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Mari Yotsuyamashita - One of the best experts on this subject based on the ideXlab platform.
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isolation and structural determination of the first 8 epi type tetrodotoxin analogs from the newt cynops ensicauda popei and comparison of tetrodotoxin analogs profiles of this newt and the Puffer Fish fugu poecilonotus
Marine Drugs, 2012Co-Authors: Yuta Kudo, Takeshi Yasumoto, Keiichi Konoki, Yuko Cho, Mari YotsuyamashitaAbstract:Identification of new tetrodotoxin (TTX) analogs from TTX-possessing animals might provide insight into its biosynthesis and metabolism. In this study, four new analogs, 8-epi-5,6,11-trideoxyTTX, 4,9-anhydro-8-epi-5,6,11-trideoxyTTX, 1-hydroxy-8-epi-5,6,11-trideoxyTTX, and 1-hydroxy-4,4a-anhydro-8-epi-5,6,11-trideoxyTTX, were isolated from the newt, Cynops ensicauda popei, and their structures were determined using spectroscopic methods. These are the first 8-epi-type analogs of TTX that have been found in a natural source. Furthermore, we examined the composition of the TTX analogs in this newt and in the ovary of the Puffer Fish, Fugu poecilonotus, using LC/MS. The results indicate that TTX and 11-deoxyTTX were present in both sources. However, 6-epiTTX and 8-epi-type analogs were detected only in the newt, while 5,6,11-trideoxyTTX was a specific and major analog in the Puffer Fish. Such considerable differences among analog compositions might reflect differences in the biosynthesis or metabolism of TTX between these animals.
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distribution of homologous proteins to Puffer Fish saxitoxin and tetrodotoxin binding protein in the plasma of Puffer Fish and among the tissues of fugu pardalis examined by western blot analysis
Toxicon, 2010Co-Authors: Mari Yotsuyamashita, Hiroe Yamaki, Natsumi Okoshi, Nao ArakiAbstract:Abstract Puffer Fish saxitoxin and tetrodotoxin binding protein (PSTBP) is a glycoprotein (200 kDa as a dimer) that we previously isolated from the plasma of Fugu pardalis ( Yotsu-Yamashita et al., 2001 ). For the study on functions of PSTBP, here we examined distribution of homologous proteins to PSTBP in the plasma of seven species of Puffer Fish, and among the tissues of F. pardalis by Western blot analysis probed with a polyclonal IgG against unglycosylated PSTBP1 expressed in Echelichia coli. One or two major positive broad bands were detected at 105–140 kDa molecular weight range in the plasma (0.5 μg protein) of all species of Puffer Fish tested, while no band was detected in the plasma (5 μg protein) of Fish other than Puffer Fish. Glycopeptidase F treated plasma of all species of Puffer Fish tested commonly showed the bands at approximately 42 kDa that was consistent to the molecular weight of unglycosylated PSTBP. These data suggest that Puffer Fish commonly possess glycoproteins homologous to PSTBP, but the sizes of N -glycan are specific to the species. Among soluble protein extracts (5 μg protein) from the tissues of F. pardalis , PSTBP was detected in all tissues examined, most prominently in heart, skin, and gall.
