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.     

Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China

A monoclonal antibody against tetrodotoxin (TTX) was produced from the hybridoma cell line T6D9, which was established by the fusion of Sp2/0 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with the TTX–keyhole limpet hemocyanin (KLH) conjugate. This monoclonal antibody belongs to the IgG1 subclass; the affinity constant of the antibody is 2.4 × 10^?8 mol l^?1. The relative cross-reactivity of the antibody with TTX was 100%, but with saxitoxin, KLH and bovine serum albumin (BSA) it was less than 1%, respectively. The titre of the antibody in ascites was 6.4 × 10⁶; the reference working concentration was 1:1.2 × 10⁵. By using this monoclonal antibody, a competitive indirect enzyme-linked immunoabsorbant assay (ELISA) for the analysis of TTX was developed. The linear portion of the dose–response curve of TTX concentration was in range 5–500 ng ml^?1. The limit of detection was 5 ng ml^?1 according 10% inhibition with TTX to anti-TTX monoclonal antibody. The concentration of TTX inhibiting 50% of antibody binding was about 50 ng ml^?1. The recoveries from TTX spiked samples were 79.5–109.5%. In addition, the toxicity of some wild puffer fish specimens captured from south-east China and the Yangzi River in Jiangsu province was determined. The results indicate that the toxicity and toxin tissue distribution vary in different species of wild puffer fish.     

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.     

河鲀毒素生物传感器的研究

基于河鲀毒素(TTX)对细胞膜Na+通道的阻断作用,建立一种检测TTX的简易组织生物传感器。组织生物传感器由pNa电极、青蛙膀胱膜、电位计等构成。在pNa电极顶端用两层醋酸纤维薄膜包裹,中间夹入青蛙膀胱膜,构成感受器。将电极插入pNa=4的NaCl溶液中,使电极输出信号趋于稳定。将TTX被测液注入感受器系统,测定感受器输出量的抑制值。每次测定只需5min。最低检出限为0.002MU/ml(3.56×10-3μg/ml)。用0.003%Na3N保存,可连续使用250h。以生物传感器和小鼠生物试验法比较,建立了工作曲线,便于在餐饮企业对原料鱼进行检测和应用。     

Toxin composition and toxicity dynamics of marine gastropod Nassarius spp. collected from Lianyungang, China

Consumption of nassariid gastropods often leads to poisoning incidents in some coastal provinces in China. To elucidate the pattern of toxicity dynamics and origin of toxins, samples of gastropod Nassarius spp. were collected from late May to early August 2007 from Lianyungang, Jiangsu province, where the poisoning incidents have been frequently reported. Toxicity was first screened with the mouse bioassay method, and tetrodotoxin and its analogues (TTXs) were analysed with high-performance liquid chromatography coupled with an ion-trap mass spectrometer (HPLC-MS(n)). The toxicity of nassariid N. semiplicatus showed an 'M'-shaped pattern of fluctuation during the sampling season. Two peaks of toxicity appeared in late May and late July. The maximum toxicity was recorded on 24 May, with the value of 846 mouse unit (MU) g(-1) of tissue (wet weight). TTX and its analogues trideoxyTTX, 4-epiTTX, anhydroTTX and oxoTTX were detected in the nassariid samples. TrideoxyTTX but not TTX was the major toxin in all the samples. No paralytic shellfish poison (PSP) was detected in the sample with the maximum toxicity by HPLC-FLD analysis. Variation of TTX content in the tissue of nassariid gastropods correlates well with the dynamics of toxicity. It is suggested that TTXs are the major toxins corresponding to the toxicity of the nassariids, and May and July are the high-risk seasons for consumption of nassariids, which is critical for the management of poisoning incidents.     

Toxin composition and toxicity dynamics of marine gastropod Nassarius spp. collected from Lianyungang,China

Consumption of nassariid gastropods often leads to poisoning incidents in some coastal provinces in China. To elucidate the pattern of toxicity dynamics and origin of toxins, samples of gastropod Nassarius spp. were collected from late May to early August 2007 from Lianyungang, Jiangsu province, where the poisoning incidents have been frequently reported. Toxicity was first screened with the mouse bioassay method, and tetrodotoxin and its analogues (TTXs) were analysed with high-performance liquid chromatography coupled with an ion-trap mass spectrometer (HPLC-MS ⁿ ). The toxicity of nassariid N. semiplicatus showed an ‘M’-shaped pattern of fluctuation during the sampling season. Two peaks of toxicity appeared in late May and late July. The maximum toxicity was recorded on 24 May, with the value of 846 mouse unit (MU) g^?1 of tissue (wet weight). TTX and its analogues trideoxyTTX, 4-epiTTX, anhydroTTX and oxoTTX were detected in the nassariid samples. TrideoxyTTX but not TTX was the major toxin in all the samples. No paralytic shellfish poison (PSP) was detected in the sample with the maximum toxicity by HPLC-FLD analysis. Variation of TTX content in the tissue of nassariid gastropods correlates well with the dynamics of toxicity. It is suggested that TTXs are the major toxins corresponding to the toxicity of the nassariids, and May and July are the high-risk seasons for consumption of nassariids, which is critical for the management of poisoning incidents.     

