Isolation and Biological Activity of 9-epiTetrodotoxin and Isolation of Tb-242B, Possible Biosynthetic Shunt Products of Tetrodotoxin from Pufferfish.

Tetrodotoxin (TTX, 1) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-epiTTX (2), and a TTX-related compound, Tb-242B (4), isolated from the pufferfish Takifugu flavipterus and Dichotomyctere ocellatus, respectively. NMR analysis suggested that 2 exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms. Compound 2 and TTX were confirmed not to convert to each other by incubation under neutral and acidic conditions at 37 °C for 24 h. Compound 4 was identified as the 9-epimer of Tb-242A (3), previously reported as a possible biosynthetic precursor of TTX. Compound 4 was partially converted to 3 by incubation in a neutral buffer at 37 °C for 7 days, whereas 3 was not converted to 4 under this condition. Compound 2 was detected in several TTX-containing marine animals and a newt. Mice injected with 600 ng of 2 by intraperitoneal injection did not show any adverse symptoms, suggesting that the C-9 configuration in TTX is critical for its biological activity. Based on the structures, 2 and 4 were predicted to be shunt products for TTX biosynthesis.

Simultaneous determination of ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam by ion-pair solid-phase extraction and hydrophilic interaction chromatography with tandem mass spectrometry.

Paralytic shellfish toxins and tetrodotoxin (puffer-fish toxin), the latter of which was recently found in bivalves from Europe, Japan, and New Zealand, are potent neurotoxins. A simple and effective clean-up procedure was developed for the simultaneous determination of ten paralytic shellfish toxins (gonyautoxins 1-6, decarbamoylgonyautoxins 2 and 3, and N-sulfocarbamoylgonyautoxins 2 and 3) and tetrodotoxin in the scallop, Mizuhopecten (Patinopecten) yessoensis, and the short-necked clam, Ruditapes philippinarum. To reduce matrix effects, 1% aqueous acetic acid extracts of the bivalves were cleaned up by ion-pair solid-phase extraction using a graphite carbon cartridge with tridecafluoroheptanoic acid as the volatile ion-pair reagent, followed by fourfold dilution. The ten paralytic shellfish toxins and tetrodotoxin were then separated on a hydrophilic interaction chromatography column and quantified by tandem mass spectrometry. The limits of detection and the limits of quantification for the ten PSTs ranged from 0.09 to 13.0 µg saxitoxin equivalents/kg and from 0.26 to 39.4 µg saxitoxin equivalents/kg, respectively. The limit of detection and the limit of quantification for tetrodotoxin ranged from 27.4 to 27.9 µg/kg and from 83.1 to 84.4 µg/kg, respectively. The proposed method yielded minimal matrix effects for the 11 analytes, thus allowing their quantification by simple external calibration. The proposed method also gave good mean recoveries of the 11 analytes ranging from 75.7 to 96.2% with relative standard deviations less than 16% at three fortification levels for the ten paralytic shellfish toxins (total concentrations of 277, 554, and 1107 µg saxitoxin equivalents/kg) and tetrodotoxin (100, 200, and 400 µg/kg) in the two bivalve samples. Finally, the proposed method was applied for the determination of the ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam samples.

First Detection of Tetrodotoxins in the Cotylean Flatworm Prosthiostomum trilineatum.

Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific Planocera lineages belonging to the suborder Acotylea. During our ongoing study of flatworm toxins, high concentrations of TTXs were detected for the first time in the flatworm Prosthiostomum trilineatum, suborder Cotylea, from the coastal area of Hayama, Kanagawa, Japan. Toxin levels were investigated by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing that this species contains comparable concentrations of toxins as seen in planocerid flatworms such as Planocera multitentaculata. This finding indicated that there may be other species with significant levels of TTXs. The distribution of TTXs among other flatworm species is thus of great interest.

Taxonomic Distribution of Tetrodotoxin in Acotylean Flatworms (Polycladida: Platyhelminthes).

Tetrodotoxin (TTX), also known as pufferfish toxin, causes a respiratory disorder by blocking neurotransmission, with voltage-gated sodium channel inhibition on muscle and nerve tissues. The toxin is widely distributed across vertebrates, invertebrates and bacteria. Therefore, it is generally thought that TTX in pufferfish accumulates via the food webs, beginning with marine bacteria as a primary producer. Polyclad flatworms in the genus Planocera are also known to be highly toxic, TTX-bearing organisms. Unlike the case of pufferfish, the source of TTX in these flatworms is unknown. In this study, taxonomical distribution patterns of TTX were investigated for acotylean flatworms from coastal waters using molecular phylogenetic analysis and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). A maximum likelihood tree based on the 28S rRNA gene sequence showed that the flatworms belonged to several different lineages among the genera Planocera, Stylochus, Paraplanocera, Discocelis, Notocomplana, Notoplana, Callioplana and Peudostylochus. After LC-MS/MS analysis, the distribution of TTX was mapped onto the molecular phylogenetic tree. TTX-bearing flatworm species were seen to be restricted to specific Planocera lineages, suggesting that the TTX-bearing flatworm species have common genes for TTX-accumulating mechanisms.

