Major Toxin Components Research Articles

Progress on the investigation and monitoring of marine phycotoxins in China

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin β-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

Target switch of centipede toxins for antagonistic switch.

Animal venoms are powerful, highly evolved chemical weapons for defense and predation. While venoms are used mainly to lethally antagonize heterospecifics (individuals of a different species), nonlethal envenomation of conspecifics (individuals of the same species) is occasionally observed. Both the venom and target specifications underlying these two forms of envenomation are still poorly understood. Here, we show a target-switching mechanism in centipede (Scolopendra subspinipes) venom. On the basis of this mechanism, a major toxin component [Ssm Spooky Toxin (SsTx)] in centipede venom inhibits the Shal channel in conspecifics but not in heterospecifics to cause short-term, recoverable, and nonlethal envenomation. This same toxin causes fatal heterospecific envenomation, for example, by switching its target to the Shaker channels in heterospecifics without inhibiting the Shaker channel of conspecific S. subspinipes individuals. These findings suggest that venom components exhibit intricate coevolution with their targets in both heterospecifics and conspecifics, which enables a single toxin to develop graded intraspecific and interspecific antagonistic interactions.

Co-Occurrence of Tetrodotoxin and Saxitoxins and Their Intra-Body Distribution in the Pufferfish Canthigaster valentini.

Pufferfish of the family Tetraodontidae possess tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs, depending on the genus or species. In the present study, to clarify the distribution profile of TTX and STXs in Tetraodontidae, we investigated the composition and intra-body distribution of the toxins in Canthigaster valentini. C. valentini specimens (four male and six female) were collected from Amami-Oshima Island, Kagoshima Prefecture, Japan, and the toxins were extracted from the muscle, liver, intestine, gallbladder, gonads, and skin. Analysis of the extracts for TTX by liquid chromatography tandem mass spectrometry and of STXs by high-performance liquid chromatography with post-column fluorescence derivatization revealed TTX, as well as a large amount of STXs, with neoSTX as the main component and dicarbamoylSTX and STX itself as minor components, in the skin and ovary. The toxins were also detected in the other tissues, but in much lower amounts than in the skin and ovary. The TTX/STX ratio varied greatly, depending on the tissue, but TTX was the major toxin component in the whole body, and STXs accounted for 25% and 13% of the total toxin amount in males and females, respectively. Like the marine pufferfish of the genus Arothron, C. valentini should be considered a pufferfish with considerable amounts of both TTX and STXs present simultaneously.

Characterization of Dinophysis acuminata from the Yellow Sea, China, and its response to different temperatures and Mesodinium prey

Abstract Dinophysis species are distributed worldwide and cause diarrheic shellfish poisoning (DSP). This paper documents the first successful culture of a Dinophysis (DAYS01) strain taken off Xiaoping Island, the Yellow Sea, China. The strain was identified as Dinophysis acuminata Claparède & Lachmann by morphological and phylogenetic analysis. The effects of temperature and different Mesodinium prey on the physiological and toxigenic characteristics of D. acuminata DAYS01 were also investigated. The results showed that the toxin concentration (toxin amount per ml of culture) increased with an increase in cell densities. Okadaic acid (OA), dinophysistoxin 1 (DTX1) and pectenotoxin 2 (PTX2) were the major toxin components of DAYS01, with the highest recorded toxin content of 5.66, 0.56 and 192.87 ng ml-1 culture medium, respectively. The prey type significantly influenced the growth yield in Dinophysis cultures. There was an effect of prey on the total toxin content, suggesting that the origin or strain of ciliate directly impacted the D. acuminata blooms and the overall toxin concentration in the system

Comparison of MBA and HPLC Post-column Oxidation Methods for the Quantification of Paralytic Shellfish Poisoning Toxins

The mouse bioassay and high performance liquid chromatography (HPLC) post-column oxidation method are different methods of quantifying paralytic shellfish poisoning toxins. In this study, we compared their ability to accurately quantify the toxicity levels in two types of field sample (oysters and mussels) with different toxin profiles for routine regulatory monitoring. A total of 72 samples were analyzed by both methods, 44 of which gave negative results, with readings under the limit of detection of the mouse bioassay (<TEX>$40{\mu}g/100g$</TEX> saxitoxin [STX] eq). In 14 oysters, the major toxin components were gonyautoxin (GTX) 1, -2, -3, -4, -5, decarbamoylgonyautoxin-2 (dcGTX2), and decarbamoylsaxitoxin (dcSTX), while 14 mussels tested positive for dcSTX, GTX2, -3, -4, -5, dcGTX2, neosaxitoxin (NEO), STX, and dcSTX. When the results obtained by both methods were compared in two matrices, a better correlation (<TEX>$r^2=0.9478$</TEX>) was obtained for mussels than for oysters (<TEX>$r^2=0.8244$</TEX>). Additional studies are therefore needed in oysters to investigate the differences in the results obtained by both methods. Importantly, some samples with toxin levels around the legal limit gave inconsistent results using HPLC-based techniques, which could have a strong economic impact due to enforced harvest area closure. It should therefore be determined if all paralytic shellfish poisoning toxins can be quantified accurately by HPLC, and if the uncertainties of the method lead to doubts regarding regulatory limits.

