Distribution Of Tetrodotoxin Research Articles

Distribution of tetrodotoxin and its analogs in the toxic flatworm Planocera multitentaculata from Honshu Island, Japan

Tetrodotoxin (TTX), one of the most potent naturally occurring neurotoxins, has been detected in both marine and terrestrial organisms. The marine polyclad flatworm Planocera multitentaculata is known to contain high levels of TTX throughout its life cycle, including in the egg and larval stages. We recently reported that P. multitentaculata from the coastal area of Hayama, Kanagawa, Japan, possesses TTX analogs including 5,6,11-trideoxyTTX, monodeoxyTTXs, dideoxyTTXs, and 11-norTTX-6(S)-ol. It is not known whether all P. multitentaculata occurring in the Japanese archipelago possess major TTX analogs, or whether regional variations in TTX profile exist. In this study, we used high-resolution liquid chromatography–mass spectrometry to investigate the content profile of TTX and its analogs in P. multitentaculata collected from several regions of the Japanese coast. We detected TTX and its major analogs 5,6,11-trideoxyTTX, dideoxyTTXs, monodeoxyTTXs, and 11-norTTX-6(S)-ol in all specimens regardless of the sampling site. In addition, their relative proportions showed a similar pattern. These results suggest that the flatworm may have mechanisms involved in maintaining the proportions of TTX and its analogs in the body. How the toxic flatworms acquire the highly concentrated toxins and maintain the proportions of TTXs is therefore of great interest and further investigation.

Levels and distribution of tetrodotoxin in the blue-lined octopusHapalochlaena fasciatain Japan, with special reference to within-body allocation

ABSTRACTThe toxins present in poisonous and venomous animals have both offensive and defensive functions. The blue-lined octopus Hapalochlaena fasciata has tetrodotoxin (TTX) in various organs, including the salivary glands, muscles and skin, and TTX is assumed to be used in both offence and defence. In this study, we explore the evolution of toxicity in H. fasciata by measuring the levels and distribution of TTX in H. fasciata specimens collected in Japan and by investigating the TTX allocation within the body. TTX was detected in all 13 adults and 3 paralarvae studied. The concentration of TTX was highest in the anterior and posterior salivary glands, and the total amount of TTX was highest in the muscles and skin. Larger individuals had significantly higher amounts of TTX, but the TTX concentrations in the muscles and skin were not correlated with body weight. There were large individual differences in TTX concentration and some individuals showed extremely low TTX levels. These individual differences may be related to regional, seasonal and sexual differences, although the origin of TTX in this species is still unclear. Furthermore, we found a strong positive correlation between TTX concentrations in the posterior salivary gland and TTX concentrations in muscles and skin, with the former being c. 20 times greater than the latter. This suggests that the allocation ratio may not result from a plastic allocation, reflecting the TTX needs of each organ or the amount of available TTX, but rather may, at least partly, be evolutionarily fixed.

Distribution of Tetrodotoxin in Pacific Oysters (Crassostrea gigas).

A potent and heat-stable tetrodotoxin (TTX) has been found to accumulate in various marine bivalve species, including Pacific oysters (Crassostrea gigas), raising a food safety concern. While several studies on geographical occurrence of TTX have been conducted, there is a lack of knowledge about the distribution of the toxin within and between bivalves. We, therefore, measured TTX in the whole flesh, mantle, gills, labial palps, digestive gland, adductor muscle and intravalvular fluid of C. gigas using liquid chromatography-tandem mass spectrometry. Weekly monitoring during summer months revealed the highest TTX concentrations in the digestive gland (up to 242 µg/kg), significantly higher than in other oyster tissues. Intra-population variability of TTX, measured in the whole flesh of each of twenty animals, reached 46% and 32% in the two separate batches, respectively. In addition, an inter-population study was conducted to compare TTX levels at four locations within the oyster production area. TTX concentrations in the whole flesh varied significantly between some of these locations, which was unexplained by the differences in weight of flesh. This is the first study examining TTX distribution in C. gigas and the first confirmation of the preferential accumulation of TTX in oyster digestive gland.

