Osamu Arakawa

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.

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.

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.

Occurrence of paralytic shellfish poison in Bangladeshi freshwater puffers.

Two species of freshwater puffer fish, Tetraodon cutcutia and Chelonodon patoca, collected from several locations in Bangladesh, showed lethal potency in mice ranging from 2.0 to 40.0 MU/g tissue as paralytic shellfish poison. In both species, toxicity of the skin was generally higher than the other tissues examined (muscle, liver and ovary). Water-soluble toxins from T. cutcutia were partially purified by activated charcoal treatment followed by column chromatographies using Bio-Gel P-2 and Bio-Rex 70. Analyses by cellulose acetate membrane electrophoresis and high-performance liquid chromatography with fluorometric detection demonstrated that the toxins were composed of saxitoxin, decarbamoylsaxitoxin, gonyautoxins 2 and 3, decarbamoylgonyautoxins 2 and 3, and three unidentified components which are possibly related to paralytic shellfish poison.

Occurrence of paralytic shellfish poisons in thai freshwater puffers

Screening tests were carried out on the toxicity of freshwater puffers Tetraodon leiurus complex and Tetraodon suvatii collected from Udonthani province, north-eastern Thailand. Toxicity was highest in the liver and varied according to the location and season of fish catch. Fish which were reared in tap water for 3 months reduced the toxicity substantially. Partial purification was achieved by an ultrafiltration technique. Toxin components were consequently identified by high-performance liquid chromatography. It was found that toxins separated from the eggs, liver, skin and muscle of these puffers were composed of saxitoxin, neosaxitoxin and decarbamoylsaxitoxin.

Occurrence of carbamoyl-N-hydroxy derivatives of saxitoxin and neosaxitoxin in a xanthid crab Zosimus aeneus

Two novel paralytic toxins were isolated from toxic specimens of a xanthid crab Zosimus aeneus inhabiting Ishigaki Island, Okinawa. The structures of two of these were deduced to be carbamoyl-N-hydroxysaxitoxin and carbamoyl-N-hydroxyneosaxitoxin based on electrophoresis, high performance liquid chromatography, electrospray ionization mass spectrometry, 1H NMR, 13C NMR and conversion experiments. They showed specific toxicities of 1700 and 1400 mouse units per mg on i.p. injection into mice.

Dimethyl sulfoxide (DMSO) blocks GABA-induced current in rat dorsal root ganglion neurons

The effects of dimethyl sulfoxide (DMSO) on gamma-aminobutyric acid (GABA)-induced Cl- currents of rat dorsal root ganglion neurons in primary culture were studied by the whole-cell patch-clamp technique. Bath application of 5 microM GABA evoked sustained inward and outward currents at membrane potentials of -60 mV and +30 mV, respectively, when the external and internal solutions both contained 142 mM Cl-. DMSO at concentrations of 0.3-3% (v/v) caused a dose-dependent inhibition of the inward current induced by 5 microM GABA at -60 mV. DMSO at 3% inhibited the GABA-induced inward and outward currents at -60 mV and +30 mV to similar extents (54% and 53% of the control, respectively), but the time-course of inhibition of the outward current at +30 mV was much faster than that of the inward current at -60 mV. These results suggest that DMSO suppressed the currents by interacting with GABA receptor-Cl- channel complex protein rather than by affecting the lipid-bilayer of the cell membrane.

Tetrodotoxin and related substances in a ribbon worm Cephalothrix linearis (Nemertean)

A ribbon worm Cephalothrix linearis (Nemertean) showed a high toxicity, of up to 22,000 MU/g proboscis and 13,600 MU/g body, in terms of tetrodotoxin. This organism secreted the toxin from the skin when wiped with gauze. The toxin was partially purified from the secretion as well as the wiped body by ultrafiltration and Bio-Gel P-2 column chromatography. Thin-layer chromatographic, electrophoretic, high performance liquid chromatographic, and gas chromatography-mass spectrometric analyses showed that the secreted toxin was composed almost exclusively of a tetrodonic acidic-like substance, whereas the remaining toxin in the wiped body consisted of the said substance and tetrodotoxin.

Properties of hemagglutinins newly separated from toxic phytoplankton.

Unique substances of the hemagglutinating activity were separated from unialgal cultures of the toxic phytoflagellates, Chattonella marina and Gymnodinium sp. Molecular masses of the substances were estimated to be 14,000 in Chattonella and 15,000 in Gymnodinium. These two substances contained phosphorus, hexose (galactose) and glycerol in the following molar ratios; 1.00:1.18:1.24 (Chattonella) and 1.00:1.07:1.10 (Gymnodinium). Hemagglutinating titres using rabbit red blood cells were inferred to be 25,000 in both preparations. In the presence of Chattonella hemagglutinin (0.10-0.50 microM), the growth of C. marina in Provasolis ES media was little affected during 24 h of incubation, whereas a marked suppression took place in growth in Gymnodinium sp. (50% inhibition, 0.12 microM) or the diatom Nitzschia closterium (0.17 microM). On the other hand, Gymnodinium hemagglutinin inhibited the growth of the three species of phytoplankton. A 50% inhibition occurred at 0.12 microM in C. marina, at 0.22 microM in N. closterium or at 0.50 microM in Gymnodinium sp.

Tetrodotoxin in two species of ribbon worm (Nemertini), Lineus fuscoviridis and Tubulanus punctatus

Extracts of two species of ribbon worm, Lineus fuscoviridis and Tubulanus punctatus, had lethal potencies in mice from 15-503 MU/g of worm and from less than 10-540 MU/g, respectively. The toxins were partially purified by ultrafiltration and column chromatography using Bio-Gel P-2 and Bio-Rex 70 (H+ form). Thin-layer chromatographic, high performance liquid chromatographic and gas chromatography-mass spectrometric analyses revealed the presence of tetrodotoxin and anhydrotetrodotoxin plus some unidentified compounds.