• Toxicological Research
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• v.32 no.3
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• pp.215-223
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• 2016

This study is to investigate the biological, biochemical and cytotoxic effects of puffer fish (Arothron stellatus) toxin extracts under in-vitro condition. Extracted toxins from various organs of puffer fish were purified by using active charcoal column, and Bio-gel-P2 column chromatography. The lethality of toxin was tested in crabs, which consists of neurotoxic compounds. The degree of the brine shrimp lethality assay was found directly proportional to the concentration of the toxin extracts, which was well supported by hemolytic assay. The experimental results suggested that the gonad was found higher toxins than the liver and muscles. The mortality rate of brine shrimp nauplii was increased with the raise of concentrations of toxin level. Among the different doses and time dependent cytotoxic effect of human cervical carcinoma (HeLa) cells were showed $4.0{\mu}g/mL$ of toxin, which was effectively inhibited cancer cell proliferation. HPLC and TLC analysis was revealed that the A. stellatus toxin contains tetrodotoxin (TTX), related compounds 4-epi TTX and anhydro-TTX. The present results suggested that the A. stellatus contain TTX as a major and anh-TTX as a minor toxin. It could be the potential candidate in the field of anticancer drug discovery against human cervical cancer cells. The present data is confirming that the puffer fish toxin as an interesting source of novel bioactive natural compounds with potent applications in pharmacology.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.40 no.5
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• pp.276-281
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• 2007

The toxicity of two species of puffer fish, Takifugu xanthopterus and T. stictonotus, collected from coastal regions of Korea, was determined using a mouse bioassay. The highest toxin scores in the muscle, skin, fins, and testis in both species were below 50 mouse units (MU) per gram, and for each organ of both species the proportion of toxic specimens containing ${\geq}10MU/g$ was less than about 10%. In T. xanthopterus, the highest toxin levels in the liver, gallbladder, and ovary exceeded 1,000 MU/g (1,275-1,910), while less than 200 MU/g (12-136) was detected in the same organs of T. stictonotus. Therefore, the toxicities of muscle, skin, and testis in both species of puffer fish were within acceptable levels for human consumption.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.41 no.5
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• pp.309-314
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• 2008

The toxicity of five species of puffer fish, Arothron firmamentum (Byeolbok), Lagocephalus gloveri (Heukmilbok), L. wheeleri (Eunmilbok), L. inermis (Minmilbok) and L. lunaris (Milbok), collected from fish markets in Korea, was determined using a mouse bioassay. In A. firmamentum, the proportion of toxic specimens containing >10 MU/g was 87.5% in the ovaries, and 10.0% in the skin; no toxicity was detected in the muscle, fin, liver, intestine and gallbladder using the mouse bioassay. The highest toxin levels were found to be 87 MU/g in the ovaries, and 13 MU/g in the skin. Toxic specimens containing >10 MU/g were not detected from samples taken from any of the organs in L. wheeleri and L. inermis. In L. gloveri, most specimens were found to be non-toxic, but toxin levels of 11-72 MU/g were detected from within the skin, fins, and intestines in one specimen. In L. lunaris, the proportion of toxic specimens was 50.0% in the ovaries, and 7.1% in the gallbladder; no toxicity was detected in the other organs by the mouse bioassay. The highest toxin levels were 75 MU/g in the ovaries, and 14 MU/g in the gallbladder. Therefore, the toxicities of edible muscle and skin in the five species of puffer fish marketed in Korea were found to be within acceptable levels for human consumption.

• Journal of Food Hygiene and Safety
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• v.15 no.1
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• pp.46-50
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• 2000

Totally, twenty seven specimens of tiger puffer, Fugu rubripes rubripes were collected at Jagalchi fish market in Pusan, Korea during January, April and September in 1995. Anatomical distribution of pufferfish toxin in tiger puffer was examined by mouse bioassay. The frequency rate of toxic specimens containing $\geq$ 10 MU/g was 14.8％ in liver; 16.7％ in gonad; and 14.8％ in skin, and no toxin was detected in muscle. The highest toxin level found was 160 MU/g in liver, 600 MU/g in gonad and 26 MU/g in skin, and each average toxin level (mean$\pm$ standard error) was 7$\pm$6, 50$\pm$35 and 5$\pm$1 MU/g, respectively. Some specimens collected in January and April were toxic, while none of the specimens collected in September showed its toxicity. Although toxicity of tiger puffer showed the seasonal variation, tested tiger puffer was evaluated as a safe seafood fur consumption, in that an acceptable level of toxin was found in the edible muscle and skin.

