria were classified into Groups I to VII, based on biological and biochemical characters. High performance liquid chromatography and gas chromatography-mass spectrometry, together with mouse bioassay for toxicity, clearly demonstrated that Group I produced tetrodotoxin and anhydrotetrodotoxin under cultivation with a medium composed of Phytone peptone (BBL) and NaCl. Some other groups also produced this toxin and/or related substances to some extent. Strains of Group I were all identified as Vibrio alginolyticus. Two strains among four produced a detectable amount of tetrodotoxin and/or anhydrotetrodotoxin, as measured by all instrumental analyses applied. Our findings suggest that some strains of V. alginolyticus are closely related to the toxification of the puffer, and probably of other species.

PUFFER POISONING: EPIDEMIOLOGY AND TREATMENT

Tetrodotoxin (TTX) intoxication resulting from the ingestion of toxic puffer is relatively common in a number of Asian countries. In Japan, 100 annual human fatality due to ingestion of toxic puffer have been reported until 1960. Poisoning incidents, however, decreased in the recent years by imposing quarantine rule and creating public awareness after extensive study of the toxicity and toxin properties of puffer. Generally, the oral paresthesia is the initial symptom of puffer poisoning. The onset of symptoms is rapid. In spite of long history of TTX intoxication, no antidotes or antitoxins to TTX is invented so far. Treatment is supportive.

Tetrodotoxin-producing bacteria from the blue-ringed octopus Octopus maculosus

Several live specimens of the blue-ringed octopus Octopus maculosus were collected from the Philippines in November 1985, and from Japan in February 1986, and the distribution of toxicity, along with toxin composition, in the posterior salivary gland and other soft parts were examined. Tetrodotoxin (TTX: 1400 mouse units g-1) was detected in the posterior salivary gland of a Japanese specimen, while not only the salivary gland but other soft parts were toxic in the Philippine specimens. The Philippine specimens contained TTX and anhydrotetrodotoxin, the Japanese specimen TTX, 4-epitetrodotoxin, and an unknown toxin. The posterior salivary gland, intestine and other parts were excised from the Philippine specimens and examined for bacterial flora. Twenty-two dominant strains were isolated and cultured in a 2xORI medium (Ocean Research Institute, Simidu and Tsukamoto 1985) at 20°C for 20 to 48 h. Cells were harvested by centrifugation, and disrupted by ultrasonication. The toxins were partially purified from the cell lyzate by ultrafiltration and Bio-Gel P-2 column-chromatography. Instrumental analyses disclosed that 16 of the 22 strains produced TTX and/or related substances. Six strains which clearly exhibited TTX productivity were identified as Alteromonas (2 strains), Bacillus (2), Pseudomonas (1) and Vibrio (1), based on biochemical and biological characteristics. Of these, one strain each of Bacillus and Pseudomonas produced TTX at a level detectable by the mouse assay.

TTX accumulation in pufferfish

Tetrodotoxin (TTX) has been detected in a variety of animals. The finding of TTX in the trumpet shell Charonia sauliae strongly suggested that its origin was its food, a TTX-bearing starfish Astropecten polyacanthus. Since then, the food chain has been consistently implicated as the principal means of TTX intoxication. To identify the primary producer of TTX, intestinal bacteria isolated from several TTX-bearers were investigated for their TTX production. The results demonstrated that some of them could produce TTX. Thus the primary TTX producers in the sea are concluded to be marine bacteria. Subsequently, detritus feeders and zooplankton can be intoxicated with TTX through the food chain, or in conjunction with parasitism or symbiosis. The process followed by small carnivores, omnivores or scavengers, and by organisms higher up the food chain would result in the accumulation of higher concentrations of TTX. Finally, pufferfish at the top of the food chain are intoxicated with TTX. This hypothesis is supported by the fact that net cage and land cultures produce non-toxic pufferfish that can be made toxic by feeding with a TTX-containing diet.

Microflora and tetrodotoxin-producing bacteria in a gastropod, Niotha clathrata

Shellfish (Niotha clathrata) were collected in both July and November from three locations in Taiwan (Pingtung, Kaohsiung and Chiai Prefecture) and assayed for anatomical distribution of tetrodotoxin (TTX) and aerobic heterotrophic bacteria. Pingtung specimens showed higher toxicity than those from Kaohsiung and Chiai, and did not show much seasonal variation. At each site, the total aerobic bacterial counts in November samples were higher than in July. The predominant genera were Vibrio, Pseudomonas, Pasteurella, Aeromonas and Plesiomonas. Vibrio comprised more than 35% of the genera, with V. alginolyticus as the major species. The viable counts of Vibrio species were higher in November than in July. However, the results did not suggest any relationship between the total count or viable count and the toxicity of the shellfish. HPLC, UV and gas chromatographic-mass spectrometric analyses demonstrated that some of the bacteria isolated, such as V. alginolyticus, V. parahaemolyticus, Pseudomonas spp. Plesiomonas sp. and Aeromonas sp., produced TTX and/or related substances.

Two new isomers of domoic acid from a red alga, Chondria armata

Isodomoic acids G and H, two new isomers of the neurotoxin domoic acid, along with isodomoic acids A, B, E and F, were isolated from a red alga, Chondria armata, collected at the southern tip of Kyushu Island. The structures of two of these were deduced to be (E, E) and (Z, E) isomers of 2-carboxy-4-(5-carboxy-l-methyl-2-hexenylidene)-3-pyrro- lidineacetic acid, based on electrospray ionization mass and [1H]nuclear magnetic resonance spectral analyses including [1H-1H]correlation spectroscopy and nuclear Overhauser effect correlation spectroscopy.

PURIFICATION AND CHARACTERIZATION OF A SULFOTRANSFERASE SPECIFIC TO N-21 OF SAXITOXIN AND GONYAUTOXIN 2+3 FROM THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

A sulfotransferase (ST) specific to N‐21 of saxitoxin (STX) and gonyautoxin 2+3 (GTX2+3) designated as N‐ST was purified to homogeneity from the cytosolic fraction of clonal‐axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum Graham GC21V, which causes paralytic shellfish poisoning. The enzyme transferred a sulfate group from 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to N‐21 in the carbamoyl group of STX and GTX2+3 to produce GTX5 and C1+2, respectively. The molecular mass of the purified enzyme was determined by SDS‐PAGE to be 59 kDa. Gel filtration chromatography showed a native molecular mass of 65 kDa, indicating that the N‐ST is a monomeric enzyme. The N‐ST was specific to only N‐21 of STX and GTX2+3, and O‐22 sulfation was not observed. Moreover, the N‐ST was not active toward neo STX and GTX1+4, which differed from STX and GTX2+3, respectively, in only N‐1 hydroxylation. When various compounds previously reported to be substrates for STs in other organisms and paralytic shellfish poisoning toxins other than STX and GTX2+3 were added to the reaction mixture, N‐ST activity was not decreased. The enzyme required PAPS as the sole source of sulfate. The enzyme was optimally active at pH 6.0 and 25° C, and its activity was enhanced by Mg2+ and Co2+. The Km values of the N‐ST for STX and GTX2+3 were 16.1 μM and 29.8 μM, respectively.