Tetrodotoxin and saxitoxin in two native species of puffer fish, Sphoeroides marmoratus and Lagocephalus lagocephalus, from NE Atlantic Ocean (Madeira Island, Portugal).

The presence in EU waters of invasive tetrodotoxin (TTX) -harbouring puffer fishes has been receiving increasingly attention due to potential new threats posed by this potent neurotoxin. The present study investigates the occurrence of tetrodotoxin, saxitoxin (STX), and their analogues in two native puffer fish species from the NE Atlantic. High TTX content was detected by LC-MS/MS in several tissues of the Guinean puffer Sphoeroides marmoratus from Madeira Island (Portugal), reaching concentrations as high as 15 mg TTX kg-1 in the digestive tract of a male specimen and 7.4 mg TTX kg-1 in gonads of a female specimen. Several TTX analogues were also detected, including the 4-epi-TTX, 4,9-Anhydro-TTX, 5- 11- deoxyTTX and 6,11-dideoxyTTX. Although at low levels, STX was detected in liver of the Oceanic puffer Lagocephalus lagocephalus. Trace levels of decarbamoylsaxitoxin (dcSTX) were also observed in L. lagocephalus. This study reports the presence of TTX and STX in native fish from EU waters, highlighting the need for a proper understating of the origin, distribution and fate of these toxins in NE Atlantic.

Toxic Flatworm Egg Plates Serve as a Possible Source of Tetrodotoxin for Pufferfish.

The pufferfish Takifugu niphobles (at present Takifugu alboplumbeus) possesses highly concentrated tetrodotoxin (TTX), an extremely potent neurotoxin that provides effective protection from predators, at least at the larval stages. However, the source of the toxin has remained unclear. Recently, DNA from the toxic flatworm Planocera multitentaculata was detected in the intestinal contents of juveniles and young of the pufferfish, suggesting that the flatworm contributes to its toxification at various stages of its life. In this study, we describe the behavior of the pufferfish in the intertidal zone that appears to contribute to its toxification before and during its spawning period: pufferfish were found to aggregate and ingest flatworm egg plates by scraping them off the surface of rocks. DNA analysis based on 28S rRNA and cytochrome c oxidase subunit I (COI) genes identified the egg plates as those of P. multitentaculata. Liquid chromatography with tandem mass spectrometry analysis revealed that the egg plates contain highly concentrated TTX. The feeding behavior of the pufferfish on the flatworm egg plates was also observed in the aquarium. These results suggest that pufferfish feed on the flatworm egg plate, which enables them to acquire toxicity themselves while providing their offspring with the protective shield of TTX.

Detection of tetrodotoxins in juvenile pufferfish Lagocephalus sceleratus (Gmelin, 1789) from the North Aegean Sea (Greece) by an electrochemical magnetic bead-based immunosensing tool.

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety.

Total Syntheses and Determination of Absolute Configurations of Cep-212 and Cep-210, Predicted Biosynthetic Intermediates of Tetrodotoxin Isolated from Toxic Newt.

Total syntheses of Cep-212 and Cep-210, predicted biosynthetic intermediates of tetrodotoxin isolated from the Japanese toxic newt, have been accomplished from geraniol by an intramolecular hetero Diels-Alder reaction as a key step in a highly stereoselective manner. The success of these syntheses enabled us to determine their absolute configurations by using a chiral normal-phase HPLC/MS analysis of the bis-dinitrobenzene derivative of natural Cep-212 and reference derivatives prepared from chemically synthesized enantiomers.

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin.

In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( Kd = 62.07 ± 19.97 nM), TTX-07 ( Kd = 44.12 ± 15.38 nM), and STX-41 ( Kd = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.

Difference in Uptake of Tetrodotoxin and Saxitoxins into Liver Tissue Slices among Pufferfish, Boxfish and Porcupinefish.

