Toxicity of puffer fish cultured in netcages

During 1990 to 2003, the toxicity of the liver in 4,515 specimens of the puffer fish Takifugu rubripes (torafugu) cultured in netcages or on land were investigated by means of mouse bioassay and liquid chromatography-mass spectrometry (LC/MS). Other tissues (skin, muscles, gonads, etc.) were also investigated in some of them. All the livers and other parts examined were found to be non-toxic. The peak corresponding to tetrodotoxin (TTX) was not detected in the samples by LC/MS analysis for TTX (< 0.1 MU/g). These results show that puffer fish fed on a non-toxic diet in netcages do not become intoxicated.     

Identification of Toxin and Fish Species in Cooked Fish Liver Implicated in Food Poisoning

ABSTRACT: The cooked fish liver retained by the victims was assayed for toxicity and mitochondrial DNA. Meanwhile, 8 live specimens of puffer Takifugu niphobles were also assayed. The toxicity of cooked fish liver was 280 ± 20 mouse units per gram (MU/g). All specimens of T. niphobles showed high toxicity (more than 850 MU/g) in the liver. The toxin from cooked fish liver and liver of T. niphobles was identified to be tetrodotoxin. The cooked fish liver and fresh liver of T. niphobles showed the same sequence genotype and the same single restriction site for Bsa I. Therefore, the species of cooked fish liver was suggested as T. niphobles.     

Toxicity of puffer fish fins

Puffer fish is prized as a Japanese traditional food and its fin is also used in the cuisine. However, whether the fin is edible or not is determined for convenience from the toxicity of skin, since little information is available about the toxicity of puffer fish fins. In the present study, we examined the toxicity of fins and skin of three toxic species, Takifugu vermicularis, T. snyderi, and T. porphyreus. The toxicity of T. vermicularis fins (< 5-52.4 MU/g) was significantly lower than that of skin (<5-1200 MU/g). HPLC analysis showed that tetrodotoxin was a major toxic principle irrespective of the toxicity value in each tissue of T. vermicularis. In the case of T. snyderi and T. porphyreus, the toxicity of fins was at almost the same level as that of the skin. The toxicity (< 10-12 MU/g) of caudal fins of T. porphyreus was apparently increased to 16.5-22.0 MU/g by drying. However, the toxin amounts in the dried fins were slightly decreased as compared with those of the non-dried fins. These results demonstrate that puffer fish with toxic skin also have toxic fins.     

Liquid chromatography-tandem mass spectrometry determination of the toxicity and identification of fish species in a suspected tetrodotoxin fish poisoning

Suspected tetrodotoxin (TTX) poisoning was associated with eating unknown fish in April 2009 in Taiwan. After ingestion of the fish, symptoms of the victim included perioral paresthesia, nausea, vomiting, ataxia, weakness of all limbs, respiration failure, and death within several hours. The toxicity in the remaining fish was determined, with the mice exhibiting symptoms of neurotoxin poisoning. The implicated fish and deceased victim tissues were analyzed for TTX by liquid chromatography-tandem mass spectrometry. The urine, bile, cerebrospinal fluid (spinal cord), pleural effusion, and pericardial effusion of the victim contained TTX. In addition, the partial cytochrome b gene of the implicated fish was determined by PCR. The DNA sequence in the partial 465-bp cytochrome b gene identified the implicated fish as Chelonodon patoca (puffer fish). These results indicate that people should avoid eating unknown fish species from fish markets where harvested fish may include toxic species.     

鲀毒鱼毒素检测及物种鉴定技术研究进展

鲀毒鱼引起的河鲀毒素(tetrodotoxin,TTX)中毒是中国沿海地区食物中毒致死的主要原因之一.河鲀毒素中毒发病迅速,至今尚无特效药,因此,通过检测TTX含量或鉴定携带TTX的物种可以更好地进行TTX中毒的风险分析、管理和控制.本文综述了鲀毒鱼TTX的检测技术以及鲀毒鱼物种鉴定的方法,以期能预警并减少鲀毒鱼引起的...     

