TTX Content Research Articles

Tetrodotoxin Content of the Small Gastropod Nassarius sufflatus

Severe tetrodotoxin (TTX) poisoning due to small gastropods has been documented in Japan. In this study, we investigated the TTX content of the muscles and viscera of Nassarius sufflatus collected off the coast of Futaoi Island, Shimonoseki, Yamaguchi Prefecture, Japan, to prevent the occurrence of TTX poisoning caused by this small gastropod. Live specimens were obtained, and their muscles and viscera were collected. Test solutions were prepared from tissues of specimens and analyzed for TTX by HPLC-fluorescence detection. TTX was detected in both tissues at concentrations ranging from <0.1 to 18.2 μg/g for muscle and <0.1 to 130.7 μg/g for viscera. These results suggested that N. sufflatus accumulates TTX not only in its viscera but also in its muscles, and that precautions should be taken to prevent food poisoning due to this gastropod.

Geographical differences in the composition of tetrodotoxin and 5,6,11-trideoxytetrodotoxin in Japanese pufferfishes and their origins

Tetrodotoxin (TTX)-bearing fish are thought to accumulate TTXs in their bodies through a food chain that begins with marine bacteria. However, the mechanism of TTXs transfer between prey and predators in the food chain remains unclear and the reasons for regional differences in pufferfish toxicity are also unknown. To investigate these matters, we collected juveniles of four species of pufferfish, Takifugu alboplumbeus, Takifugu flavipterus, Takifugu stictonotus, and Chelonodon patoca, from various locations in the Japanese Islands, and subjected them to liquid chromatography-tandem mass spectrometry analysis for TTX and its analog 5,6,11-trideoxyTTX (TDT). Concentrations of these substances tended to be higher in pufferfish juveniles collected from the Sanriku coastal area (Pacific coast of northern Japan) than in those from other locations. Juveniles had higher concentrations of TTX at all locations than of TDT. Mitochondrial cytochrome c oxidase subunit I (COI) sequences specific to the TTX-bearing flatworm, Planocera multitentaculata, were detected in the intestinal contents of up to 100% of pufferfish juveniles from various sampling sites, suggesting that P. multitentaculata was widely involved in the toxification of the juveniles in the coastal waters of Japan. A toxification experiment was conducted on three species of pufferfish juveniles (T. alboplumbeus, Takifugu rubripes and C. patoca) using TTX-bearing flatworm eggs harboring equal amounts of TTX and TDT. The TTX content of juveniles fed on flatworm eggs was found to be more than twice that of TDT, suggesting that pufferfish preferentially incorporate TTX compared to TDT.

Development of a polyclonal antibody-based indirect competitive ELISA for the determination of tetrodotoxins in marine ribbon worms (NEMERTEA) and its comparison with high performance liquid chromatography-tandem mass spectrometry

Tetrodotoxin (TTX) is a potent neurotoxin frequently occurring in marine organisms along with its numerous analogues. To determine the total TTX content, we developed an enzyme-linked immunosorbent assay (ELISA) technique utilizing polyclonal antibodies against TTX. The technique was tested using extracts of marine worms of the phylum Nemertea and confirmed by HPLC-MS/MS. It proved to be suitable for a preliminary assessment of the toxicity of marine organisms.

Difference in tetrodotoxin content between two sympatric planocerid flatworms, Planocera multitentaculata and Planocera reticulata

Planocerid flatworms and the related species (Platyhelminthes: polycladida) are known as tetrodotoxin (TTX)-bearing organisms, and they contribute to toxification of marine organisms at higher trophic levels, such as pufferfish and sea slugs. However, little is known of their biology or ecology. In this study, we therefore investigated the occurrence and toxicity of two sympatric planocerids, Planocera multitentaculata and Planocera reticulata, in intertidal zones of the central region of mainland Honshu, Japanese Islands. Planocera multitentaculata was much more abundant than P. reticulata. Body weight was greater in P. multitentaculata than in P. reticulata. Although a significant difference in TTX concentration was not observed between the two species, total TTX content per individual was greater in P. multitentaculata.