진해만 해역에서 지중해담치 (Mytilus galloprovincialis)의 마비성패독 독화 양상

This study looked at toxicity of Mediterranean mussels, Mytilus galloprovincialis, which had accumulated paralytic shellfish toxins (PST) from early March to late May 2005 at Jinhae Bay, Korea. Alexandrium sp. was observed in low densities ( STXeq. /100 g). Peak PSP (Paralytic Shellfish Poisoning) toxicity in the mussels occurred during high Alexandrium sp. cell densities in April. Mussels toxicity decreased with decline of Alexandrium sp. cell density. Major toxin components identified were , , followed by , , , and neoSTX. Trace or sporadic toxin components were STX, , , and dcSTX. Toxin component analysis from the middle to end of the study showed that -epimers (, ) were converted into -epimers (, ) and started to determine STX.

Construction of a recombinant intergenus multidomain chimeric protein for simultaneous expression of haemolysin BL of Bacillus cereus, listeriolysin O of Listeria monocytogenes and enterotoxin B of Staphylococcus aureus

Haemolysin BL (HBL) of Bacillus cereus, listeriolysin O (LLO) of Listeria monocytogenes and enterotoxin B (SEB) of Staphylococcus aureus are among the major toxin components contributing to the pathogenicity of these organisms in foodborne illnesses. In this study, an intergenus non-toxic multidomain fusion protein (r-HLE) was generated with specificity for HBL, LLO and SEB. The fusion gene (r-hle) comprising the conserved regions of hblD and the hly and entB genes was codon-optimized for expression in Escherichia coli and encoded a 50 kDa recombinant multidomain chimeric protein (r-HLE). Hyperimmune antiserum raised against r-HLE specifically reacted with the L1 (38 kDa) component of the HBL complex of B. cereus, LLO (58 kDa) of L. monocytogenes and SEB (28 kDa) of S. aureus during Western blot analysis when tested on standard strains. During testing on isolates, the antiserum again identified the appropriate toxin molecules and was highly specific to the relevant bacterial species. The antigenicity of the SEB component of the r-HLE protein was also confirmed using a commercially available TECRA kit. The described procedure of creating a single antigenic molecule carrying components of three different toxins whilst still retaining the original antigenic determinants of individual toxins will be highly advantageous in the development of rapid, reliable and cost-effective immunoassays.

A catalog for transcripts in the venom gland of the Agkistrodon acutus: Identification of the toxins potentially involved in coagulopathy

Agkistrodon acutus is a special agkistrodon halys, only distributed in Southern China, with a few exceptions in Vietnam. It is a cherished element used in traditional Chinese medicine. In order to produce a global panorama of gene expression in the Agkistrodon acutus venom gland, a non-normalized cDNA library was constructed, and 8696 high quality 5′ end expressed sequenced tags (ESTs) were sequenced and analyzed. The initial sequences were assembled into 2855 clusters. Of these clusters, only 45.60% clusters matched known sequence and 54.40% had no match to any known sequence in GenBank. Except for putative cellular proteins (1184 clusters), the remaining 118 clusters (40.16% of all ESTs) corresponded to sequences associated with diverse toxin function. According to expression abundance, the major toxin components were metalloproteinases (32.08%) and C-type lectin (5.22%), and other components including bradykinin-potentiating peptide (0.90%), serine proteases (0.51%), nucleotidase and nuclease (0.41%), phospholipase A2 (0.30%), disintegrin (0.05%), cytokine-like molecules (0.06%), and other proteins (0.63%). The majority of these components are thought to be responsible for coagulopathy after A. acutus bites. We have therefore generated a comprehensive catalog of the A. acutus venom gland described so far. Gene expression from the very specialized secretory tissue, especially for those involved in coagulopathy, can be surveyed and provide important information in finding novel toxins.

