Multi-Omics Analysis of Gene and Protein Candidates Possibly Related to Tetrodotoxin Accumulation in the Skin of Takifugu flavidus.

Pufferfish is increasingly regarded by many as a delicacy. However, the tetrodotoxin (TTX) that accumulates in its body can be lethal upon consumption by humans. TTX is known to mainly accumulate in pufferfish skin, but the accumulation mechanisms are poorly understood. In this study, we aimed to explore the possible mechanism of TTX accumulation in the skin of the pufferfish Takifugu flavidus following treatment with TTX. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we detected 37.3% of toxin accumulated in the skin at the end of the rearing period (168 h). Transcriptome and proteome analyses revealed the mechanism and pathways of TTX accumulation in the skin of T. flavidus in detail. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes analyses strongly suggest that cardiac muscle contraction and adrenergic signaling in cardiomyocyte pathways play an important role in TTX accumulation. Moreover, some upregulated and downregulated genes, which were determined via RNA-Seq, were verified with qPCR analysis. This study is the first to use multi-omics profiling data to identify novel regulatory network mechanisms of TTX accumulation in the skin of pufferfish.

Tetrodotoxin/Saxitoxins Selectivity of the Euryhaline Freshwater Pufferfish Dichotomyctere fluviatilis.

The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7-6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish.

Safety, Tolerability, Pharmacokinetics, and Concentration-QTc Analysis of Tetrodotoxin: A Randomized, Dose Escalation Study in Healthy Adults.

Tetrodotoxin (TTX) is a highly specific voltage-gated sodium channel (VGSC) blocker in clinical evaluation as a peripheral-acting analgesic for chronic pain. This study presents the first published results of the safety including cardiac liability of TTX at therapeutic-relevant concentrations in twenty-five healthy adults. Randomized, double-blind, placebo-, and positive- (moxifloxacin) controlled study evaluated single ascending doses of 15 µg, 30 µg, and 45 µg TTX over 3 periods with a 7-day washout between each period. Subcutaneous injections of TTX were readily absorbed, reaching maximum plasma concentration (Cmax) within 1.5 h. Both extent of exposure (AUC) and Cmax increased in proportion to dose. No QT prolongation was identified by concentration-QTc analysis and the upper bounds of the two-sided 90% confidence interval of predicted maximum baseline and placebo corrected QTcF (ΔΔQTcF) value did not exceed 10 ms for all tetrodotoxin doses, thereby meeting the criteria of a negative QT study. Safety assessments showed no clinically relevant changes with values similar between all groups and no subject withdrawing due to adverse events. Paresthesia, oral-paresthesia, headache, dizziness, nausea, and myalgia were the most common TEAEs (overall occurrence ≥5%) in the TTX treatment groups. TTX doses investigated in this study are safe, well-tolerated, and lack proarrhythmic proclivity.

Development and validation of a specific and sensitive liquid chromatography tandem mass spectrometry method for determination of tetrodotoxin in human urine and its pharmacokinetic study.

Tetrodotoxin (TTX) exhibits the therapeutic potential in blocking pain and in low doses can safely relieve severe pain. The urinary excretion profiles of TTX in humans have not been reported due to the extremely low lethal dose. In this study, a rapid and specific method based on protein precipitation coupled to liquid chromatography tandem mass spectrometry was developed to determine the level of TTX in human urine samples. 11-Deoxytetrodotoxin was used as an internal standard (IS). Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 320.0 → 162.1 for TTX and m/z 304.0 → 176.0 for 11-deoxyTTX. The separation of analytes was achieved on a hydrophilic interaction liquid chromatography column (250 × 4.6 mm, 5.0 µm). The mobile phase consisted of 5 mM ammonium formate in water (pH = 4.50) and 5 mM ammonium formate in acetonitrile (pH = 4.50). The flow rate was set at 0.80 mL/min in a gradient condition. Calibration plots were linear throughout the range 0.986-98.6 ng/mL of TTX in human urine. The intra-assay accuracies and precisions were within the acceptable range. The method was successfully applied to a urinary excretion study after intravenous administration of TTX to healthy volunteers. The developed method will be helpful for future pharmacological studies of TTX.