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。     

Occurrence of tetrodotoxin and paralytic shellfish poisons in a gastropod implicated in food poisoning in southern Taiwan

The toxicity of the gastropod Nassarius papillosus implicated in a food paralytic poisoning incident in Liuchiu Island, Taiwan, in October 2005 is reported. The symptoms of a victim (67 years old) were featured by general paresthesia, paralysis of phalanges and extremities, paralysis, coma, and aphasia. The remaining specimens of shell were assayed for toxicity. The range of specimen toxicity was found to be 63-474 mouse units (MU) per specimen for N. papillosus by a tetrodotoxin (TTX) bioassay. The mean (SD) toxicity of the digestive gland and other portions were 296 ± 120 and 382 ± 156 MU in N. papillosus. The toxin was partially purified from the acidic methanol extract of the gastropod by using a C18 solid-phase extraction column. The eluate was then filtered through a 3000 MW cut-off ultrafree microcentrifuge filter. It was shown that the toxin purified from gastropods analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry contained TTX 42-60 µg g^-1 (about 90%), whereas along with minor paralytic shellfish poisons (PSP) it was 3-6 µg g^-1 (about 10%).     

Tetrodotoxin poisoning due to smooth-backed blowfish Lagocephalus inermis and toxicity of L. inermis caught off the Kyushu coast, Japan

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan.     

A biodynamic understanding of dietborne metal uptake by a freshwater invertebrate

Aquatic organisms accumulate metals from dissolved and particulate phases. Dietborne metal uptake likely prevails in nature, but the physiological processes governing metal bioaccumulation from diet are not fully understood. We characterize dietborne copper, cadmium, and nickel uptake by a freshwater gastropod (Lymnaea stagnalis) both in terms of biodynamics and membrane transport characteristics. We use enriched stable isotopes to trace newly accumulated metals from diet, determine food ingestion rate (IR) and estimate metal assimilation efficiency (AE). Upon 18-h exposure, dietborne metal influx was linear over a range encompassing most environmental concentrations. Dietary metal uptake rate constants (k(uf)) ranged from 0.104 to 0.162 g g(-1) day(-1), and appeared to be an expression of transmembrane transport characteristics. Although k(uf) values were 1000-times lower than uptake rate constants from solution, biodynamic modeling showed that diet is the major Cd, Cu, and Ni source in nature. AE varied slightly among metals and exposure concentrations (84-95%). Suppression of Cd and Cu influxes upon exposure to extreme concentrations coincided with a 10-fold decrease in food IR, suggesting that feeding inhibition could act as an end point for dietary metal toxicity in L. stagnalis.     

Reduction of nickel and uranium toxicity and enhanced trichloroethylene degradation to Burkholderia vietnamiensis PR1301 with hydroxyapatite amendment

The use of hydroxyapatite (HA) to sequester metals at mixed waste sites may reduce metal toxicity and facilitate microbial degradation of cocontaminant organics. The constitutive trichloroethylene (TCE) degrader, Burkholderia vietnamiensis PR1301, grew at 34.1 and 1.7 mM Ni at pH 5 and 7, respectively, with 0.01 g mL(-1) HA compared to 17 and 0.85 mM Ni without HA. PR1 grew at 4.2 mM U at pH 5 and 7 with 0.01 g mL(-1) HA compared to 1.1 mM U without HA. A similar decrease in the toxicity of Ni and U in combination was observed with HA. The ability of PR1 to degrade TCE at 0.85, 1.7, and 3.4 mM Ni and at 0.42 and 1.1 mM U was examined. The presence of TCE resulted in a decreased tolerance of PR1 to Ni and U; however, HA facilitated TCE degradation in the presence of Ni and U, effectively doubling the metal concentrations at which TCE degradation proceeded. These studies suggest that metal sequestration via HA amendments may offer a feasible approach to reducing metal toxicity to microorganisms at mixed waste sites, thereby enhancing the degradation of cocontaminant organics.     

