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Rabbitfish belonging to the order Perciformes are well-known venomous fish that are frequently involved in human accidents. However little research has been done into either the whole venom toxicities or the structures and properties of their venom toxins. In this study, we first examined biological activities of the crude venom extract prepared from dorsal spines of Siganus fuscescens, a rabbitfish most commonly found along the coasts of Japan. As a result, the crude venom extract was shown to have mouse-lethal activity, hemolytic activity against rabbit erythrocytes, edema-forming activity and nociceptive activity, similar to the known scorpaeniform fish toxins (stonefish toxins and their analogues). Then, the primary structure of the S. fuscescens toxin was successfully elucidated by the same cDNA cloning strategy as previously employed for the toxins of some scorpaeniform fish (lionfish, devil stinger and waspfish). The S. fuscescens toxin is obviously an analogue of stonefish toxins, being composed of two kinds of subunits, an α-subunit of 703 amino acid residues and a β-subunit of 699 amino acid residues. Furthermore, the genes encoding both subunits were cloned from genomic DNA and shown to have an architecture of three exons and two introns, as reported for those of the scorpaeniform fish toxins. This study is the first to demonstrate the occurrence of stonefish toxin-like toxins in perciform fish., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Marine pufferfish of the Tetraodontidae family contain high levels of tetrodotoxin (TTX) in the liver and ovary. TTX is suggested to transfer from the liver to the ovary in female pufferfish during maturation. TTX in pufferfish eggs may act as a repellent against predators and as a sexual pheromone to attract male pufferfish. The toxification mechanism of the pufferfish ovary is poorly understood. Here we evaluated the chemical form of TTX and its related substances in the ovary of the panther pufferfish Takifugu pardalis by LC-ESI/MS. TTX and its analogs 4-epi-TTX, 4, 9-anhydroTTX, deoxyTTX, dideoxyTTX, and trideoxyTTX were detected in a low molecular weight fraction by Sephacryl S-400 column chromatography. The finding of an unknown TTX-related substance in a high molecular weight fraction from the Sephacryl S-400 column suggested the occurrence of toxin-binding protein in the ovary. The toxin-binding protein in the ovary was purified by ion-exchange HPLC, gel filtration HPLC, and SDS-PAGE. Amino acid sequencing and cDNA cloning revealed that the toxin-binding protein, TPOBP-10 (Takifugu pardalis ovary toxin-binding protein with a molecular mass of 10 kDa) was homologous with the predicted vitellogenin-1-like protein [Takifugu rubripes] subdomain, a von Willebrand factor type D domain. TPOBP-10 mRNA was highly expressed in the ovary and liver and less in other organs of female individuals based on RT-PCR. These findings reveal a novel function of the vitellogenin subdomain as binding with TTX-related substances, and its involvement in the toxification of the pufferfish ovary., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Marine pufferfish of the family Tetraodontidae accumulate high levels of tetrodotoxin (TTX). The profile of TTX accumulation is reported to differ between tiger pufferfish Takifugu rubripes juveniles and adults administered TTX. Adults mainly accumulate TTX in liver, while juveniles transfer TTX from the liver to the skin. In the present study, we investigated TTX uptake into liver tissue slices of T. rubripes juveniles (4-month-old) and adults (18-month-old) in an in vitro incubation experiment, and compared their differential gene expression profiles in the liver by suppression subtracted hybridization (SSH). The tissue culture experiment revealed that TTX uptake in the liver itself was indistinguishable between the juveniles and the adults. In SSH analysis, a total of 176 clones were upregulated in the juvenile liver, the majority of which comprised hemoglobin subunit alpha-2-like gene (53 clones), hemoglobin subunit beta-like gene (40 clones), and type-4 ice-structuring protein LS-12-like gene (20 clones). A total of 211 clones were upregulated in the adult liver, including serotransferrin-like gene (84 clones), fibrinogen beta chain-like gene (15 clones), and 14 kDa apolipoprotein gene (10 clones). Based on these and previous findings on genes related to TTX intoxication in pufferfish, serotransferrin-like gene, complement C3-like gene, water-temperature-acclimation-related-65 kDa-protein-like gene, and chymotrypsin elastase family member 2A-like gene appear to be involved in TTX toxification of the T. rubripes liver., (Copyright © 2015. Published by Elsevier Ltd.)