Strong myotoxic activity of Trimeresurus malabaricus venom: Role of metalloproteases

Trimeresurus malabaricus is an endemic snake found in the Southern region of Western Ghats section of India along with the more widely distributed species like Naja naja and Daboia russelii. T. malabaricus venom is not lethal when injected (i.p.) up to 20 mg/kg body weight in mice, but causes extensive local tissue degeneration. N. naja and D. russelii are highly toxic (i.p.) with minimum local tissue damage in experimental mice. In this study a comparative analysis of local tissue damage of T. malabaricus venom is made with N. naja and D. russelii snake venoms of the Southern regions of Western Ghats. T. malabaricus venom exhibits caseinolytic activity 16 and 24 times more than N. naja and D. russelii venom. Inhibition studies with specific protease inhibitors reveal that the major proteases belong to metalloproteases. T. malabaricus venom hydrolyses gelatin and induces strong hemorrhagic activity in mice. Both N. naja and D. russelii fail to hydrolyze gelatin even at very high concentration and did not induce any hemorrhagic activity. With D. russelii venom small hemorrhagic spot was observed at the site of injection. The hemorrhagic activity of T. malabaricus venom is completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor. The i.m. injection of T. malabaricus venom causes extensive degradation of muscle tissue within 24 h. The light microscopic observation of muscle tissue showed congestion of blood vessels and hemorrhage at the early stage followed by extensive necrosis of muscle fibers. The elevated levels of serum CK and LDH activity further supported the muscle degeneration. Such pathological symptoms were not seen with N. naja and D. russelii snake venom. The hemorrhagic and the muscle necrosis was completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor strongly suggests that the major toxin component in the T. malabaricus venom is metalloprotease and its activity can be easily neutralized using chelating agents and its use in the first aid as chelation therapy is beneficial.

Production of isodomoic acids A and B as major toxin components of a pennate diatom Nitzschia navis-varingica

In order to obtain more detailed information on the distribution of domoic acid-producing Nitzschia in Asian waters, Nitzschia-like diatoms were isolated primarily from the Philippines and established in culture for analysis by HPLC with fluorescence detection. Out of 58 isolates, 35 Nitzschia-like diatoms from the estuary areas of San Pedro Bay, Tacloban City and Manila Bay showed significant levels of domoic acid production (1.3–5.6 pg/cell). These were identified as Nitzschia navis-varingica. Two isolates from the same locality did not produce domoic acid. Of the 21 isolates from Bulacan Estuary, Manila Bay, none produced domoic acid. They did, however, produce two substances that seemed to be domoic acid derivatives. One of the strains was mass cultured and the substances were extracted, purified and analyzed by LC-MS/MS, proton and 13C NMR, and UV spectra. The produced substances were determined as isodomoic acids A and B. This is the first report of a diatom that produces isodomoic acids A and B as major toxin components.

Simultaneous production of homoanatoxin-a, anatoxin-a, and a new non-toxic 4-hydroxyhomoanatoxin-a by the cyanobacterium Raphidiopsis mediterranea Skuja

A neurotoxin, homoanatoxin-a, was identified from a toxic strain of the cyanobacterium Raphidiopsis mediterranea Skuja (strain LBRI 48) isolated from Lake Biwa, Japan, as the major toxin component (0.57% of dry cell-weight). This cyanobacterium produced anatoxin-a and a new homoanatoxin-a derivative as minor components (0.04 and 0.06%, respectively). The structure of a new compound was assigned based on the spectral data as 4-hydroxyhomoanatoxin-a, which was not toxic to mice up to 2.0 mg/kg by intraperitoneal injection. The isolation of minor components was accomplished by improved extraction and separation procedures: (1) extraction with methanol–water (4:1) from dried cells, (2) adsorption of aqueous residue on ODS column (or cartridge) after evaporation of methanol, (3) cleaning up of the column by successive elution with water and 50% methanol/water, (4) elution of a toxic fraction by 20% methanol/water containing 0.1% TFA and (5) HPLC (ODS) purification with methanol/water containing 0.05% TFA. The procedures were effective in removing impurities and concentrating alkaloidal neurotoxins. It should be noted that this is the first report demonstrating the simultaneous production of anatoxin-a and homoanatoxin-a by the same strain of cyanobacterium.

