Paralytic shellfish poison (PSP) profiles and toxification of short-necked clams fed with the toxic dinoflagellate Alexandrium tamarense

As a part of our studies on paralytic shellfish poison (PSP) accumulation kinetics in bivalves, short-necked clam Tapes japonia was experimentally contaminated with PSP by being fed with the toxic dinoflagellate Alexandrium tamarense for 2, 4, 6, 8 and 10 days, and the processes of PSP accumulation and bioconversion were investigated: the toxicity level was determined by mouse bioassay and toxin components were identified by high-performance liquid chromatography (HPLC). The strain of A. tamarense used in this study possessed a specific toxicity of 186.7 +/- 81 (mean +/- S.D., n = 5) x 10(-6) MU/cell. Total toxin concentration of this strain was 140.4 +/- 61 (mean S.D., n = 5) fmol/cell. The toxicity level of short-necked clams increased almost in parallel with the abundance of A. tamarense, reaching 1.8, 3.2, 3.8, 3.5 and 4.6 MU/g meat for 2, 4, 6, 8 and 10 days of feeding, respectively. The accumulation rates of PSP toxins, which are the ratio of the total amount of toxins accumulated in the bivalves to the estimated intake in each feeding experiment, were 7.5, 8.1, 5.7, 4.2 and 4.4% for 2, 4, 6, 8 and 10 days, respectively. At the end of each exposure period, many undigested algal cells were found in pseudofeces under microscopic observation. There was a remarkable difference in the relative proportions of the predominant toxin components between A. tamarense and short-necked clams. The most notable difference was the change in the relative amounts of C2 (carbamoyl-N-sulfo-11beta-hydroxysaxitoxin sulfate), GTX1 and GTX 4 during the first two days. In the toxic bivalves, the amount of C2, which is dominant in A. tamarense, decreased to below half a percent after being ingested. Subsequently, the amount of GTX1 in the shellfish meat reached 50.1 mol%, while that of GTX4 decreased to about half of that in A. tamarense. As for the configuration of 11-hydroxysulfate, PSP components in A. tamarense exist almost exclusively as beta-epimers (GTX3, GTX4, C2 and C4), accounting for 72.8 mol% of the total. This contrasts with the case of the short-necked clams, where the beta-epimers represented 25.8, 33.8, 30.8, 36.8 and 28.5 mol% of the total after 2, 4, 6, 8 and 10 days, respectively. PSP components seemed to be converted rapidly at an early stage of the feeding of A. tamarense.     

Accumulation of paralytic shellfish poison (PSP) and biotransformation of its components in oysters, Crassostrea gigas, fed with the toxic dinoflagellate Alexandrium tamarense

As a part of our studies on the mechanism of uptake of paralytic shellfish poison (PSP) and the kinetics of its accumulation in bivalves, oysters Crassostrea gigas were experimentally contaminated with PSP by being fed with the toxic dinoflagellate Alexandrium tamarense for 2, 4, 6, 8 and 10 days. Temporal variations in the PSP contents and their profiles in oysters during the feeding experiment were monitored by high-performance liquid chromatography (HPLC) and the toxin profile of the oysters was compared with that of A. tamarense. Toxins excreted from the infested oysters into the seawater for 2 and 10 days were recovered and analyzed by HPLC. PSP toxicity rapidly appeared in the tissues of oysters and their toxicity levels reached 0.6 (0.3), 2.2 (1.1), 1.0 (0.5), 3.4 (1.6) and 1.1 (0.5) MU/g (nmol/g) shucked meat at 2, 4, 6, 8 and 10 days, respectively. The accumulation rates of toxin, calculated from the total amount (nmol) of toxins expressed by the total cell number fed during the exposure period and the toxicity of the oysters, were 14.1, 18.7, 5.1, 14.9 and 3.2% for 2, 4, 6, 8 and 10 days. During feeding experiments, the toxin profile of oysters changed substantially, showing marked differences from the proportions found in the toxigenic dinoflagellate used as food. The toxin components in this strain existed almost exclusively as beta-epimers, which accounted for 66.3 mol% of the total. This contrasts with the case of the oysters, where the beta-epimers represented 24.8, 29.8, 25.1, 27.3 and 25.2 mol% of the total at 2, 4, 6, 8 and 10 days, respectively. The amount of gonyautoxin-1 (GTX1) accumulated in oysters increased linearly and slowly for 8 days and the maximum content of GTX1 reached 51.3 mol%. The composition of GTX group compounds recovered from the seawater in which the oysters had been reared was a little different from that within the oyster tissues.     

Matrix effect on paralytic shellfish toxins quantification and toxicity estimation in mussels exposed to Gymnodinium catenatum

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2?+?3, GTX2?+?3 and GTX1?+?4, while an enhancement was found for C1?+?2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1?+?2 (median?=?421?ng?g^?1) exceeded largely the values for the other quantified compounds (0.09–58?ng?g^?1). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1?+?2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218?µg STX equivalents kg^?1. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200–800?µg STX equivalents kg^?1 an increase was found between 44 and 28%.     

Matrix effect on paralytic shellfish toxins quantification and toxicity estimation in mussels exposed to Gymnodinium catenatum

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2 + 3, GTX2 + 3 and GTX1 + 4, while an enhancement was found for C1 + 2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1 + 2 (median = 421 ng g?1) exceeded largely the values for the other quantified compounds (0.09-58 ng g?1). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1 + 2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218 μg STX equivalents kg?1. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200-800 μg STX equivalents kg?1 an increase was found between 44 and 28%.     