Accumulation and Elimination of Tetrodotoxin in the Pufferfish Takifugu obscurus by Dietary Administration of the Wild Toxic Gastropod Nassarius semiplicata.

To investigate pufferfish accumulation, elimination, and distribution of tetrodotoxin (TTX), Takifugu obscurus was fed with wild TTX-containing gastropod Nassarius semiplicata to simulate the natural food chain. Three-month-old non-poisonous T. obscurus was fed with wild toxic N. semiplicata at three exposure dose for 28 days, and later, with toxin-free food until day 67. Three fish individuals from each treatment were sampled, and the distribution of TTX in different tissues was measured. The results showed that the accumulation ratio of TTX in the three exposure dose groups ranged from 35.76% to 40.20%. The accumulation ratio in the skin and liver was the highest amongst all tissues, accounting for more than 85% of the total TTX, whereas that in the kidney and gallbladder was the lowest (0.11–0.78%). Studies on the kinetic of TTX accumulation and elimination revealed that the skin was the tissue with the highest accumulation speed constant (8.06), while the liver, kidney, and intestinal tract showed the highest speed of TTX elimination. The time required for TTX reduction to reach the safety limit could be predicted by using standard elimination equations. Qualitative analysis by UPLC-MS/MS revealed the occurrence of seven TTX derivatives in T. obscurus; of these TTX, 5-deoxy TTX, 11-deoxy TTX, 4,9-anhydro TTX were found in all tested tissues.

Distribution of Tetrodotoxin in the New Zealand Clam, Paphies australis, Established Using Immunohistochemistry and Liquid Chromatography-Tandem Quadrupole Mass Spectrometry.

Tetrodotoxin (TTX) is one of the most potent neurotoxins known. It was originally thought to only occur in puffer fish but has now been identified in twelve different classes of freshwater and marine organisms, including bivalves. Despite being one of the world’s most studied biotoxins, its origin remains uncertain. There is contradictory evidence regarding the source of TTX and its pathway through food webs. To date, the distribution of TTX has not been examined in bivalves. In the present study, 48 Paphies australis, a TTX-containing clam species endemic to New Zealand, were collected. Thirty clams were dissected, and organs and tissues pooled into five categories (siphons, digestive gland, adductor muscles, and the ‘rest’) and analyzed for TTX using liquid chromatography-mass spectrometry (LC-MS). The micro-distribution of TTX was visualized in the remaining 18 individuals using an immunohistological technique incorporating a TTX-specific monoclonal antibody. The LC-MS analysis revealed that siphons contained the highest concentrations of TTX (mean 403.8 µg/kg). Immunohistochemistry analysis showed TTX in the outer cells of the siphons, but also in the digestive system, foot, and gill tissue. Observing TTX in organs involved in feeding provides initial evidence to support the hypothesis of an exogenous source in P. australis.

Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China

A monoclonal antibody against tetrodotoxin (TTX) was produced from the hybridoma cell line T6D9, which was established by the fusion of Sp2/0 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with the TTX–keyhole limpet hemocyanin (KLH) conjugate. This monoclonal antibody belongs to the IgG1 subclass; the affinity constant of the antibody is 2.4 × 10−8 mol l−1. The relative cross-reactivity of the antibody with TTX was 100%, but with saxitoxin, KLH and bovine serum albumin (BSA) it was less than 1%, respectively. The titre of the antibody in ascites was 6.4 × 106; the reference working concentration was 1:1.2 × 105. By using this monoclonal antibody, a competitive indirect enzyme-linked immunoabsorbant assay (ELISA) for the analysis of TTX was developed. The linear portion of the dose–response curve of TTX concentration was in range 5–500 ng ml−1. The limit of detection was 5 ng ml−1 according 10% inhibition with TTX to anti-TTX monoclonal antibody. The concentration of TTX inhibiting 50% of antibody binding was about 50 ng ml−1. The recoveries from TTX spiked samples were 79.5–109.5%. In addition, the toxicity of some wild puffer fish specimens captured from south-east China and the Yangzi River in Jiangsu province was determined. The results indicate that the toxicity and toxin tissue distribution vary in different species of wild puffer fish.