Marine pufferfish of the family Tetraodontidae accumulate a considerable amount of tetrodotoxin (TTX), mainly in the liver and ovary. The detailed distribution of TTX in pufferfish body tissues, however, remains poorly understood. Here we investigated the tissue distribution and biliary excretion of TTX in cultured pufferfish Takifugu rubripes juveniles (6-month-old, 81.5 ± 2.0 g body weight) for 24 h after intramuscular administration of 0.25 μg TTX/g body weight into the caudal muscle. The blood TTX concentration was 0.53 ± 0.15 μg/mL at 1 h, and gradually decreased to 0.05 ± 0.01 μg/mL at 24 h after administration (p < 0.05). The TTX concentration in the liver declined from 1.59 ± 0.10 μg/g at 1 h to 0.48 ± 0.21 μg/g at 24 h (p < 0.05). In contrast, the TTX concentration in the skin increased from 0.27 ± 0.04 μg/g at 1 h to 0.48 ± 0.08 μg/g at 24 h (p < 0.05). The concentration of TTX in the bile remarkably increased from 0.08 ± 0.03 μg/mL at 1 h to 0.39 ± 0.05 μg/mL at 8 h (p < 0.05) and remained at almost the same level at 24 h. These findings indicate that TTX was excreted from the liver into the gallbladder bile in the pufferfish T. rubripes juveniles., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Marine puffer fish accumulate a high level of tetrodotoxin (TTX) in the liver and ovary, but the underlying mechanism of this toxification is unclear. To elucidate the genes related to toxification of the marine puffer fish, we examined the hepatic gene expression profile of the marine puffer fish Takifugu rubripes by suppression subtractive hybridization in response to the intramuscular administration of 0.50 mg TTX/kg body weight into the caudal muscle. The accumulation of TTX in the liver reached 68 ± 4% that of the administered dose within 12 h of administration. A total of 1048 clones from the subtracted cDNA libraries were successfully sequenced. The nucleotide sequence of 92 of the 1048 clones was identified as a hepcidin precursor. Reverse transcription-polymerase chain reaction experiments revealed that hepcidin precursors were highly expressed in the TTX-administered group. In addition, complement C3 (31 clones), serotransferrin (30 clones), apolipoprotein A-1 (14 clones), high temperature adaptation protein Wap65-2 (14 clones), complement C7 (12 clones), fibrinogen beta chain (12 clones), and 70 kDa heat-shock protein 4 (11 clones) were obtained. This study confirmed that the intramuscular administration of TTX increases the gene expression of the acute-phase response proteins in the liver of puffer fish T. rubripes., (Copyright © 2010. Published by Elsevier Ltd.)

To elucidate the involvement of plasma protein binding in the disposition of tetrodotoxin (TTX) in puffer fish, we used equilibrium dialysis to measure protein binding of TTX in the plasma of the marine puffer fish Takifugu rubripes and the non-toxic greenling Hexagrammos otakii, and in solutions of bovine serum albumin (BSA) and bovine alpha-1-acid glycoprotein (AGP). TTX (100-1000 microg/mL) bound to protein in T. rubripes plasma with low affinity in a non-saturable manner. The amount of bound TTX increased linearly with the TTX concentration, reaching 3.92+/-0.42 microg TTX/mg protein at 1000 microg TTX/mL. Approximately 80% of the TTX in the plasma of T. rubripes was unbound in the concentration range of TTX examined, indicating that TTX exists predominantly in the unbound form in the circulating blood of T. rubripes at a wide range of TTX concentrations. TTX also bound non-specifically to H. otakii plasma proteins, BSA, and bovine AGP. The amount of the bound TTX in the plasma of H. otakii and BSA, respectively, was 1.86+/-0.36 and 4.65+/-0.70 microg TTX/mg protein at 1000 microg TTX/mL, and that in the bovine AGP was 8.78+/-0.25 microg TTX/mg protein at 200 microg TTX/mL., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In this study, we investigated the hepatic uptake clearance (CL(uptake)) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25mg TTX/kg body weight into the hepatic vein at 20 degrees C. The blood concentration of TTX decreased over time after the injection, from 1451+/-45 ng/mL at 10 min to 364+/-59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240+/-90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63+/-5% of the administered dose. Integration plot analysis indicated a CL(uptake) of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.

Marine puffer fish accumulates tetrodotoxin (TTX) in the liver and ovary. In this study, we examined the pharmacokinetics of TTX in Takifugu rubripes by a single administration under general anesthesia at 20 degrees C for 300 min. The blood concentration-time profile showed multiple distinct phases after injection into hepatic portal vein. The area under the blood concentration-time curve (AUC) increased linearly at the dosage of 0.25-0.75 mg TTX/kg body weight, and the total body clearance was 2.06+/-0.17 mL/min/kg body weight. The AUCs following administration into the hepatic portal vein and hepatic vein were closely similar (147+/-33 versus 141+/-1 ng.min/microL), indicating negligible hepatic first-pass effect. Comparison of the AUCs following an administration to the hepatic vein and gastrointestinal tract (0.25 mg TTX/kg body weight) elucidated the bioavailability of TTX to be 62%. There was no significant increase in the AUCs following direct injection into the gastrointestinal tract (0.50 versus 1.0 mg TTX/kg body weight). At the dosage of 0.25 mg TTX/kg body weight into the hepatic vein, hepatic portal vein or gastrointestinal tract, TTX amount in the liver accounted for 84+/-6%, 70+/-9% or 49+/-17% of the total TTX amount applied, respectively. These results demonstrate that TTX is absorbed into the systemic circulation from the gastrointestinal tract by saturable mechanism and finally accumulated in the liver within 300 min.

Although puffer fish contain tetrodotoxin (TTX) at a high concentration mainly in liver, the underlying mechanism remains to be elucidated. In the present study, uptake of TTX into the liver tissue slices of puffer fish Takifugu rubripes was investigated by in vitro incubation experiment. When T. rubripes liver slices were incubated with 0-2000microM TTX at 20 degrees C for 60min, the uptake rates exhibited non-linearity, suggesting that the TTX uptake into T. rubripes liver is carrier-mediated. The TTX uptake was composed of a saturable component (V(max) 47.7+/-5.9pmol/min/mg protein and K(m) 249+/-47microM) and a non-saturable component (P(dif) 0.0335+/-0.0041microL/min/mg protein). The uptake of TTX was significantly decreased to 0.4 and 0.6 fold by the incubation at 5 degrees C and the replacement of sodium-ion by choline in the buffer, respectively, while it was not affected by the presence of 1mM l-carnitine, p-aminohippurate, taurocholate or tetraethylammonium. The TTX uptake by black scraper Thamnaconus modestus liver slices was much lower than that of T. rubripes and independent of the incubation temperature, unlike T. rubripes. These results reveal the involvement of carrier-mediated transport system in the TTX uptake by puffer fish T. rubripes liver slices.