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examination of transformation among tetrodotoxin and its analogs in the living cultured juvenile Puffer Fish kusafugu fugu niphobles by intramuscular administration
Toxicon, 2008Co-Authors: Michiko Kono, Jun-ho Jang, Takashi Matsui, Kiyoshi Furukawa, Takuhiko Takase, Kunio Yamamori, Hideko Kaneda, Daisuke Aoki, Mari YotsuyamashitaAbstract:In Puffer Fish, tetrodotoxin (TTX) exists as the major toxin with chemically equilibrium analogs (4-epiTTX, 4,9-anhydroTTX) and chemically non-equilibrium analogs (deoxy analogs, 11-oxoTTX, 4-S-cysteinylTTX). There are two purposes to this study: 1) to search for the reason why TTX is the most major analog in Puffer Fish, even 4,9-anhydroTTX is chemically more stable, 2) to investigate whether or not chemically non-equilibrium analogs are transformed in Puffer Fish, because these were predicted to be biosynthetic intermediates. Pure TTX, 4-epiTTX, 4,9-anhydroTTX, and 11-oxoTTX were separately administrated to the cultured non-toxic juvenile Puffer Fish kusafugu, Fugu niphobles by intramuscular injection. Sixteen days after administration, TTX analogs in the whole Fish were analyzed by LC-fluorescent detection and LC/MS. By the administration of TTX, 4-epiTTX, and 4,9-anhydroTTX, 34-40% of the administrated doses of the toxins were accumulated, and 4,9-anhydroTTX has become the major toxin after inter-conversion. This result indicates discrepancy from the previous ones wherein TTX was predominantly accumulated when TTXs were administrated through diets; this suggests that dietary administration might be necessary to accumulate TTX as the major toxin, and not 4,9-anhydroTTX. Transformations from TTX to deoxy analogs or 11-oxoTTX, or from 11-oxoTTX to TTX were not detected in this study.
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two critical residues in p loop regions of Puffer Fish na channels on ttx sensitivity
Toxicon, 2008Co-Authors: Satoshi Maruta, Kaoru Yamaoka, Mari YotsuyamashitaAbstract:We previously showed that Asn-383 and Thr-1569 residues of p-loop regions in domains I and IV, respectively, of the Puffer Fish, Fugu pardialis, skeletal muscle Na(v) (fNa(v)1.4a), are anomalous to those of other species of TTX-sensitive Na(+) channels, where the aromatic residues of Phe or Tyr, and Gly are the counterparts [Yotsu-Yamashita, M., Nishimori, K., Nitanai, Y., Isemura, M., Sugimoto, A., Yasumoto, T., 2000. Binding properties of (3)H-PbTx-3 and (3)H-saxitoxin to brain membranes and to skeletal muscle membranes of Puffer Fish Fugu pardalis and the primary structure of a voltage-gated Na(+) channel alpha-subunit (fMNa1) from skeletal muscle of F. pardalis. Biochem. Biophys. Res. Commun. 267, 403-412]. The former was suggested to confer TTX resistance by using Y401N mutant of rNa(v)1.4 [Venkatesh, V., Lu, S.Q., Dandona, N., See, S.L., Benner, S., Soong, T.W., 2005. Genetic basis of tetrodotoxin resistance in PufferFishes. Curr. Biol. 15, 2069-2072]. The latter function remained to be elucidated. Thus, we further explored the function of these two residues, electrophysiologically, by evaluating the K(d) (dissociation constants) values of TTX for F385N, F385A, F385Q, G1718T, and F385N/G1718T mutants of rNa(v)1.2a, transiently expressed in HEK-293 cells. F385N caused 3000-fold increase of the K(d), while G1718T and F385N/G1718T caused 2- and 3-fold increases compared with those of WT and F385N, respectively, suggesting that G1718T further enhanced TTX resistivity caused by F385N. The K(d) for F385A and F385Q were 2- and 11-fold larger than that of F385N, respectively, suggesting that the longer side chain in the non-aromatic amino acid residue causes the larger decrease of TTX sensitivity. Despite drastic changes in the K(d), the mutations at F385 caused only small changes in the k(off) from that of WT, suggesting that the K(d) for TTX receptors are mainly determined by the k(on).