Distribution of Tetrodotoxin in Pufferfish Collected from Coastal Waters of Sihanouk Ville, Cambodia

In Cambodia, fatal food poisonings associated with the consumption of pufferfish have occurred for decades, but the causative species or toxins have never been documented. Herein, we investigated the toxicity of three pufferfish species of the genus Lagocephalus collected from the coastal waters of Sihanouk Ville, one of the main regions where poisonings have occurred. L. wheeleri and L. spadiceus were non-toxic, whereas L. lunaris was toxic and all of its body tissues exhibited toxicity levels exceeding the safety limit for human consumption (10 mouse units/g). Tetrodotoxin (TTX) was identified as the main toxin in this species; no paralytic shellfish poison(s) were detected. Consequently, we can confirm pufferfish to be a hazardous reservoir of TTX in Sihanouk Ville. It is likely that L. lunaris is one of the causative species of past pufferfish poisonings that have occurred in Cambodia.

Distribution of tetrodotoxin in the xanthid crab ( Atergatis floridus) collected in the coastal waters of Kanagawa and Wakayama Prefectures

The objective of this investigation was to study the distribution of tetrodotoxin (TTX) in the xanthid crab ( Atergatis floridus) found in the coastal waters of Kanagawa and Wakayama Prefectures of Japan using mouse assay methods. We used 32 crab samples (18 males and 14 females) and toxicity was analyzed on 13 parts of the body of each sample. The muscle of chelipeds was found to be toxic in all the samples with a wide range of toxicity (5–237 MU/g), whereas the toxicity in the muscle of the cephalothorax was found to be non-toxic (below detectable limit) in all the samples [ Narita, H., Watanabe, K., Baba, K., Ohgami, H., Ai, T.K., Igarashi, Y., Nara, M., Noguchi, T., Hashimoto, K., 1987. The toxicity of digestive gland of trampet shells inhabiting the coast of Shizuoka Prefecture. J. Food Hyg. Soc. Jpn. 28, 115–118.]. Further investigation of different parts of the chelipeds indicated that the muscle of the palm and carpus are usually toxic and that of merus and ischium are almost non-toxic. Toxicity of the muscles of palm ranged between 7 and 52 MU/g, whereas toxicity of the muscle of ischium was below detectable limit. Results from our study indicate clear contrast in the distribution of tetrodotoxin in muscles of different parts of the xanthid crabs, plausibly due to some inherent physiological mechanism. Further investigation is necessary to understand the mechanism responsible for such contrast.

248 Change in distribution of tetrodotoxin in the ovary of a marine pufferfish Takifugu vermicularis during spawning season

248 Change in distribution of tetrodotoxin in the ovary of a marine pufferfish Takifugu vermicularis during spawning season

Toxicities and distribution of tetrodotoxin in the tissues of puffer fish found in the coast of the Baja California Peninsula, Mexico

Toxicities and tetrodotoxin distribution in tissues of five puffer fish species commonly found in the littoral of Baja California Peninsula, Mexico ( Sphoeroides annulatus, S. lobatus, S. lispus, Arothron meleagris and Canthigaster punctatissima) were evaluated by bioassay and HPLC. The toxicities estimated as tetrodotoxin-equivalents of all species were more than 0.42 μg/g in at least one of the tissues tested, and the highest was found in S. lispus liver (130 μg/g).

