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

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_107 cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12 h. 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 0.5 h 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 0.5 h, 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

Alexandrium tamarense により毒化した広島湾産二枚貝に含まれる麻痺性貝毒成分

1992年4月, 広島湾産養殖マガキ, ムラサキイガイ及びアサリから高い麻痺毒性が検出された. これらをそれぞれ80%エタノール (pH 3.5) で抽出し, 脱脂後, 限外ろ過, 活性炭及びSep-Pak C18カートリッジ処理した. HPLC分析した結果, マガキ, ムラサキイガイ及びアサリに含まれる麻痺毒はいずれもゴニオトキシン-1 (GTX1) を51～55% (mol%) 含む麻痺性貝毒 (PSP) であることが判明した. その他にGTX2,3,4, 更にムラサキイガイ及びアサリには微量のサキシトキシン (STX) が検出された. また, プロトゴニオトキシン-1,2 (PX1,2) も1.6～4.5% (mol%) 含まれることが明らかとなった.In April, 1992, paralytic toxicity substantially exceeding the quarantine limit of 4MU/g edible part as paralytic shellfish poison (PSP) was detected in cultured oyster Crassostrea gigas, mussel Mytilus edulis and short-necked clam Tapes (Amygdala) japonica from Hiroshima Bay, Hiroshima Prefecture concomitantly with the appearance of the toxic dinoflagellate Alexandrium tamarense. The toxicities were 31.4MU/g for oyster, 214.6MU/g for mussel and 20.3MU/g for short-necked clam on 22nd April. Attempts were made to identify the paralytic toxin in these bivalves. They were extracted with 80% ethanol (pH 3.5), followed by defatting with dichloromethane. The aqueous layer obtained was ultrafiltered through a Diaflo YM-2 membrane (Amicon) to eliminate substances of more than 1, 000 daltons, treated with activated charcoal and then applied to a Sep-Pak C18 cartridge (Waters). The unbound toxic fraction was analyzed by HPLC. In gonyautoxin (GTX) analysis of each toxin, protogonyautoxin-1, 2 (PX1, 2; epi-GTX8, GTX8; C1, 2), GTX4, GTX1, GTX3 and GTX2 were detected. In saxitoxin (STX) analysis, a small peak of STX was detected in mussel and short-necked clam toxin, but not in the oyster toxin. Consequently, the toxin of the bivalves in Hiroshima Bay was found to be comprised of GTX1-4 as the major components, which accounted for approximately 92-95% (mole ratio) of all components, with a trace of STX. In all cases, GTX1 was the major component (approximately 51-55%; mole ratio). On the other hand, the content of PX1, 2, which are N-sulfocarbamoyl derivatives, was 1.6-4.5% (mole ratio) irrespective of the sample. It was concluded from these results that the toxin of the above bivalves collected in Hiroshima Bay in April, 1992 consisted predominantly of PSP, possibly derived from the toxic plankton A. tamarense detected there

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

Toxicity Change of Paralytic Shellfish Poison-infested Oyster during Canning, Drying and Sauce-making Processes

麻痺性貝毒により毒化したカキの食品としての有効利用を図るため, 毒性が30MU/9程度のむき身を原料として現行の加工処理による減毒試験を行った. 各処理段階の毒性と毒組成をマウス生物試験とHPLCにより測定した. 燻煙油漬け缶詰と水煮缶詰製品では, HPLCにより毒性成分の一部が検出されたが, 残存毒性はすべて2.5MU/g以下と規制値を越えることはなかった. オイスターソースでは原料 (解凍液と缶詰製造時の煮沸液, 2～4MU/g) の毒性の約90%が消失し, マウス生物試験法では不検出となった. 乾燥カキでは規制値以上の毒性 (7MU/g) があった. 缶詰とオイスターソース製造では加熱条件を十分に考慮すれば食品としての利用が可能であると考えられた.Paralytic shellfish poison-infested oysters (about 30MU/g) were smoked or boiled and then canned. Dried oyster and oyster sauce were also produced. The toxicity of canned smoked oyster decreased to under the quarantine limit (4MU/g) or to an undetectable level (<2MU/g). A similar result was obtained in canned boiled oyster. It seems possible to use contaminated oysters safely as canned foods. In the production of oyster sauce, over 90% of the toxicity was destroyed during condensation (6hrs of boiling in a water bath). As the toxicity of the starting material of oyster broth was under 5MU/g (usually 2-4MU/g), the oyster sauce produced also cleared the official quarantine limit successfully. On the other hand, the dried oyster product retained a high level of residual toxicity (7MU/g) and was still hazardous