养殖河豚鱼毒素的研究

为了探究养殖河豚鱼可食用性,本实验对养殖河豚鱼的食用安全进行了研究,提取河豚鱼的肝脏、肌肉、血液、性腺为研究对象,用液相色谱法测定TTX的含量。结果表明,养殖(红鳍东方豚、暗纹东方豚、菊黄东方豚、黄鳍东方豚)肝脏、血液无毒或弱毒,肌肉无毒,可作为食用鱼种,而野生的暗纹东方豚均有剧毒,不可食用。     

Identification of tetrodotoxin and fish species in a dried dressed fish fillet implicated in food poisoning

There were five victims of neurotoxic food poisoning from a dried dressed fish fillet in Changhua County, Taiwan, in February 2000. The toxicity of the dried dressed fish fillets was 243 mouse units per g according to a tetrodotoxin bioassay. The partially purified toxin was identified as tetrodotoxin and anhydrotetrodotoxin. The sequence of the 376-nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype as that of the toxic puffer fish Lagocephalus lunaris. The same single restriction site for Hinfl was found in the polymerase chain reaction (PCR) products from the dried dressed fish fillet and the muscle of L. lunaris, yielding two DNA fragments of 170 and 206 bp. However, no restriction site for Hinfl was found in the PCR products from other toxic puffer fishes, including Takifugu niphobles, Takifugu oblongus, and Takifugu rubripes. Therefore, the species of the dried dressed fish fillet was identified as L. lunaris and its causative agent was identified as tetrodotoxin.     

Hematological toxicity associated with tissue extract from poisonous fish Lagocephalus lagocephalus--influence on erythrocyte function in Wistar rats

The puffer fish Lagocephalus lagocephalus represents serious public health problems in the world. The relative toxicity of each organ (liver and flesh) was determined by the relation dose-death time "mouse bioassay." The average liver toxicity of the puffer fish was the highest when compared with flesh giving 14.32 and 10.88 MU/g, respectively. A mouse unit is the amount of toxin (extract of fish organ) that kills a 20 g male mouse in 30 min after intraperitoneal injection. One mouse unit is equivalent to 0.22 microg of TTX. For the rat bioassay tests, Wistar rats were daily i.p. injected, for 10 d, with extracts of liver (LT) or flesh (FT) (muscles + skin) of L. lagocephalus. Control rats received injection of NaCl (0.9%). During the experiment, a significant reduction in red blood cell number (RBC), hemoglobin (HGB) concentration, and hematocrit (HCT) was observed essentially after 10 d of treatment in the FT and LT-exposed groups. Consequently, treatment led to severe anemia and hemolytic action as indicated by a significant reduction in the total number of erythrocytes. In fact, our study revealed a significant increase in erythrocyte lipid peroxidation (LPO) in FT and LT groups compared with controls after experimental exposure. The flesh and liver tissue extracts also altered antioxidative enzymes activities: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Histopathological alterations in the spleen occurred exclusively at the end of treatment. We marked also an increase in reticulo-endothelial cells, which led to remove damaged erythrocytes.     

Liquid chromatography-mass spectrometry method to detect Tetrodotoxin and Its analogues in the puffer fish Lagocephalus sceleratus (Gmelin, 1789) from European waters

The presence of tetrodotoxin (TTX) and its analogues, 4-epiTTX, 4,9-anhydroTTX, 5-deoxyTTX, 11-deoxyTTX, 5,6,11-trideoxyTTX, 11-norTTX-6(S)-ol, and 11-norTTX-6(R)-ol was investigated for the first time in five different tissues (liver, gonads, gastrointestinal tract, muscle, and skin) of six specimens of the marine puffer fish Lagocephalus sceleratus from European waters (Aegean Sea, Greece) by using liquid chromatography coupled to electrospray ionisation mass spectrometry operating in the conventional mode in addition to low-energy collision dissociation tandem mass spectrometry (CID-MS/MS).Two isomers of 5,6,11-trideoxyTTX were detected in all specimens as the major TTX analogues, followed by 11-deoxyTTX, 11-norTTX-6(S)-ol, and TTX. However, minor amounts of 4-epiTTX, 4,9-anhydroTTX, 5-deoxyTTX, and 11-norTTX-6(R)-ol were also found in most of the tested tissues. In all puffer fish specimens, gonads, gastrointestinal tract, and liver contained the highest toxin levels, whereas muscle and skin contained lower amounts. Toxin distribution within the tissues of the six L. sceleratus specimens was different depending on fish size, area, and season where fish were caught. The LC-ESI-CID-MS/MS analysis employed is proposed as a suitable technique for determination of TTX and its analogues with a low detection limit (0.08 μg/g).     