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distribution of tetrodotoxin saxitoxin and their analogs among tissues of the Puffer Fish fugu pardalis
Toxicon, 2006Co-Authors: Jun-ho Jang, Mari YotsuyamashitaAbstract:Abstract The anatomical distribution of tetrodotoxin (TTX), saxitoxin (STX) and their analogs (TTXs, STXs) in three female and three male specimens of the marine Puffer Fish Fugu pardalis from Miyagi Prefecture, 2005, Japan, were studied. 5-DeoxyTTX, 11-deoxyTTX, and 5,6,11-trideoxyTTX were quantified by liquid chromatography/mass spectrometry (LC/MS) for the first time, and other TTXs and STXs were determined by liquid chromatography-fluorescent detection (LC-FLD). As a result, 5,6,11-trideoxyTTX was found to be the major TTX analog in all tissues tested, whereas 5-deoxyTTX and 11-deoxyTTX were minor components. Especially, in female ( n = 3 ), the ratios of 5,6,11-trideoxyTTX to total of all TTX analogs (mole/mole) in ovaries (mean±SD, 0.42±0.055) were significantly larger than those in livers (0.17±0.025) ( P 0.05 ). In contrary, the ratios of 4,9-anhydroTTX to total of all TTX analogs in livers (0.27±0.047) were significantly larger than those in ovaries (0.073±0.040) ( P 0.01 ). The ratios of TTX to total of all TTX analogs were not significantly different between ovaries (0.47±0.078) and livers (0.55±0.067). In male ( n = 3 ), all these ratios were not significantly different between livers and testis. 4-S-CysteinylTTX was detected in liver, spleen, gall, and intestine in 1–6 mole% of total of all TTX analogs, supporting our previous hypothesis that 4-S-cysteinylTTX is a metabolite of TTX.
Yuji Nagashima - One of the best experts on this subject based on the ideXlab platform.
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differential gene expression profile in the liver of the marine Puffer Fish takifugu rubripes induced by intramuscular administration of tetrodotoxin
Toxicon, 2011Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Yuji NagashimaAbstract: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.
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plasma protein binding of tetrodotoxin in the marine Puffer Fish takifugu rubripes
Toxicon, 2010Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Daisuke Tanuma, Kazuma Tsutsumi, Joongkyun Jeon, Yuji NagashimaAbstract:Abstract 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 μg/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 μg TTX/mg protein at 1000 μg 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 μg TTX/mg protein at 1000 μg TTX/mL, and that in the bovine AGP was 8.78 ± 0.25 μg TTX/mg protein at 200 μg TTX/mL.
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change in tetrodotoxin content of the Puffer Fish takifugu rubripes during seed production from fertilized eggs to juveniles
Journal of The Food Hygienic Society of Japan (shokuhin Eiseigaku Zasshi), 2010Co-Authors: Yuji Nagashima, Kuniyoshi Shimakura, Isao Mataki, Maho Toyoda, Hiroshi Nakajima, Kingo Tsumoto, Kazuo ShiomiAbstract:Changes in tetrodotoxin (TTX) content of the Puffer Fish Takifugu rubripes during seed production were examined. Two mature female Puffer Fish T. rubripes (samples 1 and 2) that contained TTX were used. The toxic eggs were artificially fertilized, and hatchlings were reared in an indoor tank for 50 days and then in a netcage at sea for an additional 48 or 38 days. The TTX content of the fertilized eggs of sample 1 was initially 13.0 microg TTX/g, transiently increased to 67.6 microg TTX/g at 4 days after hatching, and then gradually decreased to 0.28 microg TTX/g at 98 days. In contrast, the total TTX content in an individual was 0.016 microg TTX at the fertilization stage and 0.01-0.03 microg TTX at the larval stage until 30 days after hatching. Thereafter, the total TTX content increased remarkably during culture in the netcage at sea, reaching 4.80 microg TTX at 98 days. Change in the TTX content of sample 2 showed a similar tendency to that of sample 1. The present study showed that the TTX content per gram of Puffer Fish body weight decreased during progression from fertilized eggs to juveniles, whereas the total TTX content increased.
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evaluation of hepatic uptake clearance of tetrodotoxin in the Puffer Fish takifugu rubripes
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract 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 0.25 mg TTX/kg body weight into the hepatic vein at 20 °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.
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pharmacokinetics of tetrodotoxin in Puffer Fish takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract 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 °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/μL), 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.
Kazuo Shiomi - One of the best experts on this subject based on the ideXlab platform.