溶液Ca、K浓度和pH对烟草Cd毒性的影响

通过水培实验,利用逻辑斯蒂克分布模型研究了溶液中不同Ca、K浓度及pH条件下镉(Cd)对烟草的急性毒性.结果表明,随着溶液pH的升高,Cd对烟草根伸长的10%抑制浓度(EC10)及半抑制浓度(EC50)逐渐增加,也即Cd的毒性逐渐降低,溶液的pH与烟草Cd毒性EC10、EC50间呈显著的正相关关系,当溶液pH从4.52增加到8.10时,烟草根伸长的EC50增加了6.73倍.烟草Cd毒性EC10、EC50测定结果表明,当溶液Ca离子浓度 < 128.0 mg/L时,添加Ca离子浓度可以显著降低烟草Cd的毒性;与Ca不同,当溶液中K离子浓度 < 64.0 mg/L时,K离子浓度的增加并没有显著降低Cd的毒性,当溶液中K离子浓度 > 64.0 mg/L,溶液K离子浓度增加可以显著降低烟草Cd的毒性.在烟草Cd污染胁迫条件下,提高介质pH和增加介质中Ca、K离子的浓度是有效降低Cd毒性的措施之一.     

Humic acids increase dissolved lead bioavailability for marine invertebrates

The presence of dissolved organic matter (DOM), as humic acids (HA), in natural waters is assumed to decrease dissolved metal bioavailability by binding metal ions and, therefore, decreasing the free ion concentration in solution. In this study, Pb complexation by HA in artificial seawater was checked by means of square wave anodic stripping voltammetry (SWASV). Uptake and toxicity of this metal in the absence and presence of HA was tested using excised gills of Mytilus edulis and the Paracentrotus lividus embryo-larval bioassay respectively. Both Pb uptake by mussel gills and Pb toxicity to sea urchin larvae increased in the presence of HA, and this increase was higher at higher HA concentrations. Since it is shown that the presence of DOM can enhance the uptake and toxicity of lead in some important marine invertebrate species, these results challenge the general applicability of the free ion activity and related models used for deriving environmental water quality criteria for metals.     

Purification and characterization of polyphenol oxidase from Trametes sp. MS39401

Two polyphenol oxidases (EC 1.14.18.1), P-1 and P-2, were purified as electrophoretically homogeneous proteins from the culture filtrate of Trametes sp. MS39401 by acetone precipitation and column chromatographies on DEAE-Sephadex A-50, Sephadex G-150 and hydroxylapatite. P-1 was purified 34-fold with a yield of 4.2%, while P-2 was purified 37-fold with a yield of 20.7%. The molecular masses of P-1 and P-2 were estimated to be 61 kDa and 90 kDa, respectively, by gel filtration. The isoelectric points of P-1 and P-2 were 3.4 and 2.7, respectively. The optimum pH range of both enzymes was 4.5-5.0 at 45 degrees C. The optimum temperature of both enzymes was 55 degrees C at pH 5.0. P-1 was stable at pH 5.0-7.5 and temperatures up to 60 degrees C. P-2 was stable at pH 3.0-7.5 and temperatures up to 50 degrees C. The thermostability of P-1 was comparable to that of the PM1 laccase of basidiomycetes, which was reported to be the most stable among basidiomycete laccases. Both enzymes were active toward various phenolic compounds and aminophenols. However, they lacked activity toward l-tyrosine. The K(m) values for (+)-catechin were 0.19 mM for P-1 and 0.67 mM for P-2. Both enzymes were appreciably inactivated by Hg(2+) and Sn(2+). Significant activation of neither enzyme was observed in the presence of metal ions and reagents. Both enzymes were significantly inhibited by copper-chelating agents, reducing agents and N-bromosuccinimide. Carbon monoxide caused appreciable inactivation of neither enzyme, so it is suggested that P-1 and P-2 belong to the group of laccases.     

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.     

Kinetics of simultaneous photocatalytic degradation of phenolic compounds and reduction of metal ions with nano-TiO2