Tetrodotoxin and saxitoxin in two native species of puffer fish, Sphoeroides marmoratus and Lagocephalus lagocephalus, from NE Atlantic Ocean (Madeira Island, Portugal)

The presence in EU waters of invasive tetrodotoxin (TTX) -harbouring puffer fishes has been receiving increasingly attention due to potential new threats posed by this potent neurotoxin. The present study investigates the occurrence of tetrodotoxin, saxitoxin (STX), and their analogues in two native puffer fish species from the NE Atlantic. High TTX content was detected by LC-MS/MS in several tissues of the Guinean puffer Sphoeroides marmoratus from Madeira Island (Portugal), reaching concentrations as high as 15 mg TTX kg−1 in the digestive tract of a male specimen and 7.4 mg TTX kg−1 in gonads of a female specimen. Several TTX analogues were also detected, including the 4-epi-TTX, 4,9-Anhydro-TTX, 5- 11- deoxyTTX and 6,11-dideoxyTTX. Although at low levels, STX was detected in liver of the Oceanic puffer Lagocephalus lagocephalus. Trace levels of decarbamoylsaxitoxin (dcSTX) were also observed in L. lagocephalus. This study reports the presence of TTX and STX in native fish from EU waters, highlighting the need for a proper understating of the origin, distribution and fate of these toxins in NE Atlantic.

Seasonal Changes in the Tetrodotoxin Content of the Flatworm Planocera multitentaculata.

Tetrodotoxin (TTX) is a potent neurotoxin that acts specifically on voltage-gated sodium channels on excitable membranes of muscle and nerve tissues. The biosynthetic process for TTX is unclear, although marine bacteria are generally thought to be the primary producers. The marine flatworm Planocera multitentaculata is a known TTX-bearing organism, and is suspected to be a TTX supplier to pufferfish. In this study, flatworm specimens were collected from an intertidal zone in Hayama, Kanagawa, Japan, the TTX content of the flatworm was measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and seasonal changes in TTX content were investigated. No significant difference in TTX concentration of the flatworm body was found between the spawning period and other periods. However, the TTX content in individual flatworms was significantly higher in the spawning period than at other times. The TTX content rose in association with an increase in the body weight of the flatworm.

Accumulation of tetrodotoxin from diet in two species of scavenging marine snails

A feeding experiment of TTX-containing diet was conducted using the small scavenging marine snails Pliarcularia globosa and Reticunassa festiva. Seventy-five specimens of each species were divided into 15 groups of 5 individuals, of which 3 groups were directly submitted, without feeding, to toxin quantification as described below. TTX was not detected. Each of the remaining 12 groups was accommodated in a plastic case (80×70×40 mm) filled with seawater, and fed for 24 hours with ovary tissue (0.1 g) of the pufferfish Takifugu vermicularis, whose TTX content had previously been determined. Then the seawater was exchanged for fresh seawater, the snails were reared for 4 days without feeding, and then the seawater was changed again. This feeding/rearing cycle (5 days) was repeated 8 times, and 3 groups were sampled every 2 cycles. The combined viscera and combined muscle of each group were each extracted with 0.1% aqueous acetic acid, and then TTX was quantified by liquid chromatography-mass spectrometry. The estimated amount of ingested TTX was calculated by multiplying the difference between the amounts of ovary tissue supplied and remaining by the toxin content (122-126 MU/g). Similar mean values of 5.1 MU/group/cycle in P. globosa and 5.3 MU/group/cycle in R. festiva were obtained. Toxin content (TTX amount per gram of tissue) and toxin amount (TTX amount per group) during the experimental period were 0.23-2.85 MU/g and 0.05-0.96 MU/group, respectively, in P. globosa viscera. Both values increased markedly from the 2nd cycle to the 6th cycle. In contrast, no such increase in toxin content/amount was observed throughout the experimental period in P. globosa muscle (<0.05-0.86 MU/g, <0.02-0.27 MU/group), R. festiva viscera (<0.05-0.8 MU/g, <0.02-0.33 MU/group), and R. festiva muscle (<0.05-0.81 MU/g, <0.02-0.23 MU/group). The remaining ratio of TTX (percentage of total toxin amount [sum of the toxin amount of viscera and muscle] to estimated TTX ingestion amount) was less than 4% in P. globosa, and less than 2% in R. festiva after the 4th cycle, suggesting that the possibility that these two species would accumulate TTX at levels high enough to raise food hygiene issues is low.