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change in tetrodotoxin content of the Puffer Fish takifugu rubripes during seed production from fertilized eggs to juveniles
Journal of The Food Hygienic Society of Japan (shokuhin Eiseigaku Zasshi), 2010Co-Authors: Yuji Nagashima, Kuniyoshi Shimakura, Isao Mataki, Maho Toyoda, Hiroshi Nakajima, Kingo Tsumoto, Kazuo ShiomiAbstract:Changes in tetrodotoxin (TTX) content of the Puffer Fish Takifugu rubripes during seed production were examined. Two mature female Puffer Fish T. rubripes (samples 1 and 2) that contained TTX were used. The toxic eggs were artificially fertilized, and hatchlings were reared in an indoor tank for 50 days and then in a netcage at sea for an additional 48 or 38 days. The TTX content of the fertilized eggs of sample 1 was initially 13.0 microg TTX/g, transiently increased to 67.6 microg TTX/g at 4 days after hatching, and then gradually decreased to 0.28 microg TTX/g at 98 days. In contrast, the total TTX content in an individual was 0.016 microg TTX at the fertilization stage and 0.01-0.03 microg TTX at the larval stage until 30 days after hatching. Thereafter, the total TTX content increased remarkably during culture in the netcage at sea, reaching 4.80 microg TTX at 98 days. Change in the TTX content of sample 2 showed a similar tendency to that of sample 1. The present study showed that the TTX content per gram of Puffer Fish body weight decreased during progression from fertilized eggs to juveniles, whereas the total TTX content increased.
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evaluation of hepatic uptake clearance of tetrodotoxin in the Puffer Fish takifugu rubripes
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract 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 0.25 mg TTX/kg body weight into the hepatic vein at 20 °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.
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pharmacokinetics of tetrodotoxin in Puffer Fish takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract 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 °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/μL), 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.
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pharmacokinetics of tetrodotoxin in Puffer Fish takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract: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.
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involvement of carrier mediated transport system in uptake of tetrodotoxin into liver tissue slices of Puffer Fish takifugu rubripes
Toxicon, 2007Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Yuichi Sugiyama, Kazuo ShiomiAbstract: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.
Takuya Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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differential gene expression profile in the liver of the marine Puffer Fish takifugu rubripes induced by intramuscular administration of tetrodotoxin
Toxicon, 2011Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Yuji NagashimaAbstract: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.
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plasma protein binding of tetrodotoxin in the marine Puffer Fish takifugu rubripes
Toxicon, 2010Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Daisuke Tanuma, Kazuma Tsutsumi, Joongkyun Jeon, Yuji NagashimaAbstract:Abstract 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 μg/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 μg TTX/mg protein at 1000 μg 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 μg TTX/mg protein at 1000 μg TTX/mL, and that in the bovine AGP was 8.78 ± 0.25 μg TTX/mg protein at 200 μg TTX/mL.
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evaluation of hepatic uptake clearance of tetrodotoxin in the Puffer Fish takifugu rubripes
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract 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 0.25 mg TTX/kg body weight into the hepatic vein at 20 °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.
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pharmacokinetics of tetrodotoxin in Puffer Fish takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract 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 °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/μL), 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.
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pharmacokinetics of tetrodotoxin in Puffer Fish takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract: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.