The simultaneous photocatalytic degradation of phenol and 4-nitrophenol and reduction of metal ions like copper (Cu2+) and chromium (Cr6+) was studied with solution combustion synthesized nanoanatase TiO2 (CS TiO2) and commercial titania, Degussa P-25. The presence of metal ion reduces the rate of degradation of phenol and 4-nitrophenol. It was found that Cu2+ reduction to Cu+ is accelerated in the presence of phenol. In the case of Cr6+, CS TiO2 enhances the initial adsorption of Cr6+ and complete reduction is achieved within the first 10 min of UV irradiation. The presence of phenol or 4-nitrophenol also enhances the initial adsorption of Cr6+ and its reduction. The metal ion reduction in the presence of CS TiO2 is compared with that of Degussa P-25. The rate of reduction of metal ions in presence of Degussa P-25 is twice as slow as that of CS TiO2 in presence of both phenol and 4-nitrophenol. The presence of Cu2+ and Cr6+ also induces the formation of the intermediates which were not observed for the phenol-CS TiO2 system. The formation and consumption of the intermediates are modeled with a simple series reaction mechanism. A detailed dual-cycle, multistep reaction mechanism of TiO2 photocatalysis for the simultaneous degradation and reduction is proposed and the model is developed following the network reduction technique. The kinetic rate constants in the model are evaluated for the systems studied.     

Copper toxicity to bioluminescent Nitrosomonas europaea in soil is explained by the free metal ion activity in pore water

The terrestrial biotic ligand model (BLM) for metal toxicity in soil postulates that metal toxicity depends on the free metal ion activity in solution and on ions competing for metal sorption to the biotic ligand. Unequivocal evidence for the BLM assumptions is most difficult to obtain for native soil microorganisms because the abiotic and biotic compartments cannot be experimentally separated. Here, we report copper (Cu) toxicity to a bioluminescent Nitrosomonas europaea reporter strain that was used in a solid phase-contact assay and in corresponding soil extracts and artificial soil solutions. The Cu(2+) ion activities that halve bioluminescence (EC50) in artificial solutions ranged 10(-5) to 10(-7) M and increased with increasing activities of H(+), Ca(2+) and Mg(2+) according to the BLM concept. The solution based Cu(2+) EC50 values of N. europaea in six contaminated soils ranged 2 × 10(-6) to 2 × 10(-9) M and these thresholds for both solid phase or soil extract based assays were well predicted by the ion competition model fitted to artificial solution data. In addition, solution based Cu(2+) EC50 of the solid phase-contact assay were never smaller than corresponding values in soil extracts suggesting no additional solid phase toxic route. By restricting the analysis to the same added species, we show that the Cu(2+) in solution represents the toxic species to this bacterium.     

The toxification of juvenile cultured kusafugu Takifugu niphobles by oral administration of crystalline tetrodotoxin

Non-toxic cultured juvenile kusafugu were fed with diet containing crystalline tetrodotoxin (TTX) for 30 days and then fed with non-toxic diet for 170 days. During this period, 5 fish were sampled and the toxicity of each tissue was determined periodically. The amount of total accumulated toxin in the fish was 90 microg, representing 50% of the administered TTX (180 microg/fish) at the 60th day. It decreased to 54 microg (30%) at the 80th day and then remained unchanged up to the 200th day. The amount of toxin in the liver amounted to 40 microg (45% of total accumulated toxin) at the 30th day and gradually decreased to 5 microg (10%) at the 200th day. The toxin amount in the skin reached the highest level with 30 microg (30%) at the 50th day and then remained unchanged during the experimental period. The testes had almost no toxicity. Although the ovaries were immature, the toxin amounts increased as the weight of the tissues increased. With administration of crystalline TTX, all kusafugu used in the experiment became toxic and retained the toxin at the level of 30% of the administered toxin for about 5 months thereafter, while being fed with non-toxic diet.     

Toxins in toxic Taiwanese crabs

Abstract

A total of 459 specimens covering 51 species in 9 families was collected from October 1992 to May 1996 in Taiwan. All specimens were assayed for the presence of tetrodotoxin (TTX) and paralytic shellfish poison (PSP). The specimens of five xanthid crabs Zosimus aeneus, Lophozozymus pictor, Ategatopsis germaini, Atergatis floridus, and De‐mania reynaudi were found to contain potent toxins. Among them, A. germaini showed the highest toxicity. The toxin profile of each toxic crab species was as follows: 82% TTX and 18% PSP in Z. aeneus, 89% TTX and 11% PSP in L. pictor, 3% TTX and 97% PSP inA germaini, 85% TTX and 15% in A. floridus, and 88% TTX and 12% PSP in D. reynaudi. PSP was mainly composed of gonyautoxins (GTXs) 1–4 in Z. aeneus, L. pictor, and A. floridus, but GTX 3 and hydroxysaxitoxin in A. germaini, and neosaxitoxin in D. reynaudi. The PSP‐producing dinoflagellate plankton Alexandrium minutum and TTX‐producing bacteria including Vibrio alginolyticus and Vibrio parahaemolyticus were isolated and considered as the sources of the toxins.     

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.