Toxicity Equivalency Factors for Tetrodotoxin Analogues Determined with Automated Patch Clamp on Voltage-Gated Sodium Channels in Neuro-2a Cells.

Tetrodotoxin (TTX) is a potent marine neurotoxin, responsible for numerous poisoning incidents and some human fatalities. To date, more than 30 TTX analogues have been identified, but their individual toxicities and roles in poisoning remain largely unknown. In this work, the toxicity equivalency factors (TEFs) of five TTX analogues were determined by assessing the blockade of voltage-gated sodium channels in Neuro-2a cells using automated patch clamp (APC). All TTX analogues were less toxic than TTX. The derived TEFs were applied to the individual TTX analogues concentrations measured in pufferfish samples, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A comparison of these results with those obtained from APC analysis demonstrated that TEFs can be effectively used to translate LC-MS/MS analytical data into meaningful toxicological information. This is the first study to utilize APC device for the toxicological assessment of TTX analogues, highlighting its potential as a bioanalytical tool for seafood safety management and human health protection.

Structure of human NaV1.6 channel reveals Na+ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin.

The sodium channel NaV1.6 is widely expressed in neurons of the central and peripheral nervous systems, which plays a critical role in regulating neuronal excitability. Dysfunction of NaV1.6 has been linked to epileptic encephalopathy, intellectual disability and movement disorders. Here we present cryo-EM structures of human NaV1.6/ß1/ß2 alone and complexed with a guanidinium neurotoxin 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), revealing molecular mechanism of NaV1.6 inhibition by the blocker. The apo-form structure reveals two potential Na+ binding sites within the selectivity filter, suggesting a possible mechanism for Na+ selectivity and conductance. In the 4,9-ah-TTX bound structure, 4,9-ah-TTX binds to a pocket similar to the tetrodotoxin (TTX) binding site, which occupies the Na+ binding sites and completely blocks the channel. Molecular dynamics simulation results show that subtle conformational differences in the selectivity filter affect the affinity of TTX analogues. Taken together, our results provide important insights into NaV1.6 structure, ion conductance, and inhibition.

An almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an odorant for the grass puffer.

Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this TTX analog, which has been present in their body with TTX but whose function was unknown, as an odorant for chemical communication or effective TTX accumulation.

Concentrations of Tetrodotoxin (TTX) and Its Analogue 4,9-Anhydro TTX in Different Tissues of the Silver-Cheeked Pufferfish (Lagocephalus sceleratus, Gmelin, 1789) Caught in the South-Eastern Mediterranean Sea, Lebanon.

Pufferfishes are among the best-known marine organisms that accumulate marine biotoxins such as Tetrodotoxin (TTX). In the Mediterranean Sea, the silver-cheeked toadfish Lagocephalus sceleratus is the most reported TTX-bearer, causing many fatal and non-fatal cases. In Lebanon, no previous studies have measured TTX levels although the possibility of TTX-poisoning is high since L. sceleratus is caught in different sizes and can be mistaken with other small fishes. Hence, this study reports TTX and its analogue 4,9-anhydro TTX in L. sceleratus collected from Lebanese waters in the Eastern Mediterranean Sea. The results show that TTX concentrations in fish tissues varied between 0.10 and 252.97 µg/g, while those of 4,9-anhydro TTX oscillated between 0.01 and 43.01 µg/g. Internal organs of L. sceleratus were the most toxic parts of its body, with the highest TTX levels found in gonads (mainly ovaries) and liver, followed by the muscles and skin with concentrations always exceeding the safety level. Toxicity fluctuations of L. sceleratus, its expansion, ecological and economic effects were also elucidated. Based on the present findings, it has been confirmed that L. sceleratus constitutes a health, ecological and economic risks, and therefore its trade in seafood markets should be banned to avoid any potential intoxication.

Keeping Lagocephalus sceleratus off the Table: Sources of Variation in the Quantity of TTX, TTX Analogues, and Risk of Tetrodotoxication.

The invasion of the tetrodotoxin (TTX)-bearing silver-cheeked toadfish and potential poisoning due to its consumption (tetrodotoxication) threatens public safety in the Mediterranean Sea. In this study, TTX and TTX analogues of Lagocephalus sceleratus (Gmelin, 1789) were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) in fish collected off the island of Crete (Southern Mediterranean). We tested the synergistic effect of a suite of factors potentially affecting toxins' levels and tetrodotoxication risk using general and generalized linear models, respectively. The type of tissue, geographic origin (Cretan Sea, Libyan Sea), sex, and fish maturity stage were significant predictors of toxin concentrations. Mean TTX was higher in gonads and lower in muscles, higher in the Libyan Sea and in female fish, and lower in juvenile (virgin) fish. The concentration of TTX was also significantly and positively correlated with the concentration of several TTX analogues (4-epiTTX, 4,9-anhydroTTX, 11-deoxyTTX, 5,11/6,11-dideoxyTTX, 5,6,11-trideoxyTTX, 11-norTTX-6-ol). The analysis showed that fish originating from the Libyan Sea had significantly higher probability to cause tetrodotoxication in case of consumption. The variability explained by the models developed in this study was relatively low, indicating that toxin levels are hard to predict and the consumption of L. sceleratus should therefore be avoided.