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saxitoxin poisoning in green turtles chelonia mydas linked to scavenging on mass mortality of caribbean sharpnose Puffer Fish canthigaster rostrata tetraodontidae
Frontiers in Veterinary Science, 2019Co-Authors: Rocio Gonzalez Barrientos, Sara C. Mcgrath, Jonathan R. Deeds, Leanne J Flewelling, Gabriela Hernandezmora, Fernando Alegre, Theresa Field, Yajaira Salazar Chacon, Karla Rojas Arrieta, Emilia Calvo VargasAbstract:Fish within the family Tetraodontidae are potential sources of both endogenous tetrodotoxins (TTXs) and dietary derived saxitoxins (STXs). Ingestion of Fish tissues containing these toxins by other vertebrates can lead to severe illness and death. The Caribbean sharpnose Puffer (Canthigaster rostrata) is a widespread tetraodontid species within the western Atlantic. Mass settlement of juveniles into foraging habitats have been associated with large-scale Puffer Fish mortality events. In 2013, 2014, and 2017, Puffer mortality events on the southern Caribbean coast of Costa Rica were also associated with strandings of green turtles (Chelonia mydas) found to have fed on C. rostrata. Stranded sea turtles were found dead without apparent cause or alive with severe neurological signs that resolved during short periods of captivity. Puffer Fish and turtle organ samples were analyzed for both TTXs and STXs. Concentrations of TTXs were extremely low in the Fish (0.5-0.7 μg/g) and undetectable in turtle stomach contents. However, concentrations of STXs in whole Fish (16.6-47.5 μg STX-eq/g) exceeded the 0.8 μg STX-eq/g human seafood safety threshold for STXs by orders of magnitude. Saxitoxins were also detected in samples of stomach contents (ingested Fish), brain, lung, kidney, and serum from two affected turtles. Study results indicate that saxitoxicosis resulting from opportunistic foraging on C. rostrata during Fish mortality events may be a significant factor in episodic stranding of green sea turtles in this region.
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saxitoxin poisoning in green turtles chelonia mydas linked to scavenging on mass mortality of caribbean sharpnose Puffer Fish canthigaster rostrata tetraodontidae
Frontiers in Veterinary Science, 2019Co-Authors: Rocio Gonzalez Barrientos, Sara C. Mcgrath, Jonathan R. Deeds, Leanne J Flewelling, Gabriela Hernandezmora, Fernando Alegre, Theresa Field, Yajaira Salazar Chacon, Karla Rojas Arrieta, Emilia Calvo VargasAbstract:Fish within the family Tetraodontidae are potential sources of both endogenous tetrodotoxins (TTXs) and dietary derived saxitoxins (STXs). Ingestion of Fish tissues containing these toxins by other vertebrates can lead to severe illness and death. The Caribbean sharpnose Puffer (Canthigaster rostrata) is a widespread tetraodontid species within the western Atlantic. Mass settlement of juveniles into foraging habitats have been associated with large-scale Puffer Fish mortality events. In 2013, 2014, and 2017, Puffer mortality events on the southern Caribbean coast of Costa Rica were also associated with strandings of green turtles (Chelonia mydas) found to have fed on C. rostrata. Stranded sea turtles were found dead without apparent cause or alive with severe neurological signs that resolved during short periods of captivity. Puffer Fish and turtle organ samples were analyzed for both TTXs and STXs. Concentrations of TTXs were extremely low in the Fish (0.5-0.7 μg/g) and undetectable in turtle stomach contents. However, concentrations of STXs in whole Fish (16.6-47.5 μg STX-eq/g) exceeded the 0.8 μg STX-eq/g human seafood safety threshold for STXs by orders of magnitude. Saxitoxins were also detected in samples of stomach contents (ingested Fish), brain, lung, kidney, and serum from three affected turtles. Study results indicate that saxitoxicosis resulting from opportunistic foraging on C. rostrata during Fish mortality events may be a significant factor in episodic stranding of green sea turtles in this region.
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Tetrodotoxin detection in Puffer Fish by a sensitive planar waveguide immunosensor
Sensors and Actuators B: Chemical, 2017Co-Authors: Laia Reverté, Jonathan R. Deeds, Monica Campas, Betsy Jean Yakes, Panagiota Katikou, Kentaro Kawatsu, Michael J. Lochhead, Christopher T. Elliott, Katrina CampbellAbstract:Abstract A nanoarray planar waveguide biosensor was developed for the detection of tetrodotoxin (TTX). This technique, specifically designed for the first time for TTX, provided a compact versatile user friendly device that obtained a test result in ten minutes. The device consisted of nanoprinted toxin-conjugate arrays constructed in the manner of an indirect competitive immunoassay, for the analysis of Puffer Fish samples under high flow conditions. The applicability to natural samples was investigated through the study of matrix effects and toxin recovery. The biosensor enabled the detection of TTX from 0.4 to 3.29 μg g−1 Puffer Fish tissue. The sensitivity attained demonstrates this assay as a rapid and sensitive screening method to detect TTX in different species of Puffer Fish, well below the Japanese maximum permitted level (2 μg g−1) and the estimated safety level used in the EU and the US (0.8 μg g−1). Assay repeatability and reproducibility were assessed at 0.4 and 0.8 μg g−1, showing relative standard deviation (RSD) values below 15% and toxin recovery within 85–115%. The appropriate correlation of data obtained from the biosensor compared to that reported by ELISA, RBA, SPR biosensor and LC–MS/MS for the analysis of 12 Puffer Fish samples, proved the feasibility and reliability of this immunosensor to support monitoring programs and research activities.