Accumulation and Elimination of Tetrodotoxin in the Pufferfish Takifugu obscurus by Dietary Administration of the Wild Toxic Gastropod Nassarius semiplicata.

To investigate pufferfish accumulation, elimination, and distribution of tetrodotoxin (TTX), Takifugu obscurus was fed with wild TTX-containing gastropod Nassarius semiplicata to simulate the natural food chain. Three-month-old non-poisonous T. obscurus was fed with wild toxic N. semiplicata at three exposure dose for 28 days, and later, with toxin-free food until day 67. Three fish individuals from each treatment were sampled, and the distribution of TTX in different tissues was measured. The results showed that the accumulation ratio of TTX in the three exposure dose groups ranged from 35.76% to 40.20%. The accumulation ratio in the skin and liver was the highest amongst all tissues, accounting for more than 85% of the total TTX, whereas that in the kidney and gallbladder was the lowest (0.11%-0.78%). Studies on the kinetic of TTX accumulation and elimination revealed that the skin was the tissue with the highest accumulation speed constant (8.06), while the liver, kidney, and intestinal tract showed the highest speed of TTX elimination. The time required for TTX reduction to reach the safety limit could be predicted by using standard elimination equations. Qualitative analysis by UPLC-MS/MS revealed the occurrence of seven TTX derivatives in T. obscurus; of these TTX, 5-deoxy TTX, 11-deoxy TTX, 4,9-anhydro TTX were found in all tested tissues.

The Duration of Nerve Block from Local Anesthetic Formulations in Male and Female Rats.

PURPOSE: It is unknown whether there are sex differences in response to free or encapsulated local anesthetics. METHODS: We examined nerve block duration and toxicity following peripheral nerve blockade in male and female rats. We studied the local anesthetic bupivacaine (free or encapsulated) as well as tetrodotoxin, which acts on a different site of the same voltage-gated channel. RESULTS: Sensory nerve blockade was 158.5 [139-190] minutes (median [interquartile range]) (males) compared to 173 [134-171] minutes (females) (p = 0.702) following bupivacaine injection, N = 8 male, 8 female. Motor nerve blockade was 157 [141-171] minutes (males) compared to 172 [146-320] minutes (females) (p = 0.2786). Micellar bupivacaine (N = 8 male, 8 female) resulted in sensory nerve blockade of 266 [227-320] minutes (males) compared to 285 [239-344] minutes (females) (p = 0.6427). Motor nerve blockade was 264 [251-264] minutes (males) compared to 287 [262-287] minutes (females) (p = 0.3823). Liposomal bupivacaine (N = 8 male, 8 female) resulted in sensory nerve blockade of 240 [207-277] minutes (males) compared to 289 [204-348] minutes (females) (p = 0.1654). Motor nerve blockade was 266 [237-372] minutes (males) compared to 317 [251-356] minutes (females) (p = 0.6671). Following tetrodotoxin injection (N = 12 male,12 female) sensory nerve blockade was 54.8 [5-117] minutes (males) compared to 54 [14-71] minutes (females) (p = 0.6422). Motor nerve blockade was 72 [40-112] minutes (males) compared to 64 [32-143] minutes (females) (p = 0.971). CONCLUSIONS: We found no statistically significant sex differences associated with the formulations tested. In both sexes, durations of nerve block were similar between micellar and liposomal bupivacaine formulations, despite the micellar formulation containing less drug.

Tetrodotoxin in marine bivalves and edible gastropods: A mini-review.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for countless human intoxications and deaths around the world. The distribution of TTX and its analogues is diverse and the toxin has been detected in organisms from both marine and terrestrial environments. Increasing detections seafood species, such as bivalves and gastropods, has drawn attention to the toxin, reinvigorating scientific interest and regulatory concerns. There have been reports of TTX in 21 species of bivalves and edible gastropods from ten countries since the 1980's. While TTX is structurally dissimilar to saxitoxin (STX), another neurotoxin detected in seafood, it has similar sodium channel blocking action and potency and both neurotoxins have been shown to have additive toxicities. The global regulatory level for the STX group toxins applied to shellfish is 800 µg/kg. The presence of TTX in shellfish is only regulated in one country; The Netherlands, with a regulatory level of 44 µg/kg. Due to the recent interest surrounding TTX in bivalves, the European Food Safety Authority established a panel to assess the risk and regulation of TTX in bivalves, and their final opinion was that a concentration below 44 µg of TTX per kg of shellfish would not result in adverse human effects. In this article, we review current knowledge on worldwide TTX levels in edible gastropods and bivalves over the last four decades, the different methods of detection used, and the current regulatory status. We suggest research needs that will assist with knowledge gaps and ultimately allow development of robust monitoring and management protocols.

