Toxicity and Toxin Composition of the Greater Blue-Ringed Octopus Hapalochlaena lunulata from Ishigaki Island, Okinawa Prefecture, Japan.

The toxicity of the greater blue-ringed octopus Hapalochlaenalunulata, whose bite is fatal to humans, was examined to better understand and prevent deaths from accidental bites. Living specimens were collected from tide pools on Ishigaki Island, Okinawa Prefecture, Japan, in November and December of 2015, 2016, and 2017. The specimens were examined for the anatomical distribution of the toxicity, which was expressed in terms of mouse units (MU), by the standard bioassay method for tetrodotoxin (TTX) in Japan. Paralytic toxicity to mice was detected in all of the soft parts. The posterior salivary glands exhibited the highest toxicity score with a maximum level of 9276 MU/g, which was classified as "strongly toxic" (more than 1000 MU/g tissue) according to the classification of toxicity established by the Ministry of Health, Labor and Welfare of Japan, followed by the hepatopancreas (21.1 to 734.3 MU/g), gonads (not detectable to 167.6 MU/g), arms (5.3 to 130.2 MU/g), and other body areas (17.3 to 107.4 MU/g). Next, the toxin from the salivary glands was partially purified by a Sep-Pak C18 cartridge and an Amicon Ultra Centrifugal Filter with a 3000-Da cut-off, and analyzed by liquid chromatography-mass spectrometry (LC-MS) equipped with a φ2.0 × 150-mm (5 µm) TSKgel Amide-80 column (Tosoh, Tokyo, Japan) with a mixture of 16 mM ammonium formate buffer (pH 5.5) and acetonitrile (ratio 3:7, v/v) as a mobile phase. This study aimed to clarify the toxicity and the composition of TTX and its derivatives in this toxic octopus. The main toxin in this toxic octopus was identified as TTX, along with 4-epi TTX, 4, 9-anhydroTTX and 6-epi TTX. Further, the toxicity of this species is also significant from a food hygiene point of view.

Food poisonings by ingestion of cyprinid fish.

Raw or dried gallbladders of cyprinid fish have long been ingested as a traditional medicine in the Asian countries, particularly in China, for ameliorating visual acuity, rheumatism, and general health; however, sporadic poisoning incidences have occurred after their ingestion. The poisoning causes complex symptoms in patients, including acute renal failure, liver dysfunction, paralysis, and convulsions of limbs. The causative substance for the poisoning was isolated, and its basic properties were examined. The purified toxin revealed a minimum lethal dose of 2.6 mg/20 g in mouse, when injected intraperitoneally. The main symptoms were paralysis and convulsions of the hind legs, along with other neurological signs. Liver biopsy of the euthanized mice clearly exhibited hepatocytes necrosis and infiltration of neutrophils and lymphocytes, suggesting the acute dysfunction of the liver. Blood tests disclosed the characteristics of acute renal failure and liver injury. Infrared (IR) spectrometry, fast atom bombardment (FAB) mass spectrometry, and 1H- and 13C-nuclear magnetic resonance (NMR) analysis indicated, a molecular formula of C27H48O8S, containing a sulfate ester group for the toxin. Thus, we concluded that the structure of carp toxin to be 5α-cyprinol sulfate (5α-cholestane-3α, 7α, 12α, 26, 27-pentol 26-sulfate). This indicated that carp toxin is a nephro- and hepato- toxin, which could be the responsible toxin for carp bile poisoning in humans.

[Distribution of toxicity in liver of wild pufferfish Takifugu rubripes].

Livers from wild pufferfish, Takifugu rubripes, can be described as having a smooth frontal side and an upper-region that is attached to the hepatic portal vein. Based on this description, the liver can be divided into 10 parts (L1-5 and R1-5), and in this work, the lethal potency of each part was determined by mouse bioassay. Among the raw livers from 58 individuals, all 10 parts of 16 individuals, and some parts of 4 individuals showed mouse lethality, but no toxicity was detected in any part of the liver from 22 individuals. In the livers of 4 individuals that were partially toxic, the lethal potency of the toxic parts was less than 4 mouse units (MU)/g, and no part showed especially high toxicity. The remaining 16 individuals were considered non-toxic based on the assay of only one part. Among 13 frozen livers, all parts of 9 individuals were toxic, while all parts of 4 individuals were non-toxic. Liquid chromatography-mass spectrometry analysis revealed that all parts of a weakly toxic raw liver and a strongly toxic frozen liver had tetrodotoxin as the major toxin. Regarding the 16 raw and 9 frozen livers, whose parts were all toxic, the relative lethal potency of each part to the mean lethal potency of the individual (8.9-709 MU/g) was calculated, and subjected to a two-way analysis of variance with 2 factors (left/right and top/bottom) for each group of livers (raw or frozen). The analysis indicated non-significance among factors and interactions at a significance level of 5% in the frozen livers. However, in the raw livers, although no interaction between the 2 factors was detected, the right-side and the 4th-portion from the top were significantly higher than the left-side and the other portions, respectively. Therefore, we concluded that individual inspection using R4, which is the region that lies below the right-center location of the liver, is suitable for toxicity evaluation of liver to ensure the safe consumption of pufferfish.