• Journal of Haehwa Medicine
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• v.28 no.2
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• pp.1-11
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• 2019

Objective: The aim of this study is to analyze the previously published research articles related to puffer fish toxin focusing on tumor. Method: Literatures were searched in PubMed database, published since 2000, using the keyword; Puffer fish, Fugu and tetrodotoxin (TTX) with cancer or tumor. Research papers were classified by year, country, study model, used material, kind of tumor and study subject. Finally, a total of 41 studies were analyzed in this study. Results: From 2000 to 2018, the most abundant papers were published in 2009 (6 studies) and almost half of the papers were studied in United Kingdom (20 studies). The 39 studies used TTX purified from puffer fish while 2 studies used crude extract of skin and gonad of puffer fish. The most used target cell line was prostate cancer (15 studies), and the next was breast cancer (14 studies). The study methods were classified into 4 clinical studies, 2 animal studies and 35 cell-based studies. Conclusions: Our results show that the overview of cancer-related studies using puffer fish poison. This information would be helpful for the puffer fish-derived drug researches in the future.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.40 no.5
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• pp.269-275
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• 2007

The toxicity of two species of puffer fish, Takifugu pardalis and T. niphobles, collected from the coastal regions of Korea was determined using a mouse bioassay. In T. pardalis collected at Tongyeong, the proportion of toxic specimens containing ${\geq}10MU/g$ exceeded 90% for the skin, fins, liver, intestine, ovary, and gallbladder, 11.1% for the testis, and 6.9% for the muscle. In each of the organs, the highest toxin levels were several tens (14-39) of mouse units (MU) per gram in the muscle, testis, and eyeball, but thousands (1,444-5,755) of MU per gram in the skin, liver, intestine, ovary, and gallbladder. The organs of T. pardalis exhibited remarkable variation in toxicity. In T. niphobles, the proportion of toxic specimens exceeded 90% for the ovary and skin, 60-80% for the fins, liver, intestine, and gallbladder, and 4.5% for the muscle; no toxicity was detected in the testis or eyeball using the mouse bioassay. The highest toxin levels were thousands (2,291-7,777) of MU per gram in the liver, intestine, ovary, and gallbladder, hundreds(146-328) of MU per gram in the skin and fins, and 18 MU/g in the muscle. Takifugu niphobles toxicity also exhibited remarkable regional variation. The toxicity in the edible muscle of T. pardalis and T. niphobles was at acceptable levels for human consumption, while the toxicity of the skin of both species of puffer fish was very high, so that care must be taken when used for human consumption.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.1
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• pp.1-7
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• 2009

The toxicity of two species of puffer fish, Takifugu poecilonotus (Heuinjeombok) and T. vermicularis (Gukmaeribok) collected from the coastal regions of Korea was determined using a mouse bioassay. In the T. poecilonotus collected in Jeju and Tongyeong, the proportion of toxic specimens containing ${\ge}10$ mouse units (MU) per gram exceeded 95% for the skin, liver, ovary, and fin, and approximately 30% for the testis and muscles. In each of the organs, the highest toxin levels were 79 MU/g in the muscle, hundreds (158-365) of MU per gram in the fin, intestine, testis, and gallbladder, but thousands (1,147-2,406) of MU per gram in the skin, liver, and ovary. In T. vermicularis collected from Incheon and Gunsan, the proportions of toxic specimens were 100% for the gallbladder, and 56-68% for the skin, fin, liver, and intestine however, no toxic muscle specimens were noted. The highest toxin scores were below 10 mouse units (MU) per gram in the muscle, 20-94 MU/g in the skin and fin, 319 MU/g in the intestine, and thousands (1,548-4,624) of MU per gram in the liver, gonad, and gallbladder. The toxicity in the muscle of T. vermicularis was deemed acceptable for human consumption, whereas the toxicities in the muscle of T. poecilonotus and the skin of both species of puffer fish were significantly high, such that special attention may be required when the fish is intended for human consumption.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.39 no.6
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• pp.447-453
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• 2006