Contrasting Toxin Selectivity between the Marine Pufferfish Takifugu pardalis and the Freshwater Pufferfish Pao suvattii.

To clarify the differences in toxin selectivity between marine and freshwater pufferfish, we conducted experiments in artificially reared nontoxic specimens of Takifugu pardalis (marine) and Pao suvattii (freshwater) using tetrodotoxin (TTX) and paralytic shellfish poison (PSP; decarbamoylsaxitoxin (dcSTX) or saxitoxin (STX)). T. pardalis specimens were administered feed homogenate containing TTX or dcSTX (dose of toxin, 55.2 nmol/fish) and P. suvattii specimens were administered feed homogenate containing TTX + STX (dose of each toxin, 19.2 nmol/fish) by oral gavage. The toxin content in the intestine, muscle, skin, liver, and gonads was quantified after 24 and 48 or 72 h. In T. pardalis, TTX administered into the intestine was absorbed into the body and transferred and retained mainly in the skin and liver, while dcSTX was hardly retained in the body, although it partly remained in the intestine. In strong contrast, in P. suvattii, little TTX remained in the body, whereas STX was absorbed into the body and was transferred and retained in the ovary and skin. The findings revealed that TTX/PSP selectivity differs between the marine species T. pardalis and the freshwater species P. suvattii. T. pardalis, which naturally harbors TTX, selectively accumulates TTX, and P. suvattii, which naturally harbors PSP, selectively accumulates PSP.

Polymer-tetrodotoxin conjugates to induce prolonged duration local anesthesia with minimal toxicity.

There is clinical and scientific interest in developing local anesthetics with prolonged durations of effect from single injections. The need for such is highlighted by the current opioid epidemic. Site 1 sodium channel blockers such as tetrodotoxin (TTX) are extremely potent, and can provide very long nerve blocks but the duration is limited by the associated systemic toxicity. Here we report a system where slow release of TTX conjugated to a biocompatible and biodegradable polymer, poly(triol dicarboxylic acid)-co-poly(ethylene glycol) (TDP), is achieved by hydrolysis of ester linkages. Nerve block by the released TTX is enhanced by administration in a carrier with chemical permeation enhancer (CPE) properties. TTX release can be adjusted by tuning the hydrophilicity of the TDP polymer backbone. In vivo, 1.0-80.0 µg of TTX released from these polymers produced a range of durations of nerve block, from several hours to 3 days, with minimal systemic or local toxicity.

Hollow Silica Nanoparticles Penetrate the Peripheral Nerve and Enhance the Nerve Blockade from Tetrodotoxin.

The efficacy of tetrodotoxin (TTX), a very potent local anesthetic, is limited by its poor penetration through barriers to axonal surfaces. To address this issue, we encapsulated TTX in hollow silica nanoparticles (TTX-HSN) and injected them at the sciatic nerve in rats. TTX-HSN achieved an increased frequency of successful blocks, prolonged the duration of the block, and decreased the toxicity compared to free TTX. In animals injected with fluorescently labeled HSN, the imaging of frozen sections of nerve demonstrated that HSN could penetrate into nerve and that the penetrating ability of silica nanoparticles was highly size-dependent. These results demonstrated that HSN could deliver TTX into the nerve, enhancing efficacy while improving safety.

Enhanced Triggering of Local Anesthetic Particles by Photosensitization and Photothermal Effect Using a Common Wavelength.

On-demand pain relief systems would be very helpful additions to the armamentarium of pain management. Near-infrared triggered drug delivery systems have demonstrated the potential to provide such care. However, challenges remain in making such systems as stimulus-sensitive as possible, to enhance depth of tissue penetration, repeatability of triggering, and safety. Here we developed liposomes containing the local anesthetic tetrodotoxin and also containing a photosensitizer and gold nanorods that were excitable at the same near-infrared wavelength. The combination of triggering mechanisms enhanced the photosensitivity and repeatability of the system in vitro when compared with liposomes with a single photoresponsive component. In vivo, on-demand local anesthesia could be induced with a low irradiance and short irradiation duration, and liposomes containing both photosensitizer and gold nanorods were more effective than those containing just one photoresponsive component. Tissue reaction was benign.