鼠李糖乳酸杆菌对河豚肝脏中河豚毒素毒性的影响

以野生河豚肝脏中的河豚毒素（tetrodotoxin,TTX）为研究对象,研究活化与热灭活处理的鼠李糖乳杆菌对其毒性的消减作用。将活化的与热灭活处理的鼠李糖乳杆菌作为材料对TTX进行脱除,采用酶联免疫吸附剂测定（enzyme-linked immunosorbnent assay,ELISA）、层析试纸检测和小鼠生物法分析TTX消减率的变化,采用气相色谱-质谱分析发酵处理后的肝脏二十碳五烯酸（eicosapentaenoic acid,EPA）与二十二碳六烯酸（docosahexaenoic acid,DHA）含量变化。竞争性ELISA结果显示加热灭活后的鼠李糖乳杆菌对TTX的消减率高达82.16%;而未经灭活处理的鼠李糖乳杆菌对TTX的消减率为70.05%,表明热灭活处理的鼠李糖乳杆菌消减效果更佳。此外,经鼠李糖乳杆菌发酵7 d后野生河豚肝脏的TTX消减率为93.27%,气相色谱-质谱检测结果显示发酵后肝脏内的EPA与DHA含量分别减少了11.93%、22.50%。综上所述,鼠李糖乳杆菌能消减河豚肝脏中的TTX,经鼠李糖乳杆菌发酵处理后消减效果更佳,本研究结果可为河豚内脏组织的毒性消减提供参考。     

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.     

一起雪卡毒素中毒事件中裸胸鳝毒性的研究

目的研究厦门一起引发食物中毒的裸胸鳝毒性及种类,以预防雪卡毒素中毒。方法对中毒事件进行流行病学调查,采集裸胸鳝样品共5份,采用小鼠生物法、雪卡毒素免疫膜检测试剂盒检测毒性大小,高效液相色谱-串联质谱法测定太平洋雪卡毒素(P-CTX-1),同时提取中毒鱼肉组织中的mtDNA,PCR技术扩增细胞色素b(Ctyb)后测序鉴定中毒鱼种。结果小鼠生物法试验检测5份样品,鱼肉毒性为无毒性(ND)～0.052 MU/g,内脏毒性为ND～0.23 MU/g;高效液相色谱-串联质谱法未检出P-CTX-1。5种鱼种经鉴定均为裸胸鳝属(Gymnothorax),其中中毒鱼种经基因测序为波纹裸胸鳝(Gymnothorax undulates)。结论引发该起食物中毒的是一种含有雪卡毒素的波纹裸胸鳝。     

Identification of tetrodotoxin in a marine gastropod (Nassarius glans) responsible for human morbidity and mortality in Taiwan

The toxicity of the gastropod Nassarius glans was investigated. This gastropod was implicated in an incident of food paralytic poisoning on Tungsa Island, Taiwan, in April 2004. Six victims consumed both digestive glands and muscle. These tissues contained high concentrations of toxin; their highest toxicity scores were 2,048 and 2,992 MU/g, respectively, based on the tetrodotoxin (TTX) bioassay. The toxin was purified from these gastropods and analyzed by high-performance liquid chromatography, which revealed TTX and related compounds 4-epi TTX and anhydro-TTX; paralytic shellfish poisons were not found. The urine and blood samples from patients were cleansed using a C18 Sep-Pak cartridge column and 3,000 molecular weight cutoff Ultrafree microcentrifuge filters, and the eluate was filtered and analyzed by liquid chromatography and mass spectrometry. The detection limit for TTX was 1 ng/ml. The standard curves were linear in the range 30 to 600 ng/ml for urine and 1 to 30 ng/ml for blood. TTX was detected in all urine samples but in only three of four blood samples tested. Thus, the causative agent of gastropod food poisoning was identified as TTX.     

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer

Tetrodotoxin (TTX), often referred to as the ‘puffer fish’ poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01–7.5 µg ml^–1; R² ≥ 0.9968) as well as for matrix-matched standards (0.05–37.50 µg g^–1; R² ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01–7.5 µg ml^–1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.     

