Pufferfish toxin (tetrodotoxin, TTX) is a potent neurotoxin of low molecular weight, which was first isolated in 1950 as a crystalline prism from toxic pufferfish by Yokoo [1]. Its structure (Figure 1) was elucidated by three groups in 1964 [2–4]. TTX inhibits nerve and muscle conduction by selectively blocking sodium channels [5], resulting in respiratory paralysis that causes death. The lethal potency is 5000 to 6000 MU/mg [1 MU (mouse unit) is defined as the amount of toxin required to kill a 20-g male mouse within 30 min after intraperitoneal administration], and the minimum lethal dose (MLD) for humans is estimated to be approximately 10000 MU ( 2 mg) [6]. Various TTX derivatives have so far been separated from pufferfish and/or some other TTX-bearing organisms [7,8].

Figure 1 Structure of TTX.

Many years ago when TTX was believed to be found exclusively in pufferfish, it was controversial whether TTX in the fish was endogenous (produced by the pufferfish itself) or exogenous (taken from the outside and accumulated). Subsequent findings, however, such as the distribution of TTX among many other organisms [9], the TTX intoxication of the trumpet shell following the ingestion of toxic starfish [10], TTX production by marine bacteria [11], and the facts pufferfish become non-toxic when artificially reared with non-toxic diets [12–14] and that such non-toxic pufferfish become toxic when fed TTX-containing diets [15–17], have elucidated that the main mechanism of TTX accumulation in pufferfish is the food chain, consisting of several steps and starting with marine bacteria as a primary source of TTX.

In the present paper, the detailed mechanisms of the distribution and accumulation of TTX are described. We also present some case reports of human intoxication, such as via pufferfish poisoning in Japan, and food poisoning due to the ingestion of small gastropods, which was recently reported in China and Taiwan.

Table 1 Toxicity of pufferfish

Lagocephalus wheeleri and L. gloveri of the same family are usually regarded as non-toxic species, although they occasionally show weak toxicity [23]. All species of the Diodontidae and Ostraciontidae families are TTX-free, though the skin of Ostraciontidae contains an ichthyotoxic and hemolytic substance called pahutoxin [24,25]. Food poisoning cases due to ingestion of the liver of Ostraciontid boxfish sometimes occur in Japan, but the causative agent is a toxin with delayed hemolytic activity, possibly palytoxin or a similar substance, and not TTX or pahutoxin [26]. Thai brackish water pufferfish Tetraodon nigroviridis and T. steindachneri, and freshwater pufferfish T. fangi, T. leiurus, and T. suwatii are also toxic. The toxin of brackish water species was identified as TTX [27,28], but in the freshwater species, saxitoxins (STXs), toxins that belong to the paralytic shellfish poison (PSP) family, were detected as the main toxic principles [29,30]. Freshwater pufferfish, Colomesus asellus from Brazil [31] and T. turgidus from Cambodia [32], also possess PSP. Recently, some marine pufferfish (Arothron spp.) from the Philippines [33], and Arothron firmamentum from Japanese waters [34] were also reported to possess STXs as the major toxins. Furthermore, palytoxin-like substance(s), in addition to a small amount of PSP, were detected in a Bangladeshi freshwater pufferfish belonging to the genus Tetraodon [35].

The generally large individual and regional variations in pufferfish toxicity [20] are too great to be explained by endogenous factors alone, i.e., differences in physiologic conditions among individual pufferfish, suggesting their exogenous intoxication.

Our recent immunohistochemical investigations using a monoclonal anti-TTX antibody clarified the intratissue microdistribution of TTX in the skin and ovary of TTX-bearing pufferfish [38–40]. Species-specificity was also recognized in the microdistribution, i.e., TTX was localized in the nucleus, yolk vesicles, and yolk granules of oocytes in the ovary of the marine species T. vermicularis, whereas it was detected in the connective tissues and nucleus of some oocytes in C. patoca [39]. TTX-bearing glands were observed in the skin of T. vermicularis, and TTX-bearing secretory cells (succiform cells) were observed in brackish water species such as C. patoca, T. steindachneri, and T. nigroviridis [38–40].