Toxicity of two species of puffer fish, Takifugu porphyreus and Takifugu rubripes, collected from coastal regions of Korea, was determined using a mouse bioassay, In T. porphyreus, the proportion of toxic specimens containing ${\ge}$ 10 MU/g was 58.3% for the ovary, 32.6% for the skin, 12.0% for the gallbladder, 11.6% for the liver and intestine, and 9.3% for the fin; no toxicity was detected in the muscle and testis using the mouse bioassay. The highest toxin levels were 531 MU/g in the liver, 253 MU/g in the intestine, 136 MU/g in the gallbladder, 118 MU/g in the skin, 116 MU/g in the ovary, and 108 MU/g in the fin. The skin, which is used for human consumption, showed significantly high toxicity with an average of $11{\pm}3\;(mean{\pm}SE) MU/g$. Takifugu porphyreus toxicity also exhibited remarkable regional variation. In T. rubripes, the proportion of toxic specimens was 25.0% for the ovary, 15.8% for the liver, 11.1% for the gallbladder, and 5.3% for the fin and intestine; no toxicity was detected in the muscle, skin, or testis. Among the organs, the highest toxin levels were 228 MU/g in the ovary, followed by 112 MU/g in the liver, 28 MU/g in the gallbladder, 18 MU/g in the intestine, 11 MU/g in the fin, and 8 MU/g in the skin. Thus, we found acceptable toxin levels in the edible muscle and skin of T. rubripes and in the muscle of T. porphyreus. However, the skin of T. porphyreus, which showed significantly high toxicity, requires special attention when used for human consumption.

• Journal of Food Hygiene and Safety
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• v.27 no.1
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• pp.37-41
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• 2012

The current standard for testing tetrodotoxin (TTX) in foodstuffs is the mouse bioassay (MBA) in Korea as in many other countries. However, this test suffers from potential ethical concerns over the use of live animals. In addition, the mouse bioassay does not test for a specific toxin thus a sample resulting in mouse incapacitation would need further confirmatory testing to determine the exact source toxin (e.g., TTX, STX, brevotoxin, etc.). Furthermore, though the time of death is proportional to toxicity in this assay, the dynamic range for this proportional relationship is small thus many samples must be diluted and new mice be injected to yield a result that falls within the quantitative dynamic range. Therefore, in recent years, there have been many efforts in this field to develop alternative assays. High performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) has been emerged as one of the most promising options. A LC-MS-MS method involves solid-phase extraction (SPE) and followed by analysis using an electrospray in the positive ionization mode and multiple reactions monitoring (MRM). To adopt LC-MS-MS method as alternative standard for testing TTX, we performed a validation study for the quantification of TTX in puffer fish. This LC-MS-MS method showed good sensitivity as limits of detection (LOD) of $0.03{\sim}0.08{\mu}g/g$ and limits of quantification (LOQ) of $0.10{\sim}0.25{\mu}g/g$. The linearity ($r^2$) of tetrodotoxin were 0.9986~0.9997, the recovery were 80.9~103.0% and the relative standard deviations (RSD) were 4.3~13.0%. The correlation coefficient between the mouse bioassay and LC/MS/MS method was higher than 0.95.

• Fisheries and Aquatic Sciences
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• v.24 no.11
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• pp.360-369
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• 2021

Paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) are neurotoxins that display pharmacological activity that is similar to that of specific sodium channel blockers; they are the principle toxins involved in shellfish and puffer fish poisoning. In Korea, puffer fish is a very popular seafood, and several cases of accidental poisoning by TTX have been reported. Therefore, it is necessary to determine whether puffer fish poisoning incidents are caused by PSTs or by TTX. In this study, we used mouse bioassay (MBA) and liquid chromatograph-tandem mass spectrometry (LC-MS/MS) to determine the presence of PSTs and TTX in puffer fish from an area near Mireuk-do, Tong-Yeong on the southern coast of Korea from January through March, 2014. The toxicity of PSTs and TTX extracts prepared from three organs of each specimen was analyzed by MBA. Most of the extracts killed mice with typical signs of TTX and PSTs. The LC-MS/MS analysis of seven specimens of Takifugu pardalis and Takifugu niphobles, each divided into muscles, intestines, and liver, were examined for TTX. In T. pardalis, the TTX levels were within the range of 1.3-1.6 ㎍/g in the muscles, 18.8-49.8 ㎍/g in the intestines, and 23.3-96.8 ㎍/g in the liver. In T. niphobles, the TTX levels were within the range of 2.0-4.5 ㎍/g in the muscles, 23.9-71.5 ㎍/g in the intestines, and 28.1-114.8 ㎍/g in the liver. Additionally, the toxicity profile of the detected PSTs revealed that dcGTX3 was the major component in T. pardalis and T. niphobles. When PSTs were calculated as saxitoxin equivalents the levels were all less than 0.5 ㎍/g, which is below the permitted maximum standard of 0.8 ㎍/g. These findings indicate that the toxicity of T. pardalis and T. niphobles from the southern coast of Korea is due mainly to TTX and that PSTs do not exert an effect.