从一起河鲀中毒调查探讨河鲀食用安全性的风险管理措施

目的通过一起河鲀中毒事件的流行病学调查和人群关于河鲀安全性的知识、态度、行为(KAP)调查,探讨河鲀食用安全的风险管理措施。方法通过病例搜索,调查病例家庭成员进食史及发病情况,采用回顾性队列研究分析发病的高危因素,制作调查问卷调查了解当地村民有关河鲀中毒的知识、态度和行为。结果共搜索到14例病例,其中1例死亡。症状主要为头晕(100%)、四肢无力(79%)、四肢肌肉麻痹和行走困难(71%)、口舌肢端麻痹(71%)等。食用河鲀是发病的危险因素,加工过程保留鱼肝共同煮食可增加中毒的危险(RR=1.8,95%CI=1.1~3.1)。KAP调查发现70%(33/47)的村民近3年曾进食过河鲀,其中88%(29/33)来源于市场或流动摊贩;98%(46/47)的村民知道河鲀有毒,94%(44/47)的村民知道国家法律禁止食用河鲀鱼,但仍有94%(44/47)的人认为其味道鲜美值得品尝。结论食用混杂于无毒河鲀中的有毒河鲀是导致本次食物中毒的主要原因。居民对河鲀安全性知晓率高但依从性差。     

SURVEY OF CIGUATERA AND TETRODOTOXIN OF BLOWFISH IN THAILAND

Twenty kinds of ciguatoxic fishes such as grouper, moray eel, barracuda, catfish and snapper, etc. were collected from fish markets in Bangkok, Chanthaburi and Chonburi. Liver and meat were extracted with ether and the presence of ciguatoxin was checked by the mouse test. Ciguatoxin was not detected in any of the samples. On the other hand, water-extracted fractions under acetic acidic conditions from meat, skin, intestine, liver and ovary of five kinds of puffer collected from Chanthaburi, Samut Sakorn and Trat caused mouse paralysis or death. The ovaries of the green rough-backed blowfish and the starry blowfish, which are very popular in Thailand, contained large amounts of tetrodotoxin. The starry blowfish contained 1482.0 mouse units of tetrodotoxin per gram of ovary.     

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 ± 4.0 and 87.1 ± 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.     

Tetrodotoxin in gastropods (snails) implicated in food poisoning in Northern Taiwan

The toxin in the gastropods (snails) Zeuxis sufflatus and Niotha clathrata implicated in a food poisoning incident in northern Taiwan in April 2001 was studied. The symptoms exhibited by four victims were general paresthesia, paralysis of the phalanges and the extremities, paralysis, coma, vomiting, and aphasia. The remaining gastropods were assayed for toxicity in the form of tetrodotoxin (TTX). The ranges of specimen toxicity were 345 to 1,640 mouse units (MU) for Z sufflatus and 190 to 643 MU for N. clathrata. The toxicities of the digestive gland and for other parts of the gastropod were 1,120 +/- 477 MU and 497 +/- 238 MU, respectively, for Z sufflatus and 683 +/- 113 MU and 289 +/- 169 MU, respectively, for N. clathrata. The toxin from the methanolic extract of the gastropods was partially purified by ultrafiltration and Bio-Gel P-2 column chromatography. Cellulose acetate membrane electrophoresis, thin-layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry analyses demonstrated that the toxin consisted of TTX. It was concluded that the causative agent of the food poisoning in question was TTX.     

养殖河豚鱼的加工及河豚毒素消减技术研究概述

河豚鱼肉质鲜嫩,味道鲜美,享誉古今中外。我国东南沿海地区、日本等许多国家和地区均有吃河豚的习惯,但河豚鱼的内脏、表皮、卵巢等部位含有河豚毒素,因食用河豚鱼导致的中毒事件时有发生。因此,有效消减控制河豚毒素对河豚鱼产业发展有重要意义。该文主要介绍了我国河豚鱼食用历史和消费现状,对养殖河豚鱼深加工制品种类做详细阐述,重点探讨了河豚毒素消减方法和消减机理的研究情况,以期对河豚毒素的消减控制提供参考。     

Identification of Tetrodotoxin and Fish Species in an Adulterated Dried Mullet Roe Implicated in Food Poisoning

ABSTRACT: There was 1 victim of neurotoxic food poisoning from an adulterated dried mullet roe in Kaohsiung, Taiwan, in March 2001. The victim exhibited typical neurotoxic symptoms. The residue of dried mullet roe retained by the victim was assayed for toxicity and mitochondrial DNA. Its toxicity was 3450 mouse units per gram. The toxin was partially purified and identified as tetrodotoxin and derivative. The sequence of the 376‐nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype and the same restriction site for SapI as that of the toxic puffer fish Lagocephalus lunaris.