Tetrodotoxin poisoning due to pufferfish and gastropods, and their intoxication mechanism.

Marine pufferfish generally contain a large amount of tetrodotoxin (TTX) in their skin and viscera, and have caused many incidences of food poisoning, especially in Japan. Edible species and body tissues of pufferfish, as well as their allowable fishing areas, are therefore clearly stipulated in Japan, but still 2 to 3 people die every year due to pufferfish poisoning. TTX is originally produced by marine bacteria, and pufferfish are intoxicated through the food chain that starts with the bacteria. Pufferfish become nontoxic when fed TTX-free diets in a closed environment in which there is no possible invasion of TTX-bearing organisms. On the other hand, TTX poisoning due to marine snails has recently spread through Japan, China, Taiwan, and Europe. In addition, TTX poisoning of dogs due to the ingestion of sea slugs was recently reported in New Zealand. TTX in these gastropods also seems to be exogenous; carnivorous large snails are intoxicated by eating toxic starfish, and necrophagous small-to-medium snails, the viscera of dead pufferfish after spawning. Close attention must be paid to the geographic expansion and/or diversification of TTX-bearing organisms, and to the sudden occurrence of other forms of TTX poisoning due to their ingestion.

[Toxicity and toxin profiles of xanthid crabs collected around Nakanoshima in the Tokara Islands, Japan].

A total of 36 specimens of 5 xanthid crab species, Zosimus aeneus (n=16), Xanthias lividus (n=4), Leptodius sanguineus (n=3), Daira perlata (n=10) and Eriphia sebana (n=3), were collected around Nakanoshima Island, which is located at the northeastern part of the Tokara Islands, Kagoshima Prefecture, Japan in May and July 2000, and their toxicity was determined by mouse bioassay. Nine of 16 Z. aeneus specimens and all of 4 X. lividus specimens showed lethal potency to mice (2.1-11 MU/g, 2.8-8.6 MU/g, respectively), whereas all the other species were non-toxic (less than 2.0 MU/g). LC/MS analyses indicated that the toxin of the Z. aeneus specimens was mainly composed of tetrodotoxin (41% of total toxicity), and 11-oxotetrodotoxin contributed to the remaining toxicity. The toxin of the X. lividus specimens, however, was apparently not tetrodotoxin. In HPLC-FLD analyses, no paralytic shellfish poison component was detected in either of the two toxic species.

[Survey of food poisoning incidents in Japan due to ingestion of marine boxfish and their toxicity].

From 1990 to 2008, 9 food poisoning incidents due to ingestion of marine boxfish occurred in Nagasaki, Miyazaki, Mie and Kagoshima Prefectures, Japan, and a total of 13 persons were poisoned. Their main symptom was severe muscle pain arising from rhabdomyolysis, which was usually accompanied by the discharge of black urine and abnormal elevation of serum creatine phosphokinase. Twelve out of the 13 victims recovered in a few days to two months, while one died after approximately 2 weeks. Since the symptoms were very similar to those caused by parrotfish "aobudai" Scarus ovifrons poisoning, the causative substance was considered to be parrotfish toxin, i.e., a palytoxin-like substance. Epidemic surveys after the incidents in Miyazaki and Nagasaki identified the leftovers as "hakofugu" Ostracion immaculatus. During screening tests to clarify the toxicity of boxfish from Western Japan, 47 of 129 specimens (36.4%) of O. immaculatus, and 7 of 18 specimens (38.9%) of "umisuzume" Lactoria diaphana were found to show acute and/or delayed lethal activity to mice (0.5-2.0 mouse unit/g). Among the tissues tested, the frequency of toxicity was highest in the viscera excluding liver (28.6% in O. cubicus, 33.3% in L. diaphana), followed by muscle (10.9%, 5.6%) and liver (6.2%, 5.6%). From the above results, we conclude that O. cubicus and L. diaphana inhabiting the coast of Japan sometimes contain toxic substance(s), which can sporadically cause food poisonings very similar to parrotfish poisoning.