Variety of PSP toxin profiles in various culture strains of Alexandrium tamarense and change of toxin profile in natural A. tamarense population

Paralytic shellfish poisoning (PSP) toxin profiles were compared between clonal and axenic culture strains of Alexandrium tamarense prepared from cysts. The cysts were collected from two stations in northern Japan. The major toxin components of A. tamarense were C2 and GTX4, however, the proportions of C2 and GTX4 varied largely 0.7–78.8 mol% and 79.4–8.5 mol%, respectively. Some culture strains contained significantly higher proportion of neoSTX than other strains. These results indicate that strains with various toxin profiles exist in the same region, and suggest that the comparison of the toxin profiles between strains at different localities is considerably difficult. A drastic change of the toxin profile was observed in natural planktonic populations containing A. tamarense. This may be explained by the presence of a lot of planktonic populations with various toxin profiles growing around the sea area.

Frequent occurrence of paralytic shellfish poisoning toxins as dominant toxins in marine puffer from tropical water

Considerably high toxicity was detected in marine puffers collected from Masinloc Bay, Philippines. The toxicity was detected in the liver, intestine, muscle and skin. Noteworthy, the specimens, the muscle of which showed high toxicity, appeared in high frequency, indicating that puffers from this area is not safe for human consumption. These puffer specimens contained paralytic shellfish poisoning (PSP) toxins, often as major toxin components, the profile of which was similar to that of freshwater puffers reported from tropical areas. These results indicate that PSP toxins are common in tropical puffers both from marine and freshwater.

Variants of Shiga-like toxin II constitute a major toxin component in Escherichia coli O157 strains from patients with haemolytic uraemic syndrome.

The prevalence and genotype of Shiga-like toxins (SLTs) in Escherichia coli (O)157 strains from patients in Germany with haemolytic uraemic syndrome (HUS) were investigated. This was done by PCR amplification of the B-subunit genes with two primer pairs--one complementary to slt-IB, and the other homologous to both slt-IIB and slt-IIvB sequences. To distinguish between slt-II and slt-IIv, the amplified DNA was digested with restriction endonucleases HaeIII and FokI. Of the 38 strains examined, 17 harboured sequences for slt-IIv; four contained only slt-IIv, three carried both slt-IIv and slt-I, and 10 strains had slt-IIv and slt-II. A further three genotypes (slt-I, slt-II, slt-I/slt-II) were found in the remaining 21 strains resulting in a total of six slt genotypes. To determine whether the slt genes were expressed, and whether genotypes correlated with phenotypes, all strains were subjected to cytotoxicity assays and colony ELISA. All 38 strains displayed cytotoxic activity to Vero cells in similar quantities. The SLT-I-specific monoclonal antibody (MAb)13C4 reacted with all 10 strains in which slt-I sequences were identified. Colony blot ELISA with the SLT-II specific MAb11E10 detected 27 of 28 strains with slt-II sequences, but did not react with any of the seven strains that carried slt-IIv, or slt-I and slt-IIv. The high SLT variability shown here has diagnostic implications and may well have consequences for the host response in infections associated with these pathogens.

Use of HPLC to demonstrate variation of venom toxin composition in the Thailand cobra venoms Naja naja kaouthia and Naja naja siamensis

The composition of the venoms of Naja naja kaouthia and Naja naja siamensis from different commercial sources has been investigated using both ion-exchange and reverse-phase high-pressure liquid chromatography (RP-HPLC) in order to investigate variation in toxin contents. The venoms contained identical major toxin components, although in different relative concentrations. The venom collected separately from the left and right glands of individual snakes were virtually the same as judged by RP-HPLC. The cytotoxin CT-II, which was previously only reported to be present in Naja naja siamensis venom, was detected in all the venoms investigated. Two long neurotoxin homologues have also been isolated.

レンサ球菌の発赤毒素について

Separation and identification of erythrogenic toxin produced by group A hemolytic streptococci, which included strain NY 5, T 18 and five isolated strains from scarlet fever patients, were carried out by DEAE-Sephadex column equilibrated to 0.05 M Tris-HCl buffer (pH 8.6). Linear gradient elution was carried out by 0.05M Tris-HCl buffer (pH 8.6) containing 0.4M NaCl.A and B toxin produced by strain NY 5 was respectively eluted from the column by 0.14M and 0M NaCl, and C toxin produced by strain T 17 was eluted by 0.07M NaCl. Concentrated culture filtrates of five isolated strains were chromatographed with this procedure and separated toxin fractions were identified serologically. In this procedure the eluate by 0 M, about 0.08M, and about 0.15M NaCl was respectively neutralized by anti-B, abti-C, and anti-A rabbit serum. Furthermore, unknown toxin type eluted by 0.25M NaCl was recognized in one of isolated strains. From these results, NaCl concentration in the eluted toxin fraction is usable for presumptive identification of streptococcal erythrogenic toxin type. All of these isolated strains produced C toxin and it was major toxin component, and except for one strain they produced one or more other minor toxin type.