Tetrodotoxin and Its Analogues in Cephalothrix cf. simula (Nemertea: Palaeonemertea) from the Sea of Japan (Peter the Great Gulf): Intrabody Distribution and Secretions.

Some nemertean species from the genus Cephalothrix accumulate tetrodotoxin (TTX) in extremely high concentrations. The current study is the first to provide high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) data on tetrodotoxin and its analogues (TTXs) profile and concentration in different regions and organs of Cephalothrix cf. simula, and its secretions produced in response to stimulation. Different specimens of C. cf. simula possessed 7-11 analogues, including nine previously found in this species and two new for nemerteans-4,9-anhydro-8-epi-5,6,11-trideoxyTTX and 1-hydroxy-8-epi-5,6,11-trideoxyTTX. The study of the toxins' distribution in different regions and organs of nemerteans revealed the same qualitative composition of TTXs throughout the body but differences in the total concentration of the toxins. The total concentration of TTXs was highest in the anterior region of the body and decreased towards the posterior; the ratio of the analogues also differed between regions. The data obtained suggest a pathway of TTXs uptake in C. cf. simula and the role of toxins in the life activity of nemerteans.

Structures of N-Hydroxy-Type Tetrodotoxin Analogues and Bicyclic Guanidinium Compounds Found in Toxic Newts.

The biosynthesis of tetrodotoxin (TTX, 1), a potent neurotoxin widely distributed in marine and terrestrial metazoans, remains unresolved. A significant issue has been identifying intermediates and shunt products associated with the biosynthetic pathway of TTX. We investigated TTX biosynthesis by screening and identifying new TTX-related compounds from Cynops ensicauda popei and Taricha granulosa. Mass spectrometry (MS)-guided screening identified two new N-hydroxy TTX analogues in newts: 1-hydroxy-8-epiTTX (2) and 1-hydroxy-8-epi-5,11-dideoxyTTX (3, previously reported as 1-hydroxy-5,11-dideoxyTTX). We prepared a new analogue, 8-epi-5,11-dideoxyTTX (4), from 3 via N-OH reduction and confirmed the presence of 4 in T. granulosa using hydrophilic interaction liquid chromatography (HILIC)-LCMS. The presence of 8-epi-type TTX analogues in both Cynops and Taricha supports a branched biosynthetic pathway of terrestrial TTX, which produces 6- and 8-epimers. In addition, new bicyclic guanidinium compounds Tgr-238 (5) and Tgr-240 (6) were identified as putative shunt products of our proposed TTX biosynthesis pathway. A structural analysis of Cep-228A (7), another bicyclic compound, was performed using NMR. Based on the structures of 5-7 and their analogues, we propose a model of the shunt and metabolic pathways of the terrestrial TTX biosynthesis.

The voltage-gated sodium channel inhibitor, 4,9-anhydrotetrodotoxin, blocks human Nav1.1 in addition to Nav1.6.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in neurons. The human genome includes ten human VGSC α-subunit genes, SCN(X)A, encoding Nav1.1-1.9 plus Nax. To understand the unique role that each VGSC plays in normal and pathophysiological function in neural networks, compounds with high affinity and selectivity for specific VGSC subtypes are required. Toward that goal, a structural analog of the VGSC pore blocker tetrodotoxin, 4,9-anhydrotetrodotoxin (4,9-ah-TTX), has been reported to be more selective in blocking Na+ current mediated by Nav1.6 than other TTX-sensitive VGSCs, including Nav1.2, Nav1.3, Nav1.4, and Nav1.7. While SCN1A, encoding Nav1.1, has been implicated in several neurological diseases, the effects of 4,9-ah-TTX on Nav1.1-mediated Na+ current have not been tested. Here, we compared the binding of 4,9-ah-TTX for human and mouse brain preparations, and the effects of 4,9-ah-TTX on human Nav1.1-, Nav1.3- and Nav1.6-mediated Na+ currents using the whole-cell patch clamp technique in heterologous cells. We show that, while 4,9-ah-TTX administration results in significant blockade of Nav1.6-mediated Na+ current in the nanomolar range, it also has significant effects on Nav1.1-mediated Na+ current. Thus, 4,9-ah-TTX is not a useful tool in identifying Nav1.6-specific effects in human brain networks.

Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin.

A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production of the specific polyclonal antibody, which reacted well with TTX and its analogs, 4-epiTTX, 11-oxoTTX and 5,6,11-trideoxyTTX, except for 4,9-anhydroTTX. The enzyme-linked immunosorbent assay (ELISA) system using this specific antibody was also developed in the present study. This newly developed polyclonal antibody with analytical procedures using direct one-step ELISA is useful to detect TTX and its analogs in toxic organisms and also disclose the mechanisms involved in their metabolic pathways and accumulation of TTX.

Public Health Risks Associated with Tetrodotoxin and Its Analogues in European Waters: Recent Advances after The EFSA Scientific Opinion.

Tetrodotoxin (TTX) and its analogues are naturally occurring toxins responsible worldwide for human intoxication cases and fatalities, mainly associated with pufferfish consumption. In the last decade, TTXs were detected in marine bivalves and gastropods from European waters. As TTXs are not regulated or monitored at EU level, their unexpected occurrence in shellfish raised concerns as a food safety hazard and revealed the necessity of a thorough assessment on the public health risks associated with their presence. For this reason, the European Food Safety Authority (EFSA) was requested by the European Commission to provide a scientific opinion, finally adopted in March 2017, according to which a provisional concentration below 44 µg TTX equivalents/kg shellfish meat, based on a large portion size of 400 g, was considered not to result in adverse effects in humans. The EFSA expert panel, however, recognized a number of shortcomings and uncertainties related to the unavailability of sufficient scientific data and provided relevant recommendations for future research to overcome these data gaps identified in order to further refine the risk assessment on TTXs. The present review aims to summarize the knowledge obtained towards addressing these recommendations in the two years following publication of the EFSA opinion, at the same time highlighting the points requiring further investigation.

Toxin and toxicity identification of mangrove horseshoe crab Carcinoscorpius rotundicauda collected from South China.

The presence of paralytic shellfish poisoning (PSP), diarrhetic Shellfish Poisoning (DSP), tetrodotoxin (TTX) and its analogues (11-oxoTTX, 4.9-anhydro-11-oxoTTX, 4.9-anhydroTTX, 5-deoxyTTX, 5.11-dideoxyTTX, 5.6.11-trideoxyTTX and 4.9-anhydro-5.6.11-trideoxy TTX) were initially investigated in Carcinoscorpius rotundicauda collected from south China with Liquid chromatography-tandem mass spectrometry (LC-MS-MS) and mouse bioassay. The TTX toxicity was 10.8 ±â€¯3.9 MU/g muscle, 6.3 ±â€¯0.6 MU/g viscera and 6.3 ±â€¯0.6 MU/g eggs in mean value. Merely dcGTX2 and dcSTX were detected in ten Specimens, ranging from 0.01 to 0.77 µg/g. Analyses suggested that these Carcinoscorpius rotundicauda contain TTX and its analogues as the major toxin and PSPs as the minor, respectively. Besides, no DSPs were found.

Tetrodotoxin and its analogues profile in nemertean species from the sea of Japan.

For the first time search for tetrodotoxin (TTX) and its analogues in the extracts of nemerteans using HPLC-MS/MS was performed. TTX analogues were detected in two nemertean species in addition to TTX: 7 analogues were detected in the extract of Cephalothrix simula, 3 analogues - in the extract 11-norTTX of Kulikovia manchenkoi. Presence of 5-deoxyTTX, 11-deoxyTTX, 5,6,11-trideoxyTTX and -6(R)-ol in nemerteans was shown for the first time.

Insight into tetrodotoxin blockade and resistance mechanisms of Nav 1.2 sodium channel by theoretical approaches.

Nav 1.2, a member of voltage-gated sodium channels (Nav s) that are responsible for the generation and propagation of action potentials along the cell membrane, and play a vital role in the process of information transmission within the nervous system and muscle contraction, is preferentially expressed in the central nervous system. As a potent and selective blocker of Nav s, tetrodotoxin (TTX) has been extensively studied in biological and chemical sciences, whereas the detailed mechanism by which it blocks nine Nav 1 channel subtypes remain elusive. Despite the high structural similarity, the TTX metabolite 4,9-anhydro-TTX is 161 times less effective toward the mammalian Nav 1.2, which puzzled us to ask a question why such a subtle structural variation results in the largely binding affinity difference. In the current work, an integrated computational strategy, including homology modeling, induced fit docking, explicit-solvent MD simulations, and free energy calculations, was employed to investigate the binding mechanism and conformational determinants of TTX analogs. Based on the computational results, the H-bond interactions between C4-OH and C9-OH of TTX and the outer ring carboxylates of the selectivity-filter residues, and the cation-π interaction between the primary amine of guanidinium of TTX and Phe385 determine the difference of their binding affinities. Moreover, the computationally simulations were carried out for the D384N and E945K mutants of hNav 1.2-TTX, and the rank of the predicted binding free energies is in accordance with the experimental data. These observations provide a valuable model to design potent and selective neurotoxins of Nav 1.2 and shed light on the blocking mechanism of TTX to sodium channels.

Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels.

Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel ß-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other ß-subunit genes (Scn2b - Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with ß1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with ß1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with ß1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with ß1-subunits. These results suggest that ß1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with ß1-subunits in a concentration-dependent manner.

Rapid screening and multi-toxin profile confirmation of tetrodotoxins and analogues in human body fluids derived from a puffer fish poisoning incident in New Caledonia.

In August 2014, a puffer fish poisoning incidence resulting in one fatality was reported in New Caledonia. Although tetrodotoxin (TTX) intoxication was established from the patients' signs and symptoms, the determination of TTX in the patient's urine, serum or plasma is essential to confirm the clinical diagnosis. To provide a simple cost-effective rapid screening tool for clinical analysis, a maleimide-based enzyme-linked immunosorbent assay (mELISA) adapted for the determination of TTX contents in human body fluids was assessed. The mELISA was applied to the analysis of urine samples from two patients and a response for the presence of TTX and/or structurally similar analogues was detected in all samples. The analysis by LC-MS/MS confirmed the presence of TTX but also TTX analogues (4-epiTTX, 4,9-anhydroTTX and 5,6,11-trideoxyTTX) in the urine. A change in the multi-toxin profile in the urine based on time following consumption was observed. LC-MS/MS analysis of serum and plasma samples also revealed the presence of TTX (32.9 ng/mL) and 5,6,11-trideoxyTTX (374.6 ng/mL) in the post-mortem plasma. The results provide for the first time the TTX multi-toxin profile of human samples from a puffer fish intoxication and clearly demonstrate the implication of TTX as the causative agent of the reported intoxication case.

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Nav 1.1 to Nav 1.7).

BACKGROUND AND PURPOSE: The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav 1.1-Nav 1.7. EXPERIMENTAL APPROACH: Each VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings. KEY RESULTS: The inhibitory effects of TTX on Nav 1.1-Nav 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav 1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav 1.7 T1425M and Nav 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively. CONCLUSIONS AND IMPLICATIONS: The reduced binding of chiriquitoxin to Nav 1.7 was attributed to its C11-OH and/or C12-NH2 , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.

An in silico perspective on the toxicodynamic of tetrodotoxin and analogues - A tool for supporting the hazard identification.

Tetrodotoxin (TTX) is a potent neurotoxin naturally found in terrestrial and marine animals targeting the voltage-gated sodium channels. Historically, TTX has raised food safety concerns mainly in the Asian countries due to the consumption of the typical pufferfish-derived delicacy fugu. However, the diffusion of TTX is getting wider today, reasonably threating in a close future a broader number of consumers than before. The understanding of TTX group toxicity is still incomplete as most of the analogues and metabolites found together with TTX are largely understudied. This prevents the establishment of a solid background for risk assessment and additional data have been claimed to timely foster the assessment of TTX toward a group-based approach. However, the high costs in sourcing TTX analogues make practically unfeasible the wide-scale assessment using experimental trials. The toxicological assessment in silico may succeed in extending data on compounds poorly affordable, hierarchizing compound to focus experiments and supporting the hazard identification. Therefore, the present work investigated the toxicodynamic of TTX, analogues and metabolites by using a molecular modeling approach. In the framework of the hazard identification, the model analyzed TTX analogues never tested before assessing qualitatively their potential toxicity in comparison to TTX. While the analogues from TTX bearing species appeared to be less toxic than TTX, some human metabolites showed a better interaction with the toxin binding site. Such results suggest that human metabolism may partially fail in preventing the interaction with the biological target. Therefore, the identification and assessment of human metabolites should be done to support the decision making process from a more informed perspective.

Dietary administration of tetrodotoxin and its putative biosynthetic intermediates to the captive-reared non-toxic Japanese fire-bellied newt, Cynops pyrrhogaster.

The origin of tetrodotoxin (TTX) in amphibians has long been disputed. In this study, TTX and its putative biosynthetic intermediates or shunt compounds (4,9-anhydro-10-hemiketal-5-deoxyTTX and Cep-242) were dietary administered to the captive-reared, non-toxic Japanese fire-bellied newt, Cynops pyrrhogaster. After 4 weeks, the ingested compounds were detected mainly in the newt body using liquid chromatography-mass spectrometry (LC-MS), while these compounds were not converted into other TTX analogues in newts.

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.

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.