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.

[The toxicity of starfishes, Astropecten genus, inhabiting the coast of Toyama Bay].

Toxicity study was conducted using an official method (mouse assay) on 77 individual starfishes of the Astropecten genus collected in 5 sites inside Toyama Bay during the period from July 2002 to January 2003. Three kinds of starfishes were toxic, i.e., Astropecten polyacanthus, A. scoparius, and A. latespinosus meissner. Of A. polyacanthus. Those collected in the winter and spring were highly toxic, and there were regional differences in toxicity. The toxicity of A. latespinosus meissner, which inhibits only Himi, was less than that of A. polyacanthus. The toxicity of A. scoparius was weak. Toxic components were extracted and analyzed by LC/MS. Tetrodotoxin and related substances were confirmed to be present in all 3 kinds of starfishes.

[Toxicity of puffer fish fins].

Puffer fish is prized as a Japanese traditional food and its fin is also used in the cuisine. However, whether the fin is edible or not is determined for convenience from the toxicity of skin, since little information is available about the toxicity of puffer fish fins. In the present study, we examined the toxicity of fins and skin of three toxic species, Takifugu vermicularis, T. snyderi, and T. porphyreus. The toxicity of T. vermicularis fins (< 5-52.4 MU/g) was significantly lower than that of skin (<5-1200 MU/g). HPLC analysis showed that tetrodotoxin was a major toxic principle irrespective of the toxicity value in each tissue of T. vermicularis. In the case of T. snyderi and T. porphyreus, the toxicity of fins was at almost the same level as that of the skin. The toxicity (< 10-12 MU/g) of caudal fins of T. porphyreus was apparently increased to 16.5-22.0 MU/g by drying. However, the toxin amounts in the dried fins were slightly decreased as compared with those of the non-dried fins. These results demonstrate that puffer fish with toxic skin also have toxic fins.

Tetrodotoxin poisoning.

Tetrodotoxin (TTX) is one of the most potent and oldest known neurotoxins. The poisoning cases due to ingestion of TTX-containing marine animals, especially for puffer, have frequently occurred in Asia since a long time ago. This chapter describes various topics on TTX poisoning including the tendency of poisoning incidents, typical case report, treatment and prevention, biology distribution, original source, infestation mechanism, detection methods, characteristics of chemistry and pharmacology, and therapeutic application. Furthermore, the protocols for how to make puffer safe to eat and how to prevent puffer products made from toxic puffers have been suggested. Finally, the biological significance and neurophysiological role of TTX have been elucidated and TTX may act as an important drug like anesthetic in future.

Accumulation and elimination profiles of paralytic shellfish poison in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Gymnodinium catenatum.

The paralytic shellfish poison (PSP)-producing dinoflagellate Gymnodinium catenatum (Gc) was fed to the short-necked clam Tapes japonica, and the accumulation, transformation and elimination profiles of PSP were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested most of the Gc cells (4 x 10(6) cells) supplied as a bolus at the beginning of the experiment, and accumulated a maximal amount of toxin (181 nmol/10 clams) after 12 hr. The rate of toxin accumulation at that time was 16%, which rapidly decreased thereafter. During the rearing period, a variation in toxin composition, derived presumably from the transformation of toxin analogues in the clams, was observed, including a reversal of the ratio of C2 to C1, and the appearance of carbamate (gonyautoxin (GTX) 2, 3) and decarbamoyl (dc) derivatives (decarbamoylsaxitoxin (dcSTX) and dcGTX2, 3), which were undetectable in Gc cells. The total amount of toxin contained in clams and residue (remaining Gc cells and/or excrement in the rearing tank) gradually declined, and only about 1% of the supplied toxin was detected at the end of the experiment.

Accumulation and depuration profiles of PSP toxins in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Alexandrium catenella.