In the toxic marine pufferfish, the liver generally shows very high toxicity throughout the year except in the spawning season, during which the ovary becomes highly toxic by accumulating TTX transferred from the liver [20]. TTX in the eggs spawned from the ovary could be involved in protecting the eggs from predators. On the other hand, when toxic pufferfish encounter enemies, their bodies swell to two or three times their usual size and TTX is excreted from their skin to repel the enemies [41,42]. These observations suggest that the pufferfish accumulate TTX as a biologic defense agent.

Table 2 Distribution of TTX in animals other than pufferfish [9].

It is quite unlikely that these TTX-bearers belonging to particular species of different phyla possess a common gene that codes for TTX production. The trumpet shell Charonia sauliae accumulates TTX by ingesting toxic starfish [10], supporting the hypothesis that the TTX of pufferfish is not endogenous, but comes via the food chain. The exact origin of TTX in the food chain, however, remains unknown. Because the ecologic environments of TTX-bearing animals seem to have no common factor other than being closely related to an aquatic system, bacteria, omnipresent organisms that commonly inhabit the aquatic system, are implicated as the primary source of TTX.

Figure 2 HPLC of TTX fraction from a Vibrio strain (left) and of authentic TTX (right) [74].

To test this hypothesis, we collected more than 6000 specimens of the marine pufferfish Takifugu rubripes that were cultured with non-TTX-containing diets in such environments (net cages on the sea or land aquaria located in eight prefectures of Japan) and examined the toxicity of their livers and some other body parts, including gonads and muscle, using an accepted TTX bioassay [14]. All specimens were non-toxic, i.e., their toxicity was under the detection limit (2–10 MU/g) (Tables 3 and 4). Furthermore, liquid chromatography/mass spectrometry analysis demonstrated that none of the 20 liver specimens from Nagasaki and 5 liver specimens from Yobuko contained detectable amounts of TTX, i.e., their TTX content was below 0.1–0.4 MU/g.

Table 3 Toxicity of liver of the pufferfish T. rubripes cultured in net cages [14]

Table 4 Toxicity of liver of the pufferfish T. rubripes cultured on land

When non-toxic cultured pufferfish T. rubripes were reared on TTX-containing diets, however, they became toxic within 40 days when fed 4 MU TTX/g body mass/day, and in 100 days when fed 0.5 MU TTX/g body mass/day. Thereafter, the toxicity continuously increased up to the end of experimental period (Figure 3) [79]. In contrast, three non-pufferfish species were not intoxicated with TTX, even after culturing for 139 days when fed 4 MU TTX/g body mass/day. These species seem not only to exclude, but also to detoxify, TTX.

Figure 3 TTX infestation to the non-toxic cultured pufferfish T. rubripes and non-toxic three species of fish by feeding TTX-containing livers of wild pufferfish. : Cultured pufferfish; TTX dosage of 0.5 MU/g body mass/day. : cultured pufferfish; (more ...)

In a similar feeding experiment using a PSP [consisting of STX and neosaxitoxin (neoSTX)]-containing diet for 60 days, non-toxic pufferfish were also intoxicated with PSP. Their toxicity scores, however, were only 21% to 53% of those of the fish fed with the same dosage of TTX as controls (unpublished data). Thus, the TTX-bearing marine pufferfish tend to selectively accumulate more TTX than PSP.

These results clearly demonstrate that the TTX of toxic pufferfish is not endogenous, but is derived from the food chain (Figure 4). Another possibility is that the TTX is produced by symbiotic or parasitic bacteria that are directly accumulated inside the puffer body and not obtained via the food chain. The amount of TTX produced by bacteria, however, appears to be too little to account for the TTX accumulation in pufferfish, therefore bioconcentration likely has a large role in the accumulation of TTX in pufferfish. Furthermore, based on the observation that 6000 specimens of pufferfish reared over 1 year in net cages or land aquaria were non-toxic, the TTX produced in the intestines of pufferfish by marine bacteria must be a minor contributor, or rather negligible, to account for the TTX accumulation in pufferfish.

Figure 4 Proposed mechanism of TTX accumulation in marine animals.

