Aqua-BioScience Monographs, Vol. 10, No. 3, pp. 41–80 (2017) www.terrapub.co.jp/onlinemonographs/absm/ Toxins of Pufferfish—Distribution, Accumulation Mechanism, and Physiologic Functions Osamu Arakawa1*, Tomohiro Takatani1, Shigeto Taniyama1 and Ryohei Tatsuno2 1Graduate School of Fisheries and Environmental Sciences Nagasaki University 1-14 Bunkyo-machi, Nagasaki, Nagasaki 852-8521, Japan 2Department of Food Science and Technology National Fisheries University, Japan Fisheries Research and Education Agency 2-7-1 Nagatahonmachi, Shimonoseki, Yamaguchi 759-6595, Japan *e-mail: [email protected] Abstract Received on April 30, 2015 Our many years of studies have provided a lot of information regarding distribution, Accepted on accumulation mechanism, and physiologic functions of the natural toxins harbored by September 12, 2016 Online published on pufferfish. We detected tetrodotoxin (TTX) and/or its derivatives in several species of October 17, 2017 aquatic organisms including marine bacteria. This, along with the fact that pufferfish became non-toxic when they were reared with non-toxic feed, indicated that the toxification Keywords of pufferfish is of exogenous origin. Subsequently, we revealed by various TTX adminis- • tetrodotoxoin tration experiments using non-toxic cultured pufferfish that the TTX administered into • paralytic shellfish poison the muscle or digestive tract rapidly transferred to the liver and skin, and that the toxin • saxitoxin transfer to the gonads was largely different between male and female, suggesting the • palytoxin • pufferfish involvement of maturation in the internal kinetics of TTX. On the other hand, we visual- • Takifugu rubripes ized micro-distribution of TTX in the tissues of various TTX-bearing organisms using an • food poisoning immunohistochemical technique, giving a crucial insight to elucidate physiologic func- tions of TTX including the function as a defensive or offensive agent. Furthermore, we found that Southeast Asian freshwater pufferfish possess paralytic shellfish poison (PSP) as a main toxin, and that boxfish and Bangladeshi freshwater pufferfish bear a palytoxin (PLTX)-like toxin, and cause a rhabdomyolysis, which overturned the common sense that pufferfish toxin equals TTX. 1. Introduction unit) is defined as the amount of toxin required to kill a 20-g male mouse within 30 min after intraperitoneal Among the food poisonings occurring in Japan, poi- administration], and the minimum lethal dose (MLD) soning due to marine toxins, particularly of pufferfish for humans is estimated to be approximately 10000 MU poisoning due to tetrodotoxin (TTX) is the most fre- (ª2 mg) (Noguchi and Ebesu 2001). quent in terms of the fatalities. TTX is a potent neuro- The main symptoms of human intoxication include toxin of low molecular weight, whose unique struc- numbness of lips, tongue and the limbs, paresthesia, ture was determined by three groups in 1964 (Goto et dysarthria, respiratory distress, and death is caused by al. 1965; Tsuda et al. 1964; Woodward 1964). Various respiratory failure in the most critical cases (Noguchi TTX derivatives have so far been separated from and Ebesu 2001). According to Toda et al. (2012), 651 pufferfish, newts, frogs, and/or some other TTX-bear- incidents of TTX poisoning due to pufferfish have oc- ing organisms (Yotsu-Yamashita 2001; Yotsu- curred in Japan, involving 976 patients and 56 deaths Yamashita et al. 2013) (Fig. 1). TTX inhibits the con- during the 22 years from 1989 to 2010. Many cases duction of action potential by selectively plugging so- occurred during the winter season in coastal prefec- dium channels on the nerve/muscle membrane at ex- tures of the Seto Inland Sea, with “komonfugu” tremely low concentrations (Narahashi 2001). The le- Takifugu poecilonotus, “mafugu” Takifugu porphyreus, thal potency is 5000 to 6000 MU/mg [1 MU (mouse “higanfugu” Takifugu pardalis, “shosaifugu” Takifugu © 2017 TERRAPUB, Tokyo. All rights reserved. doi:10.5047/absm.2017.01003.0041 42 O. Arakawa et al. / Aqua-BioSci. Monogr. 10: 41–80, 2017 Fig. 1. Structures of TTX and typical TTX derivatives. snyderi, “kusafugu” Takifugu niphobles, and “torafugu” tom of eating small necrophagous marine snails, and Takifugu rubripes as the causative species. Recently, TTX poisoning due to the snails has frequently oc- the non-edible pufferfish “dokusabafugu” curred (Arakawa et al. 2010; Hwang and Noguchi Lagocephalus lunaris, which usually inhabits tropical 2007; Noguchi et al. 2011a). At least 28 incidents were or subtropical waters, has been frequently mixed up recorded during 1985–2004 in China, and 9 incidents with edible species in Japanese coastal waters, posing during 1994–2006 in Taiwan, involving 233 patients a serious food hygiene problem. This