Although pufferfish of the family Tetraodontidae contain high levels of tetrodotoxin (TTX) mainly in the liver, some species of pufferfish, boxfish of the family Ostraciidae, and porcupinefish of the family Diodontidae do not. To clarify the mechanisms, uptake of TTX and saxitoxins (STXs) into liver tissue slices of pufferfish, boxfish and porcupinefish was examined. Liver tissue slices of the pufferfish (toxic species Takifugu rubripes and non-toxic species Lagocephalus spadiceus, L. cheesemanii and Sphoeroides pachygaster) incubated with 50 µM TTX accumulated TTX (0.99-1.55 µg TTX/mg protein) after 8 h, regardless of the toxicity of the species. In contrast, in liver tissue slices of boxfish (Ostracion immaculatus) and porcupinefish (Diodon holocanthus, D. liturosus, D. hystrix and Chilomycterus reticulatus), TTX content did not increase with incubation time, and was about 0.1 µg TTX/mg protein. When liver tissue slices were incubated with 50 µM STXs for 8 h, the STXs content was <0.1 µg STXs/mg protein, irrespective of the fish species. These findings indicate that, like the toxic species of pufferfish T. rubripes, non-toxic species such as L. spadiceus, L. cheesemanii and S. pachygaster, potentially take up TTX into the liver, while non-toxic boxfish and porcupinefish do not take up either TTX or STXs.

A novel function of vitellogenin subdomain, vWF type D, as a toxin-binding protein in the pufferfish Takifugu pardalis ovary.

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.

Rapid in vivo determination of tetrodotoxin in pufferfish (Fugu) muscle by solid-phase microextraction coupled to high-performance liquid chromatography tandem mass spectrometry.

Tetrodotoxin (TTX) is one of the most toxic substances of non-protein in nature. In present study, a rapid and sensitive method based on in vivo solid-phase microextraction (SPME) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to detect the endogenous TTX in pufferfish (Fugu). Fiber evaluation experiments demonstrated that, compared with the commercial PDMS and PA fibers, the home-made electrospun PS@PDA-GA fibers exhibited much better extraction performance towards to TTX in water (120 times and 20 times, respectively), under the optimized conditions. Then, the home-made SPME fibers were employed to extract TTX in spiked homogeneous fish muscle samples, and a LC-MS-MS was used for the analysis. The reproducibilities (RSDs of inter and intra fiber were 12.1%, and 7.9% respectively), linear range (10-1000ngg-1, R2=0.9963) and sensitivity (the LOD was 2.3ngg-1) of the method were found to be excellent and satisfactory for further in vivo experiments. Especially the LOD of the established method is lower than the National Standard Method of China (GB/T 23217-2008, LOD 50ngg-1). Subsequently, the method was successfully applied to detect the TTX in the dorsal muscle of living pufferfish, and the accuracy was verified with traditional liquid extraction (LE) method. In general, this is the first study to detect TTX in pufferfish by in vivo sampling method, which provided a promising alternative method for the studies of TTX, and also advanced the implementation of SPME for more in vivo studies.

Tetrodotoxin in Asian newts (Salamandridae).

Tetrodotoxin (TTX) and its analogues occur in a wide range of marine animals but also in terrestrial vertebrates such as frogs, toads and newts. Despite numerous studies on TTX in New World newts (Notophthalmus viridescens, Taricha spp.), few data only exist for Asian newts. Methanolic extracts of newts from China (Cynops orientalis, Pachytriton labiatus, Paramesotriton chinensis), Vietnam (Paramesotriton deloustali, P. guangxiensis), and Laos (Laotriton laoensis) were analyzed by liquid-chromatography-fluorescent detection (LC-FLD) and mass-spectrometry (LC-MS). In all species, variable amounts of TTX were detected, in most specimens also TTX-analogues like 6-epiTTX, in C. orientalis 11-oxoTTX, confirming the presence of these toxins in modern Asian newts.

Seasonal changes in the tetrodotoxin content of the pufferfish Takifugu niphobles.

To investigate seasonal changes in the whole body content and tissue distribution of tetrodotoxin (TTX) in the pufferfish Takifugu niphobles, wild individuals were collected from the coastal waters around Miura Peninsula from November 2010 to May 2012, and their tissues were subjected to LC-MS/MS analysis. Fish that were sexually mature were classified as being in the maturation period (April), the spawning period (May-July) or the "ordinary period" (i.e., other months). In both sexes, gonad somatic index rapidly increased during the maturation period and then decreased during the spawning period. Whole body TTX content was significantly higher during the maturation/spawning period than in the ordinary period. Through all seasons, TTX was localized in the skin or ovary in females and in the skin or liver in males: the difference in TTX localization between females and males was particularly evident during the spawning period.

Tetrodotoxin, an Extremely Potent Marine Neurotoxin: Distribution, Toxicity, Origin and Therapeutical Uses.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many human intoxications and fatalities each year. The origin of TTX is unknown, but in the pufferfish, it seems to be produced by endosymbiotic bacteria that often seem to be passed down the food chain. The ingestion of contaminated pufferfish, considered the most delicious fish in Japan, is the usual route of toxicity. This neurotoxin, reported as a threat to human health in Asian countries, has spread to the Pacific and Mediterranean, due to the increase of temperature waters worldwide. TTX, for which there is no known antidote, inhibits sodium channel producing heart failure in many cases and consequently death. In Japan, a regulatory limit of 2 mg eq TTX/kg was established, although the restaurant preparation of "fugu" is strictly controlled by law and only chefs qualified are allowed to prepare the fish. Due to its paralysis effect, this neurotoxin could be used in the medical field as an analgesic to treat some cancer pains.