During our studies on the toxicity and toxin profiles of the Japanese newt Cynops pyrrhogaster [83,84], we observed that the eggs of the newt retained a certain amount of TTX inherited from their parents, which gradually disappeared in the larval stage. In the juvenile stage in a terrestrial environment, the newt abruptly begins to accumulate TTX again, and becomes highly toxic in the adult stage. When artificially reared with non-toxic diets for 5 years after hatching, however, the newt became non-toxic (< 2 MU/g). The non-toxic newt, when orally administered TTX or 6-epitetrodotoxin (6-epiTTX), accumulated approximately 50% of the administered toxins. These observations strongly suggest that the TTX of C. pyrrhogaster is exogenous, i.e., the newt begins to accumulate TTX after metamorphosis, with their food organisms as its origin.

Daly et al. [85] examined the intoxication mechanism of frogs. They hatched eggs of the Panamanian frog Atelopus varius which possesses both bufadienolides (a type of steroid) and TTX in the skin, and artificially reared them for 25 to 45 months. In the skin of the adult frogs, bufadienolides were detected at the same level as in wild specimens, but TTX was not detected, leading them to conclude that bufadienolides are endogenous and the TTX is exogenous, possibly derived from food organisms or environmental factors such as symbiotic bacteria.

Table 5 Resistance of TTX- and non-TTX-bearing organisms.

The mechanism of TTX resistance in pufferfish and rewts has been explained based mainly on the TTX-resistant sodium channels found in the animals, in which the aromatic amino acid commonly located in the p-loop region of domain I in TTX-sensitive sodium channels is replaced by a nonaromatic amino acid, resulting in their extremely low affinity to TTX [88–91]. Garter snakes and clams can also acquire similar sodium channel mutation-based TTX/PSP resistance by interacting with their toxic food organisms, TTX-bearing newts and PSP-producing dinoflagellates, respectively [92,93]. On the other hand, the shore crab Hemigrapsus sanguineus possesses TTX-binding proteins in its hemolymph, which confer TTX resistance on this non-toxic crab [94,95]. Pufferfish and newts may be equipped not only with TTX-resistant sodium channels, but also with other mechanisms whereby they mask and/or modulate TTX molecules inside their body.

Table 6 Edible part of pufferfish in Japan

Table 7 Pufferfish poisoning incidents in Japan.

In this case, toxicity scores of the leftover liver and flesh were determined to be 715-4260 and less than 5 MU/g, respectively, and TTX was identified as the toxic principle [96]. The victim was estimated to have ingested more than 10000 MU, the amount equivalent to the MLD of TTX in humans, and the cause of his death was concluded to be TTX contained in the liver of wild T. poecilonotus.

According to the victims, the causative fish, which had been collected from the coastal area of Chunghua Prefecture, might be the marine pufferfish Takifugu niphobles. They retained a small piece (about 11 g) of the cooked fish liver, which, along with eight live specimens of T. niphobles, was then assayed for toxicity. The results indicated that the toxicity of the cooked fish liver was 280 MU/g, and all the T. niphobles specimens had high toxicity (>850 MU/g) in their liver [100]. The toxin of both samples was identified as TTX. Mitochondrial DNA of the leftover and T. niphobles specimens showed the same sequence genotype and the same single restriction site for BsaI, indicating that T. niphobles was responsible for the intoxication caused by TTX [101].

In Bangladesh, a food poisoning incident due to ingestion of the roe of the marine pufferfish Takifugu oblongus occurred in November 1998, affecting eight people and resulting in five deaths [105]. Their symptoms were dyspnea, numbness of the lips, paralysis, and stomachache followed by vomiting, which appeared 2 hours after ingestion. Two victims became unconscious within 5 hours of exposure. On the way to the hospital, two of the eight patients died and the remaining six were admitted. Three of the six patients died in the hospital and three recovered.

Small-sized brackish water and freshwater pufferfish have also occasionally caused food poisoning incidents, including fatal cases in Asian countries such as Thai, Bangladesh, and Cambodia [32,106,107], though the causative toxin is PSP or palytoxin-like toxin in freshwater species.