5HT4Receptors Couple Positively to Tetrodotoxin-Insensitive Sodium Channels in a Subpopulation of Capsaicin-Sensitive Rat Sensory Neurons

The distribution of tetrodotoxin (TTX)-sensitive and -insensitive Na+ currents and their modulation by serotonin (5HT) and prostaglandin E2 (PGE2) was studied in four different types of dorsal root ganglion (DRG) cell bodies (types 1, 2, 3, and 4), which were previously identified on the basis of differences in membrane properties (). Types 1 and 2 DRG cells expressed TTX-insensitive Na+ currents, whereas types 3 and 4 DRG cells exclusively expressed TTX-sensitive Na+ currents. Application of 5HT (1-10 microM) increased TTX-insensitive Na+ currents in type 2 DRG cells but did not affect Na+ currents in type 1, 3, or 4 DRG cells. The 5HT receptor involved resembled the 5HT4 subtype. It was activated by 5-methoxy-N,N-dimethyltryptamine (10 microM) but not by 5-carboxyamidotryptamine (1 microM), (+)-8-hydroxydipropylaminotetralin (10 microM), or 2-methyl-5HT (10 microM), and was blocked by ICS 205-930 with an EC50 of approximately 2 microM but not by ketanserin (1 microM). PGE2 (4 or 10 microM) also increased Na+ currents in varying portions of cells in all four groups. The effect of 5HT and PGE2 on Na+ currents was delayed for 20-30 sec after exposure to 5HT, suggesting the involvement of a cytosolic diffusible component in the signaling pathway. The agonist-mediated increase in Na+ current, however, was not mimicked by 8-chlorophenylthio-cAMP (200 microM), suggesting the possibility that cAMP was not involved. The data suggest that the 5HT- and PGE2-mediated increase in Na+ current may be involved in hyperesthesia in different but overlapping subpopulations of nociceptors.

Distribution of tetrodotoxin in Taiwan puffer Lagocephalus wheeleri and L. gloveri: D.-F. Hwang, C.-Y. Kao, S.-C. Tsai and S.-S. Jeng (Department of Marine Food Science, National Taiwan Ocean University, Keelung, Taiwan, R.O.C.).

Distribution of tetrodotoxin in Taiwan puffer Lagocephalus wheeleri and L. gloveri: D.-F. Hwang, C.-Y. Kao, S.-C. Tsai and S.-S. Jeng (Department of Marine Food Science, National Taiwan Ocean University, Keelung, Taiwan, R.O.C.).

Distribution of tetrodotoxin, 6- epitetrodotoxin, and 11-deoxytetrodotoxin in newts

Tetrodotoxin was detected in all nine species of newts tested, 6- epitetrodotoxin in six species, and 11-deoxytetrodotoxin in five species. Only one species lacked the analogues, thus suggesting that the analogues were common metabolites among newts. Toxin levels were high in Taricha granulosa and Notophthalmus viridescens followed by Cynops spp. Distinct differences in toxin contents and profiles existed among species and tissues

Distribution of tetrodotoxin in various organs of the starfish Astropecten polyacanthus

Tissues of various organs of the starfish Astropecten polyacanthus collected in the Hiroshima Prefecture from April 1985 to June 1986 were analyzed for lethal potency by the assay method for tetrodotoxin. The ovary showed the highest potency (47 to 1 450 MU g-1), followed by the digestive organs (<10 to 960 MU g-1) and the exoskeleton including spines and tube-feet (<10 to 170 MU g-1). The pyloric caecum and testis were less toxic. Overall toxicity was remarkably higher in females (2 060±382 MU, mean±SE) than in males (1 106±214 MU).

Distribution of tetrodotoxin in the tissues of the flatworm Planocera multitentaculata (Platyhelminthes)

K. Miyazawa, J. K. Jeon, T. Noguchi, K. Ito and K. Hashimoto. Distribution of tetrodotoxin in the tissues of the flatworm Planocera multitentaculata (Platyhelminthes). Toxicon 25, 975 – 980, 1987.—Tetrodotoxin distribution in various tissues of the flatworm Planocera multitentaculata was examined. The oviduct was most toxic (2000 – 3240 MU/g), followed by digestive organs (900 – 1400 MU/g), oviduct-free genital organs (430 – 590 MU/g), etc. The mucus secreted also showed some lethal potency. On the other hand, the eggs laid, whether in laboratory aquarium or on coastal rocks, exhibited an extremely high potency up to 10,700 MU/g. It was suggested from these findings that flatworms possess tetrodotoxin as a defense or alarm substance against predators.