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.

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.

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.

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.

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.

[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.

Phenotypic mismatches reveal escape from arms-race coevolution.

Because coevolution takes place across a broad scale of time and space, it is virtually impossible to understand its dynamics and trajectories by studying a single pair of interacting populations at one time. Comparing populations across a range of an interaction, especially for long-lived species, can provide insight into these features of coevolution by sampling across a diverse set of conditions and histories. We used measures of prey traits (tetrodotoxin toxicity in newts) and predator traits (tetrodotoxin resistance of snakes) to assess the degree of phenotypic mismatch across the range of their coevolutionary interaction. Geographic patterns of phenotypic exaggeration were similar in prey and predators, with most phenotypically elevated localities occurring along the central Oregon coast and central California. Contrary to expectations, however, these areas of elevated traits did not coincide with the most intense coevolutionary selection. Measures of functional trait mismatch revealed that over one-third of sampled localities were so mismatched that reciprocal selection could not occur given current trait distributions. Estimates of current locality-specific interaction selection gradients confirmed this interpretation. In every case of mismatch, predators were "ahead" of prey in the arms race; the converse escape of prey was never observed. The emergent pattern suggests a dynamic in which interacting species experience reciprocal selection that drives arms-race escalation of both prey and predator phenotypes at a subset of localities across the interaction. This coadaptation proceeds until the evolution of extreme phenotypes by predators, through genes of large effect, allows snakes to, at least temporarily, escape the arms race.

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.

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.

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.

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.

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.

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.

Pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique.

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.

Evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes.

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

Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver.

Non-toxic cultured juvenile kusafugu Fugu niphobles were fed with a diet containing highly toxic natural komonfugu Fugu poecilonotus liver until the 30th day (8.0 microg of TTX and 3.7 microg of 4,9-anhydroTTX/fish/day), and then fed with a non-toxic diet until the 240th day. During the 30-240th day, five or six fish were periodically sampled six times, and the contents of TTX and 4,9-anhydroTTX in each tissue were determined. The total TTX and 4,9-anhydroTTX accumulated in all tissues tested was not significantly changed during the experimental period, both being kept at 70% of administrated doses. However, in the liver, the TTX content accounted to be 120 microg (50% of administrated) on the 30th day, and then it gradually decreased to 50 microg until the 240th day, while 4,9-anhydroTTX content was kept at approximately 40 microg (40% of administrated) during all the experimental periods. In contrast to the liver, in the skin, TTX and 4,9-anhydroTTX were 40 and 5 microg, respectively, on the 30th day, and then gradually increased to 80 and 24 microg, respectively, until the 240th day. In the intestine, TTX and 4,9-anhydroTTX contents were kept at 25 and 12 microg, respectively, during all the experimental periods. According to these results, we assumed that a part of TTX accumulated in the liver was slowly transferred to the skin.

Involvement of carrier-mediated transport system in uptake of tetrodotoxin into liver tissue slices of puffer fish Takifugu rubripes.

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.

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.

[Experimental approach to murder by aconite poisoning from the viewpoint of medicolegal toxicology].

An autopsy case performed by the author in 1986 had been gradually revealed to be a murder using aconite poisons. The puffer fish toxin was certified afterwards to be co-administered together with aconite alkaloids in this case. In order to investigate this murder case, animal experiments were done using mice to clarify the metabolism of aconitine and tetrodotoxin, and to examine the influences of tetrodotoxin on aconite poisoning. We also examined biological effects under the chronic intoxication of aconitine, and the elimination and degradation of aconitine in dead body. For this purpose we have developed technical methods using GC/MS and LC/MS for the quantification of these toxins in biological materials.