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Tetrodotoxin poisoning outbreak from imported dried Puffer Fish - Minneapolis, Minnesota, 2014
Morbidity and Mortality Weekly Report, 2015Co-Authors: Jon B. Cole, William G. Heegaard, Sara C. Mcgrath, Jonathan R. Deeds, Sara M HandyAbstract:On June 13, 2014, two patients went to the Hennepin County Medical Center Emergency Department in Minneapolis, Minnesota, with symptoms suggestive of tetrodotoxin poisoning (i.e., oral paresthesias, weakness, and dyspnea) after consuming dried Puffer Fish (also known as globeFish) purchased during a recent visit to New York City. The patients said two friends who consumed the same Fish had similar, although less pronounced, symptoms and had not sought care. The Minnesota Department of Health conducted an investigation to determine the source of the product and samples were sent to the Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition for chemical and genetic analysis. Genetic analysis identified the product as Puffer Fish (Lagocephalus lunaris) and chemical analysis determined it was contaminated with high levels of tetrodotoxin. A traceback investigation was unable to determine the original source of the product. Tetrodotoxin is a deadly, potent poison; the minimum lethal dose in an adult human is estimated to be 2-3 mg. Tetrodotoxin is a heat-stable and acid-stable, nonprotein, alkaloid toxin found in many species of the Fish family Tetraodontidae (Puffer Fish) as well as in certain gobies, amphibians, invertebrates, and the blue-ringed octopus. Tetrodotoxin exerts its effects by blocking voltage-activated sodium channels, terminating nerve conduction and muscle action potentials, leading to progressive paralysis and, in extreme cases, to death from respiratory failure. Because these Fish were reportedly purchased in the United States, they pose a substantial U.S. public health hazard given the potency of the toxin and the high levels of toxin found in the Fish. Language: en
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Public health response to Puffer Fish (tetrodotoxin) poisoning from mislabeled product.
Journal of food protection, 2009Co-Authors: Nicole J. Cohen, Jonathan R. Deeds, Eugene S Wong, Robert Hanner, Haile F. Yancy, Kevin D White, Trevonne M. Thompson, Michael Wahl, Tu D Pham, Frances M GuichardAbstract:Tetrodotoxin is a neurotoxin that occurs in select species of the family Tetraodontidae (Puffer Fish). It causes paralysis and potentially death if ingested in sufficient quantities. In 2007, two individuals developed symptoms consistent with tetrodotoxin poisoning after ingesting home-cooked Puffer Fish purchased in Chicago. Both the Chicago retailer and the California supplier denied having sold or imported Puffer Fish but claimed the product was monkFish. However, genetic analysis and visual inspection determined that the ingested Fish and others from the implicated lot retrieved from the supplier belonged to the family Tetraodontidae. Tetrodotoxin was detected at high levels in both remnants of the ingested meal and Fish retrieved from the implicated lot. The investigation led to a voluntary recall of monkFish distributed by the supplier in three states and placement of the supplier on the U.S. Food and Drug Administration's Import Alert for species misbranding. This case of tetrodotoxin poisoning highlights the need for continued stringent regulation of Puffer Fish importation by the U.S. Food and Drug Administration, education of the public regarding the dangers of Puffer Fish consumption, and raising awareness among medical providers of the diagnosis and management of foodborne toxin ingestions and the need for reporting to public health agencies.