Binding and Pharmacokinetics of the Sodium Channel Blocking Toxins (Saxitoxin and the Tetrodotoxins).

Tetrodotoxin (TTX) found in diverse variety of animals including puffer fishes, some newts, frogs and limited number of non-vertebrate species (6 different phyla). The saxitoxin (STX) and the TTX are small molecules composed of 7,8,9 guanidinium and 1,2,3 guanidinium groups, respectively in their structures. These groups provide positive charge to the molecules and are believed to interact with negatively charged Glu755 and Asp400 residues in domain II and I of the sodium channel strongly. The pharmacokinetic studies (absorption, distribution and accumulation) reported on Takifugu rubripes, Takifugu pardalis, Takifugu niphobles, Takifugu vermicularis, Takifugu snyderi, etc. revealed that higher concentration of TTX is accumulated in liver than in the skin or other tissues. Although TTX is also accumulated in the skin of various marine species (secretory glands) and the excess of TTX are emitted through skin which acts as a defence agent for those species. STX showed high toxicity on crab and other animals, due to its accumulation in the tissues and resistance to the sodium channel proteins. It concluded that TTX and STX based toxicities are developed on the species by the absorption, distribution and accumulation of toxins in tissues. Also the ingestion of these species (marine species) as food may allow transferring toxin to the human being.

Biliary excretion of tetrodotoxin in the cultured pufferfish Takifugu rubripes juvenile after intramuscular administration.

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

DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish.

Pufferfish accumulate tetrodotoxin (TTX) at high levels in liver and ovary through the food chain. However, the mechanisms underlying TTX toxification in pufferfish have been poorly understood. In order to search gene candidates involved in TTX accumulation in the torafugu pufferfish Takifugu rubripes, a custom 4x44k oligonucleotide microarray slide was designed by the Agilent eArray program using oligonucleotide probes of 60 bp in length referring to 42,724 predicted transcripts in the publicly available Fugu genome database. DNA microarray analysis was performed with total RNA samples from the livers of two toxic wild specimens in comparison with those from a nontoxic wild specimen and two nontoxic cultured specimens. The mRNA levels of 1108 transcripts were more than 2-fold higher in the toxic specimens than in the nontoxic specimens. The levels of 613 transcripts were remarkably high, and 16 transcripts encoded by 9 genes were up-regulated more than 10-fold. These genes included those encoding forming structural filaments (keratins) and those related to vitamin D metabolism and immunity. It was also noted that the levels of the transcripts encoding serpin peptidase inhibitor clade C member 1, coagulation factor X precursor, complement C2, C3, C5, C8 precursors, and interleukin-6 receptor were high in the toxic liver samples.

Investigating diet as the source of tetrodotoxin in Pleurobranchaea maculata.

The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species.

Comparison of the localization of tetrodotoxin between wild pufferfish Takifugu rubripes juveniles and hatchery-reared juveniles with tetrodotoxin administration.

To reveal the accumulation profile of tetrodotoxin (TTX) in pufferfish Takifugu rubripes juveniles, we compared the localization of TTX in various tissues among wild juveniles and hatchery-reared juveniles with or without TTX administration using immunohistochemical technique with anti-TTX monoclonal antibody. Immuno-positive reaction was observed in hepatic tissue, basal cell of skin and olfactory, olfactory epithelium, optic nerve and brain (optic tectum, cerebellum, medulla oblongata) of wild juveniles (body length: BL, 4.7-9.4 cm). TTX was detected in the same tissues as wild juveniles and epithelial cell layer of intestine of hatchery-reared juveniles (BL, 5.0-5.3 cm) to which TTX was orally administrated. No positive reaction was observed from the tissues of hatchery-reared juveniles without TTX administration. These results suggest that orally administrated TTX to the non-toxic cultured juveniles is accumulated in the same manner of wild juveniles. In addition, our study revealed that pufferfish accumulates TTX in the central nervous system.