A toxic dinoflagellate responsible for paralytic shellfish poisoning (PSP), Alexandrium catenella (Ac) was fed to the short-necked clam Tapes japonica, and the accumulation and depuration profiles of PSP toxins were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested more than 99% of the Ac cells (4 x 10(7)cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12h. The rate of toxin accumulation at that time was 23%, which rapidly decreased thereafter. Composition of the PSP toxin accumulated in the clams obviously different from that of Ac even 0.5h after the cell supply, the proportion of C1+2 being much higher than in Ac, although the reason remains to be elucidated. In contrast, a higher ratio of gonyautoxin (GTX)1+4 than in Ac was detected in the toxin profiles of clam excrements. The variation in toxin composition derived presumably from the transformation of toxin analogues in clams was observed from 0.5h, such as reversal of the ratio of C1 to C2, and appearance of carbamate (saxitoxin (STX), neoSTX and GTX2, 3) and decarbamoyl (dc) derivatives (dcSTX and dcGTX2, 3), which were undetectable in Ac cells. The total amount of toxin distributed over Ac cells, clams and their excrements gradually declined, and only 1% of supplied toxin was detected at the end of experiment.

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.

Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land.

Marine pufferfish (family Tetraodontidae) are believed to accumulate tetrodotoxin (TTX) mainly in liver and ovary through the food chain by ingesting TTX-bearing organisms such as starfish, gastropods, crustacean, flatworms, ribbonworms, etc. Consequently, it is hypothesized that non-toxic pufferfish can be produced if they are cultured with TTX-free diets in netcages at sea or aquaria on land, where the invasion of TTX-bearing organisms is completely shut off. To confirm this hypothesis, more than 5000 specimens of the pufferfish ("torafugu", Takifugu rubripes) cultured in such manners for 1-3 years were collected from several locations in Japan during 2001-2004, and toxicity of their livers and some other parts was examined according to the Japanese official mouse assay method for TTX. In addition, typical specimens were submitted to LC/MS analysis. The results showed that all the livers and other parts tested were 'non-toxic' in both of the mouse assay (less than 2 MU/g) and LC/MS analysis (less than 0.1 MU/g). Thus, it is undoubtedly confirmed that pufferfish are intoxicated through the food chain, and non-toxic pufferfish can be successfully produced by netcage or land culture. The livers from these fish can be used with safety as a Japanese traditional food "fugu-kimo" (puffer liver).

[Toxicity of puffer fish cultured in netcages].

During 1990 to 2003, the toxicity of the liver in 4,515 specimens of the puffer fish Takifugu rubripes (torafugu) cultured in netcages or on land were investigated by means of mouse bioassay and liquid chromatography-mass spectrometry (LC/MS). Other tissues (skin, muscles, gonads, etc.) were also investigated in some of them. All the livers and other parts examined were found to be non-toxic. The peak corresponding to tetrodotoxin (TTX) was not detected in the samples by LC/MS analysis for TTX (< 0.1 MU/g). These results show that puffer fish fed on a non-toxic diet in netcages do not become intoxicated.

Immunoenzymatic visualization of tetrodotoxin (TTX) in Cephalothrix species (Nemertea: Anopla: Palaeonemertea: Cephalotrichidae) and Planocera reticulata (Platyhelminthes: Turbellaria: Polycladida: Planoceridae).

Tetrodotoxin (TTX) was localized as brown color in different tissues of an undescribed species of the nemertean genus Cephalothrix (phylum Nemertea) and a turbellarian Planocera reticulata (phylum Platyhelminthes) on light microscopy by means of a monoclonal anti-TTX antibody. In the Cephalothrix sp., TTX was recognized in the vesicles apically arranged in the bacillary cells in the epidermis, basal lamina, the granular cells in the proboscis epithelium, rhynchocoel epithelium, and the vesicles in the basal portion of the intestinal wall near the blood vessels and rhynchocoel. The excretory system and the ovum also showed positive reaction of TTX antigen-antibody. On the other hand, the hermaphrodite flatworm P. reticulata exhibited TTX antigen-antibody complex only in their ovum. To our knowledge, this is the first experimental effort on micro-distribution of TTX in invertebrates.

Occurrence of saxitoxins as a major toxin in the ovary of a marine puffer Arothron firmamentum.

Eleven male and 14 female specimens of a marine puffer Arothron firmamentum were collected from Oita and Iwate Prefectures, Japan. The toxicity assay using mouse showed that only ovary and skin of the female specimens were toxic, the toxicity scores being 5-740 as paralytic shellfish poison and <5-30 MU/g as tetrodotoxin (TTX), respectively. The toxin extracts from the both tissues were then treated with cartridge columns, and subjected to high performance liquid chromatography and liquid chromatography-mass spectral analyses. In the analyses, saxitoxin (STX) and decarbamoylSTX (dcSTX) were identified as the major toxins in the ovary, while the skin contained only TTX.