Change in the transfer profile of orally administered tetrodotoxin to non-toxic cultured pufferfish Takifugu rubripes depending of its development stage

To investigate the effects of growth (organ development) on tetrodotoxin (TTX) dynamics in the pufferfish body, TTX-containing feed homogenate was administered to 6- and 15-month old non-toxic cultured specimens of the pufferfish Takifugu rubripes at a dose of 40 mouse units (MU) (8.8 μg)/20 g body weight by oral gavage. After 24 h, the specimens were killed and the skin tissues (dorsal and ventral), muscle, liver, digestive tract, and gonads were separated. TTX content (μg/g) in each tissue, determined by liquid chromatography/mass spectrometry, revealed that the TTX distribution profile, particularly the TTX content of the liver, greatly differed between the two ages; the TTX score of 15-month old fish (3.3 μg/g) was nearly 5-fold that of 6-month old fish (0.68 μg/g). The total remaining TTX amount per individual (relative amount to the given dose) was 31% in 6-month old fish, of which 71% was in the skin, and 84% in 15-month old fish, of which 83% was in the liver. The gonadosomatic index (GSI) and hepatosomatic index (HSI) scores, and histologic observations of the gonads and liver suggest that although there is little difference in maturation stage between these two ages, there are clear distinctions in the developmental stage of the liver. The results suggest that the TTX dynamics in T. rubripes are linked to the development of the liver, i.e., the TTX taken up into the pufferfish body via food organisms is eliminated or transferred mainly to the skin in young fish with an undeveloped liver, but as the fish grow and the liver continues to develop, most of the TTX is transferred to and accumulated in the liver.

Toxin composition and toxicity dynamics of marine gastropod Nassarius spp. collected from Lianyungang, China

Consumption of nassariid gastropods often leads to poisoning incidents in some coastal provinces in China. To elucidate the pattern of toxicity dynamics and origin of toxins, samples of gastropod Nassarius spp. were collected from late May to early August 2007 from Lianyungang, Jiangsu province, where the poisoning incidents have been frequently reported. Toxicity was first screened with the mouse bioassay method, and tetrodotoxin and its analogues (TTXs) were analysed with high-performance liquid chromatography coupled with an ion-trap mass spectrometer (HPLC-MS n ). The toxicity of nassariid N. semiplicatus showed an ‘M’-shaped pattern of fluctuation during the sampling season. Two peaks of toxicity appeared in late May and late July. The maximum toxicity was recorded on 24 May, with the value of 846 mouse unit (MU) g−1 of tissue (wet weight). TTX and its analogues trideoxyTTX, 4-epiTTX, anhydroTTX and oxoTTX were detected in the nassariid samples. TrideoxyTTX but not TTX was the major toxin in all the samples. No paralytic shellfish poison (PSP) was detected in the sample with the maximum toxicity by HPLC-FLD analysis. Variation of TTX content in the tissue of nassariid gastropods correlates well with the dynamics of toxicity. It is suggested that TTXs are the major toxins corresponding to the toxicity of the nassariids, and May and July are the high-risk seasons for consumption of nassariids, which is critical for the management of poisoning incidents.

Transfer profile of intramuscularly administered tetrodotoxin to artificial hybrid specimens of pufferfish, Takifugu rubripes and Takifugu niphobles

Tetrodotoxin (TTX) was intramuscularly administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu niphobles to investigate toxin accumulation in hybrids, and TTX transfer/accumulation profiles in the pufferfish body. In the test fish administered 146 MU TTX in physiologic saline, TTX rapidly transferred from the muscle via the blood to other organs. Toxin transfer to the ovary rapidly increased to 53.5 MU/g tissue at the end of the 72-h test period. The TTX content in the liver and skin was, at most, around 4–6 MU/g tissue, and in the testis it was less than 0.01 MU/g tissue. On the other hand, based on the total amount of toxin per individual (% of the administered toxin), the skin and the liver contained higher amounts (20–54% and 2–24%, respectively), but the amount in the liver rapidly decreased after 8–12 h, and fell below the level in the ovary after 48 h. These findings suggest that part of the TTX is first taken up in the liver and then transferred/accumulated in the skin in male specimens and in the ovary in female specimens.

Change in Tetrodotoxin Content of the Puffer Fish Takifugu rubripes during Seed Production from Fertilized Eggs to Juveniles

Changes in tetrodotoxin (TTX) content of the puffer fish Takifugu rubripes during seed production were examined. Two mature female puffer fish T. rubripes (samples 1 and 2) that contained TTX were used. The toxic eggs were artificially fertilized, and hatchlings were reared in an indoor tank for 50 days and then in a netcage at sea for an additional 48 or 38 days. The TTX content of the fertilized eggs of sample 1 was initially 13.0 microg TTX/g, transiently increased to 67.6 microg TTX/g at 4 days after hatching, and then gradually decreased to 0.28 microg TTX/g at 98 days. In contrast, the total TTX content in an individual was 0.016 microg TTX at the fertilization stage and 0.01-0.03 microg TTX at the larval stage until 30 days after hatching. Thereafter, the total TTX content increased remarkably during culture in the netcage at sea, reaching 4.80 microg TTX at 98 days. Change in the TTX content of sample 2 showed a similar tendency to that of sample 1. The present study showed that the TTX content per gram of puffer fish body weight decreased during progression from fertilized eggs to juveniles, whereas the total TTX content increased.

Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver

Non-toxic cultured juvenile kusafugu Fugu niphobles were fed with a diet containing highly toxic natural komonfugu Fugu poecilonotus liver until the 30th day (8.0 μg of TTX and 3.7 μg of 4,9-anhydroTTX/fish/day), and then fed with a non-toxic diet until the 240th day. During the 30–240th day, five or six fish were periodically sampled six times, and the contents of TTX and 4,9-anhydroTTX in each tissue were determined. The total TTX and 4,9-anhydroTTX accumulated in all tissues tested was not significantly changed during the experimental period, both being kept at 70% of administrated doses. However, in the liver, the TTX content accounted to be 120 μg (50% of administrated) on the 30th day, and then it gradually decreased to 50 μg until the 240th day, while 4,9-anhydroTTX content was kept at approximately 40 μg (40% of administrated) during all the experimental periods. In contrast to the liver, in the skin, TTX and 4,9-anhydroTTX were 40 and 5 μg, respectively, on the 30th day, and then gradually increased to 80 and 24 μg, respectively, until the 240th day. In the intestine, TTX and 4,9-anhydroTTX contents were kept at 25 and 12 μg, respectively, during all the experimental periods. According to these results, we assumed that a part of TTX accumulated in the liver was slowly transferred to the skin.

In vitro accumulation of tetrodotoxin in pufferfish liver tissue slices

The liver tissue slices of pufferfish accumulate in vitro tetrodotoxin (TTX), when incubated with minimum essential medium containing TTX. In the case of Takifugu rubripes liver slices incubated at a concentration of 25 μg TTT/ml, TTX of 3.9 μg/g was first detected at 2 h and increased to 15 μg/g at 48 h. The TTX content accumulated was not decreased, even when the slices were further incubated without TTX for additional 48 h. Another species of pufferfish T. paradalis also showed similar trend in TTX accumulation, except they accumulated higher concentration of TTX (36.4 μg/g at 48 h) than T. rubripes. On the contrary, in the cases of the liver slices from parrot-bass Oplegnathus fasciatus, green ling Hexagrammos otakii and filefish Thamnaconus modestus incubated at a concentration of 25 μg TTX/ml, TTX of 3–4 μg/g was detected even at 0.5 h. However, no significant change in TTX contents was recognized during the incubation for 48 h. Further incubation of the filefish liver slices without TTX for additional 48 h did not decrease the TTX content. It is unlikely that the liver slices of filefish as well as pufferfish rapidly excrete TTX. These results suggest that the difference in the accumulation of TTX between pufferfish and filefish livers is ascribable to the difference not in the TTX excreting ability but in the ability to take up TTX.

The binding of tetrodotoxin to nerve membranes.

1. The% reduction in size of the externally recorded action potential produced by concentrations of tetrodotoxin (TTX) in the range 6-300 nM was determined for the small non-myelinated fibres of the rabbit cervical vagus nerve and of the walking leg nerves of crab and lobster. The concentration of TTX for 50% reduction was around 80 nM for rabbit vagus and 14 nM for crab nerve.2. Bio-assay procedures were devised to measure the amount of TTX taken up by a nerve when it was exposed to a very small volume of a solution whose TTX content was just great enough to produce 100% block of conduction. The extracellular space of each nerve was determined with [(14)C]sugar so that an allowance could be made for extracellular dilution.3. The TTX binding by rabbit, crab and lobster nerve was respectively 0.064, 0.053 and 0.036 p-mole/mg wet weight of nerve.4. The binding of saxitoxin was measured in rabbit vagus nerve, and found to be much the same as that of TTX.5. Control experiments on rabbit sciatic nerve, where the area of excitable membrane was much smaller, showed that there was relatively little unspecific binding of TTX.6. In view of the evidence presented here and elsewhere that the blocking of sodium conductance by TTX involves the attachment of only one TTX molecule at each sodium site, and that unspecific binding of TTX does not cause serious errors, these results suggest that in 1 mum(2) of nerve membrane the number of sodium sites is 75 for rabbit, 49 for crab, and 36 for lobster nerve.