Tetrodotoxin and Its Analogues in the Pufferfish Arothron hispidus and A. nigropunctatus from the Solomon Islands: A Comparison of Their Toxin Profiles with the Same Species from Okinawa, Japan.

Pufferfish poisoning has not been well documented in the South Pacific, although fish and other seafood are sources of protein in these island nations. In this study, tetrodotoxin (TTX) and its analogues in each organ of the pufferfish Arothron hispidus and A. nigropunctatus collected in the Solomon Islands were investigated using high resolution LC-MS. The toxin profiles of the same two species of pufferfish from Okinawa, Japan were also examined for comparison. TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh. Due to higher TTX concentrations (51.0 and 28.7 µg/g at highest) detected in the skin of the two species from the Solomon Islands (saxitoxin was <0.02 µg/g), these species should be banned from consumption. Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus. TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.

Tetrodotoxin detection and species identification of pufferfish in retail roasted fish fillet by DNA barcoding in China.

This study identifies the pufferfish species and detects tetrodotoxin (TTX) in roasted fish fillet samples collected in Beijing, Qingdao and Xiamen, China. The cytochrome c oxidase I (COI) gene was used as the target gene for identification of the pufferfish species in the samples. Enzyme-linked immunosorbent assay (ELISA) screened the TTX levels in samples that had been detected as containing pufferfish by DNA barcode. A total of 125 samples were identified by DNA barcodes; 32 (26%) samples contained pufferfish composition and, among them, 26 (81%) were the highly toxic species Lagocephalus lunaris. All 32 samples containing the pufferfish composition were positive for TTX with levels ranging from 100 to 63,800 ng g(-1). Most of the 32 samples contained the highly toxic L. lunaris. Based on the results, we suggest that the monitoring of roasted fish fillet should be strengthened and the processing procedures should be standardised to minimise TTX poisoning caused by pufferfish.

Immunoaffinity chromatography purification and ultrahigh performance liquid chromatography tandem mass spectrometry determination of tetrodotoxin in marine organisms.

A highly selective and sensitive method was developed for the determination of tetrodotoxin (TTX) in marine organisms by immunoaffinity chromatography (IAC) purification coupled with ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). An IAC column was prepared and used to cleanup the extracted samples. The operating conditions of the IAC column were optimized, and the capacity of new IAC column was found to be 1106 ng mL(-1), which was sufficient for TTX determination. The MS/MS conditions and UPLC mobile phase were also studied to optimize the operation conditions. Fortified marine organism samples at levels of 0.3-5.0 ng g(-1) were utilized, and the average recoveries were 86.5-103.6% with intra- and inter-day relative standard deviations less than 7.22 and 9.88%, respectively. The limits of detection and quantification were 0.1 and 0.3 ng g(-1), respectively. The method was later successfully applied for the determination of TTX in 100 marine organism samples collected from local markets.

First identification of tetrodotoxin (TTX) in the flatworm Stylochoplana sp.; a source of TTX for the sea slug Pleurobranchaea maculata.

High concentrations of the neurotoxin tetrodotoxin (TTX) were detected by liquid chromatography-mass spectrometry (LC-MS) in the Platyhelminthes Stylochoplana sp. from Pilot Bay (Tauranga, New Zealand). This is the first detection of TTX in this genus. Concentrations were monitored from March to November (2013) and found to significantly decrease from a peak in July (avg. 551 mg kg(-1)) to November (avg. 140 mg kg(-1)). Stylochoplana sp. co-occurred with TTX-containing Pleurobranchaea maculata (Opisthobranchia). A Stylochoplana sp.-specific real-time PCR assay was developed targeting the mitochondrial cytochrome c oxidase subunit I gene to determine if P. maculata consumed Stylochoplana sp. Positive Stylochoplana sp. signals were obtained for 7 of 19 P. maculata tested. Mass calculations indicate Stylochoplana sp. could supply Pilot Bay P. maculata with the TTX required to account for the concentrations reported in previous studies (ca. 1.04 mg TTX per individual) based on an ingestion rate of one individual every 2-3 days throughout their lifetime. However, due to the lack of Stylochoplana sp. in areas with dense P. maculata populations, and high concentration (ca. 1400 mg kg(-1)) of TTX detected in some individuals, it is unlikely that Stylochoplana sp. represent the sole source of TTX in P. maculata.