[Postmortem distribution of tetrodotoxin in tissues and body fluids of guinea pigs].

OBJECTIVE: To investigate the postmortem distribution of tetrodotoxin in tissues and body fluids of guinea pig, and to provide method and evidence for forensic identification and clinical diagnosis and treatment. METHODS: Guinea pigs were intragastric administrated with 100, 50, 15 microg/kg tetrodotoxin, respectively. The poisoning symptoms were observed. The samples of heart, liver, spleen, lung, kidney, brain, stomach, intestines, bile, heart blood and urine were collected. The concentrations of tetrodotoxin in tissues and body fluids were measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: After administrated with tetrodotoxin, all guinea pigs came out poisoning signs including tachypnea, weary and dead finally. Tetrodotoxin concentrations in lung, stomach, intestines and urine were higher, followed by blood, heart and brain. The concentration in bile was the lowest. CONCLUSION: Postmortem distribution of tetrodotoxin in guinea pig is uneven. The concentration in the lung, stomach, intestines, urine and heart blood are higher, those tissues could be used for diagnosis of tetrodotoxin poisoning.

Transfer profile of orally and intramuscularly administered tetrodotoxin to artificial hybrid specimens of the pufferfish Takifugu rubripes and Takifugu porphyreus.

Tetrodotoxin (TTX) was administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu porphyreus to investigate toxin accumulation in hybrids and TTX transfer/accumulation profiles in the pufferfish body. In test fish administered TTX-containing feed homogenate at a dose of ∼400 MU/fish by oral gavage using a syringe (OGA group), the toxin content (MU/g tissue) of the digestive tract rapidly decreased and that of the liver increased from 1 to 24 h after administration. From 24 to 120 h, the toxin content of the liver decreased gradually, and the toxin appeared in the skin. On the other hand, intramuscularly administered TTX (400 MU/fish) was rapidly transferred to the liver and skin via the blood, and only a little toxin remained in the muscle even at 1 h (IMA group). The total amount of toxin remaining in the whole body (% of administered toxin) was 31-45% in the OGA group, and 42-74% in the IMA group; the scores in the OGA group were generally lower than those in the IMA group. In both OGA and IMA groups, the greatest amount of toxin accumulated in the liver (23-52%) after 8 h, followed by the skin (11-21%) after 72 h. The TTX administration experiment, especially using the oral gavage administration method, revealed that skins and livers of 'torama' pufferfish hybrid are endowed with TTX-accumulating ability, but the muscles are not, and that TTX taken up from toxic feed to the pufferfish body is transferred first to the liver and then to the skin via the blood.

Transfer profile of intramuscularly administered tetrodotoxin to artificial hybrid specimens of pufferfish, Takifugu rubripes and Takifugu niphobles.

Tetrodotoxin (TTX) was intramuscularly administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu niphobles to investigate toxin accumulation in hybrids, and TTX transfer/accumulation profiles in the pufferfish body. In the test fish administered 146 MU TTX in physiologic saline, TTX rapidly transferred from the muscle via the blood to other organs. Toxin transfer to the ovary rapidly increased to 53.5 MU/g tissue at the end of the 72-h test period. The TTX content in the liver and skin was, at most, around 4-6 MU/g tissue, and in the testis it was less than 0.01 MU/g tissue. On the other hand, based on the total amount of toxin per individual (% of the administered toxin), the skin and the liver contained higher amounts (20-54% and 2-24%, respectively), but the amount in the liver rapidly decreased after 8-12 h, and fell below the level in the ovary after 48 h. These findings suggest that part of the TTX is first taken up in the liver and then transferred/accumulated in the skin in male specimens and in the ovary in female specimens.

Tissue distribution of tetrodotoxin in the red-spotted newt Notophthalmus viridescens.

High levels of tetrodotoxin (TTX) and of its analogue 6-epi-tetrodotoxin have been detected in skin and liver extracts of the red-spotted newt Notophthalmus viridescens assayed by a post-column fluorescent-HPLC system. Using a monoclonal antibody-based immunoenzymatic technique, TTX was localized in the granular glands of the epidermis as well as in the muscle layer between the muscle fibres, but also in most organs suggesting that TTX is stored in the whole body of the newt and is secreted by skin glands.