Tetrodotoxin (TTX) was intramuscularly administered to non-toxic cultured specimens of the pufferfish Takifugu rubripes to investigate TTX transfer/accumulation profiles in the pufferfish body. In two groups of test fish administered either 50 MU/individual of TTX standard (purified TTX; PTTX) or crude extract of toxic pufferfish ovary (crude TTX; CTTX), TTX rapidly transferred from the muscle via the blood to other organs. The toxin transfer profiles differed between groups, however, from 4 to 72 h. In the PTTX group, little TTX was retained in the liver, and most (> 96%) of the toxin remaining in the body transferred/accumulated in the skin after 12 h, whereas in the CTTX group, a considerable amount of toxin (15%-23% of the administered toxin or 28%-58% of the remaining toxin) was transferred/retained in the liver for up to 24 h, despite the fact that 89% of the remaining toxin transferred/accumulated in the skin at the end of rearing period (168 h). The total amount of toxin remaining in the entire body at 1 to 4 h was approximately 60% of the administered toxin in both groups, which decreased at 8 to 12 h, and then increased again to approximately 60% to 80% at 24 to 168 h. Immunohistochemical observation revealed that the toxin accumulated in the skin was localized at the basal cells of the epidermal layer.

The toxicity of two species of wild Cambodian freshwater pufferfish of the genus Tetraodon, T. turgidus and Tetraodon sp., was investigated. Tetraodon sp. was non-toxic.The toxicity of T. turgidus localized mainly in the skin and ovary. Paralytic shellfish toxins (PSTs), comprising saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) account for 85% of the total toxicity. Artificially-reared specimens of the same species were non-toxic. When PST (dcSTX, 50 MU/individual) was administered intramuscularly into cultured specimens, toxins transferred via the blood from the muscle into other body tissues, especially the skin. The majority (92.8%) of the toxin remaining in the body accumulated in the skin within 48 h. When the same dosage of TTX was similarly administered, all specimens died within 3 to 4 h, suggesting that this species is not resistant to TTX. Toxin analysis in the dead specimens revealed that more than half of the administered TTX remained in the muscle and a small amount was transferred into the skin. The presence of both toxic and non-toxic wild specimens in the same species indicates that PSTs of T. turgidus are derived from an exogenous origin, and are selectively transferred via the blood into the skin, where the toxins accumulate.

From October 2006 to December 2007, wild specimens of the pufferfish Takifugu poecilonotus (93 females, 45 males) were collected from the Ariake Sea. Tissue toxicity was examined by mouse bioassay, and tetrodotoxin (TTX) content in the blood plasma by enzyme-linked immunosorbent assay. The relationship between toxicity and maturation was investigated based on changes in the gonadosomatic index: December-March in females and November-March in males, the 'maturation period'; April, 'just after spawning'; and the other months, the 'ordinary period'. Toxicity of both sexes was high throughout the year, but sharply declined in April. In all tissues examined (skin, liver, and ovary) other than testis, toxicity exceeded 1000 MU/g or 10,000 MU/individual in many individuals. Seasonal profiles of tissue toxicity differed markedly between sexes. In females, liver toxicity was high during the ordinary period, and ovary toxicity was high during the maturation period. In males, little maturation-associated change in the toxin distribution was observed. Plasma TTX levels were similar between the sexes (1.59-15.1 MU/ml), and fluctuated largely throughout the year without corresponding changes in tissue toxicity. The percentage of TTX binding to high molecular-weight substances in the plasma varied in association with maturation; the binding ratio fluctuated at relatively low levels during the ordinary period, and stabilized at a high level during the maturation period.

In Cambodia, fatal food poisonings associated with the consumption of pu#erfish have occurred for decades, but the causative species or toxins have never been documented. Herein, we investigated the toxicity of three pu#erfish 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 pu#erfish to be a hazardous reservoir of TTX in Sihanouk Ville. It is likely that L. lunaris is one of the causative species of past pu#erfish poisonings that have occurred in Cambodia.

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