It is astonishing that there continues to be many victims, as well as deaths, due to the ingestion of commercially cooked small gastropods in China, which may become a serious public health issue. Moreover, this type of poisoning has spread from the coast of Zheijiang to other areas of the East China Sea and Yellow Sea. Very recently (July 2007) in Nagasaki, Japan, a food poisoning incident occurred due to the same type of small gastropod, Alectrion glans. An investigation revealed that a large amount of TTX (600 MU/g) remained in the leftover cooked gastropods, including muscles and digestive glands (unpublished data). Although the TTX accumulation mechanism in the gastropods remains uncertain, we suspect that the gastropods might become toxic by feeding on the toxic viscera of pufferfish that died after spawning.

The leftover digestive gland had a toxicity score of 17000 MU, and the responsible toxin was identified as TTX [47]. Thus, it was estimated that the victim might have ingested an amount of TTX almost equivalent to the MLD. TTX was also identified as the causative agent in the leftovers from similar TTX poisoning cases in Wakayama Prefecture in 1982 and Miyazaki Prefecture in 1987 [115].

Poisonings due to the ingestion of eggs of the horseshoe crab Carcinoscorpius rotundicauda have occasionally been reported in Thailand [116]. The symptoms of the victims were mostly similar to those caused by TTX or PSP. C. rotundicauda toxin consists mainly of TTX and anhydroTTX [59,117,118], but sometimes contains STX and neoSTX [119]. Hence, the responsible toxin for horseshoe crab poisonings is considered to be TTX and/or PSP.

As described above, a large variety of organisms besides pufferfish possess TTX. By knowing the inter- or intra-tissue distribution of TTX, it is possible to estimate the functions of TTX in these organisms. For example, marine pufferfish (Table 1) and flatworms [9,120] retain high toxicity in their eggs, and pufferfish and newts are equipped with TTX-secreting glands or cells in their skin [38–40,121], suggesting that TTX is present as a defensive substance to protect their eggs or themselves from external enemies. In this context, we recently found that the immunity of cultured pufferfish is significantly activated when they are reared with TTX-containing diets [122], though the mechanism remains to be clarified. On the other hand, blue-ringed octopus and ribbon worms possess TTX in the post salivary gland and proboscis, respectively [54,120], indicating that they utilize TTX to capture their prey.

The TTX accumulation mechanism in pufferfish can be explained by bioconcentration via the food chain starting from bacteria. It remains to be elucidated how TTX molecules are absorbed, transferred, and retained/eliminated after entering into the pufferfish body via the toxic food organisms. We recently found that when intramuscularly administered to the non-toxic cultured specimens of TTX-bearing marine pufferfish, TTX is rapidly transferred to the skin and liver through the blood (unpublished data). A similar transfer is also observed when PSP is administered to the non-toxic cultured specimens of PSP-bearing freshwater pufferfish [32]. On the other hand, Nagashima et al. [123,124] reported that liver tissue slices of pufferfish accumulate considerable amounts of TTX in vitro when incubated with TTX-containing medium. These phenomena were not observed in non-toxic fish, suggesting the presence of a specific mechanism in the pufferfish liver or skin whereby the fish selectively and/or actively accumulate TTX/PSP. TTX-binding proteins exist in the blood plasma of pufferfish [125,126], which may be implicated in the transportation of TTX. In this connection, Lee et al. [127] identified novel genes that are possibly related to TTX accumulation in pufferfish.

Not only pufferfish, but various other animals induce TTX intoxication in humans. The symptoms in the patients appear shortly after ingestion of the causative food, and the time to fatality is also very short (average 6 hours). There are currently no antidotes or antitoxins to TTX, and there is no treatment for the seriously TTX-intoxicated patient except for artificial respiration. A monoclonal anti-TTX antibody was recently developed [128], but it can be used only as a reagent for research, and is not expected to have clinical efficacy. There is a possibility that TTX poisoning may increase or diversify further in the future due to changes in the marine environment and the increase in international commerce of fishery products.

TTX has a very important role in neurophysiology, and in other fields of study as a research reagent (a specific sodium channel blocker). In China, it is used as a clinical medicine (an analgesic for terminal cancer patients). In Japan, TTX was previously clinically applied as an analgesic for neuralgia and rheumatism. A thorough understanding of the biochemical properties and mechanisms of TTX accumulation will facilitate the development of technology to use TTX or TTX-bearing organisms effectively as reagents and medicines, or to produce non-toxic cultured pufferfish whose liver can be used as a poison-free food.