pufferfish, which and 24 deaths in total. As described later, a poisoning bears a very similar appearance to the almost nontoxic due to the similar marine snail “kinshibai” Nassarius species “shirosabafugu” Lagocephalus spadiceus, also glans also occurred in Kyushu, Japan, in 2007 and possesses high levels of TTX in their muscles, caused 2008, respectively (Taniyama et al. 2009a). In Spain, 5 poisoning incidents with 11 patients due to mistaken the European carnivorous trumpet shell Charonia ingestion in Kyushu and Shikoku Islands during 2008– lampas lampus caused human TTX intoxication in the 2009. same year (2007) (Noguchi et al. 2011a; Radriguez et In Taiwan and China, including Hong Kong, although al. 2008). In New Zealand, 15 dogs were suddenly consumption of pufferfish is officially prohibited, and poisoned at the beaches adjacent to Hauraki Gulf in not eaten as frequently as in Japan, there have also been 2009, all exhibiting similar symptoms, and 5 of them many food poisoning cases due to the ingestion of wild died. McNabb et al. (2010) detected a very high level pufferfish (Noguchi and Arakawa 2008). In countries of TTX in the grey side-gilled sea slug outside of East Asia, people generally do not have a Pleurobranchaea maculate found in tide pools near the custom of eating pufferfish, but poisoning due to acci- beach, and claimed that the dogs were poisoned with dental ingestion of pufferfish occasionally occur all TTX by contact with the sea slugs. over the world, including Australia (Isbister et al. On the other hand, pufferfish poisonings due to other 2002), Brazil (Silva et al. 2010), Thailand (Brillantes toxins than TTX have also occurred. In the United et al. 2003), and Bangladesh. Particularly in Bangla- States, Floridian Sphoeroides pufferfish caused 28 desh, 3 large pufferfish poisoning incidents occurred cases of food poisonings during 2002 to 2004, whose in 2008, involving 141 patients and 17 deaths (Islam main toxic principle was subsequently identified not et al. 2011). Recently, toxic Indo-Pacific pufferfish, as TTX, but as paralytic shellfish poison (PSP) including “senninfugu” Lagocephalus sceleratus, mi- (Landsberg et al. 2006). As described later, poisonings grated from the Red Sea via the Suez Canal to the due to PSP-bearing freshwater pufferfish sometimes Mediterranean Sea, and poisoning by this species have occur in Southeast Asian countries (Kungsuwan et al. occurred in the eastern Mediterranean (Bentur et al. 1997; Ngy et al. 2008; Zaman et al. 1997). Since 1990, 2008). a series of poisonings due to ingesting boxfish, a fam- Pufferfish is not the only cause of TTX poisonings. ily of pufferfish, have occurred in western Japan, in- In China and Taiwan, people have a time-honored cus- volving 13 patients and 1 death (Taniyama et al. doi:10.5047/absm.2017.01003.0041 © 2017 TERRAPUB, Tokyo. All rights reserved. O. Arakawa et al. / Aqua-BioSci. Monogr. 10: 41–80, 2017 43 2009b). The boxfish poisoning differs from usual pufferfish poisoning, but is very similar to parrotfish poisoning (a unique variety of food poisoning that has sporadically occurred in Japan) in terms of causing rhabdomyolysis as the main symptom (Arakawa et al. 2010). In Bangladesh, a similar poisoning is frequently caused by freshwater pufferfish. With the change in marine environment, poisonings due to pufferfish or pufferfish toxins seem to be diver- sified, and geographically expanded. Although the Japanese have been eating pufferfish since ancient times, and have created a unique food culture for pufferfish, the fundamental understanding not only on the toxin itself, but also on the organisms that possess it is essential to avoid poisonings, and ensure the safety and reliability for eating pufferfish. Over many years, we have accumulated varied information on the char- acteristics and distribution of pufferfish toxins, and their accumulation mechanism and physiological func- tions in bearing organisms. The present article sum- marized the results. 2. Distribution of TTX in aquatic organisms and toxification mechanism of pufferfish 2-1. Detection of TTX or its derivatives in several aquatic organisms TTX was long believed to be present only in pufferfish. Since Mosher et al. (1965) identified a toxin from the eggs of the California newt Taricha torosa as TTX, however, TTX has been detected in a wide vari- ety of animals, such as the goby “tsumugihaze” Yongeichthys criniger, atelopid frogs (Atelopus spp.), the blue-ringed octopus Hapalochlaena maculosa, the carnivorous gastropod “boshubora” Charonia sauliae, starfish of genus Astropecten, the xanthid crab “subesubemanjugani” Atergatis floridus, and flatworms (Hwang and Noguchi 2007; Miyazawa and Noguchi Fig. 2. ESI/MS spectra of TTX (upper), TTX-U1, and TTX- U2