First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.     

麻痹性贝类毒素受体蛋白Saxiphilin的克隆与表达

目的麻痹性贝类毒素受体蛋白Saxiphilin可特异性结合麻痹性贝类毒素,本文采用杆状病毒表达载体系统对Saxiphilin进行体外表达研究。方法从牛蛙肝脏克隆得到ssziphilin基因(N端含有自身分泌信号肽),利用特异引物使其C端带上组氨酸标签。结果使用杆状病毒表达载体系统构建带有目的基因的重组病毒,用病毒感染草地贪夜蛾(Spodoptera frugiperda)细胞Sf9以表达Saxiphilin,通过优化细胞培养基中胎牛血清含量及感染时间,确定使用无血清培养基培养,重组病毒感染Sf9细胞72 h时,培养基中可溶性目的蛋白表达量最大。结论通过镍柱纯化得到Saxiphilin蛋白,以期将此蛋白进一步用于麻痹性贝类毒素的检测。     

上海市售贝类产品中麻痹性贝类毒素污染状况调查及其评价

目的 调查上海市售贝类产品中麻痹性贝类毒素污染状况。方法 2010年8月~2011年7月间, 在上海水产品批发市场进行5种贝类样品采集, 每月抽取样品24份, 全年共288份。采用生物法(SC/T 3023-2004)对其进行了麻痹性贝类毒素的检测, 其中虾夷扇贝的肠腺和肌肉(扇贝柱)进行分开测定。结果 缢蛏、菲律宾蛤仔、牡蛎、文蛤、虾夷扇贝肠腺和肌肉中麻痹性贝类毒素的含量范围分别为ND~121.5 MU/100 g、ND~113.4 MU/100 g、ND~177.7 MU/100 g、ND~124.6 MU/100 g、261.7~3363.5 MU/100 g和ND。全年麻痹性贝类毒素的平均含量分别在98.5±10.5 MU/100 g、78.6±9.3 MU/100 g、50.4±10.1 MU/100 g、40.6±14.8 MU/100 g、1242.2±974.3 MU/100 g和0。按照目前我国贝类产品主要出口国家和国际组织对麻痹性贝类毒素的限量要求进行评价, 仅仅是虾夷扇贝肠腺中麻痹性贝类毒素超标, 超标率为98%, 因此在食用扇贝时应去除其肠腺; 而其余贝类产品中麻痹性贝类毒素均在限量规定范围内。结论 上海市售贝类产品对食用的安全性不产生负面影响。     

Occurrence of paralytic shellfish poison (PSP)-producing dinoflagellate Alexandrium tamarense in Hiroshima Bay, Hiroshima Prefecture, Japan, during 1993-2004 and its PSP profiles

To assess levels of shellfish intoxication by the paralytic shellfish poison (PSP)-producing dinoflagellate Alexandrium tamarense, potential health risks to human shellfish consumers and the possible need for regulatory intervention, yearly variations of maximum cell density of this species were examined from 1993 to 2004 in Kure Bay and Kaita Bay, which are located within Hiroshima Bay, Hiroshima Prefecture, Japan. The seawater temperature was determined concomitantly. In Kure Bay, maximum concentrations of 1,400 and 1,300 cells/mL at 0 and 5 m depths were observed on 21 and 24 April 1997. In Kaita Bay, remarkably high concentrations above 1,000 cells/mL of A. tamarense were observed in two out of three years investigated. These facts suggest that the environment in both bays is favorable for the propagation of A. tamarense. The temperature range at which the natural population of A. tamarense blooms was generally from 12 to 16 degrees C. Four strains (ATKR-94, -95, -97 and -01) from Kure Bay and one strain (ATKT-97) from Kaita Bay were established. The strain ATKR-94, cultured in modified SW-2 medium at 15 degrees C for 15 days, showed a specific toxicity of 33.8 x 10(-6) MU/cell. The toxins in all five strains exist almost exclusively as beta-epimers (C2 (PX2 or GTX8), GTX3, dcGTX3 and GTX4), which accounted for 54.9 to 73.0 mol% of the total. The corresponding a-epimers (C1 (PX1 or epi-GTX8), GTX2, dcGTX2 and GTX1) accounted for 6.0 to 28.9 mol%. The toxin profiles of ATKR-97 and ATKT-97 were characterized by unusually high proportions of low-potency sulfocarbamoyl toxin, which comprised 62.4 and 68.2 mol%, respectively, of total toxins. In the toxic bivalves, the low-toxicity sulfocarbamoyl components, major components of A. tamarense, were present in amounts of only a few percent, suggesting that in vivo conversion of PSP occurs after ingestion. A comparison of the toxin profiles of the causative dinoflagellate and contaminated bivalves showed that PSP components exist in the bivalves in the form of alpha-epimers, presumably owing to accumulation or storage of the toxins.     

2018—2020年河北省市售贝类中麻痹性贝类毒素污染状况调查分析

目的 了解2018—2020年河北省市售贝类中麻痹性贝类毒素(paralytic shellfish poison,PSP)污染状况.方法 2018年8月—2020年5月间,对河北省市售的7种双壳贝类,共508份进行检测分析.样品经0.5％乙酸水提取,石墨化碳黑固相萃取柱净化,采用高效液相色谱-串联质谱法进行检测.结果...     

舟山海域麻痹性贝类毒素污染情况及其2种检测方法比较

目的检测舟山东极与嵊泗枸杞2个海域养殖贻贝中的麻痹性贝类毒素(paralytic shellfish poison,PSP),比较小鼠生物测定法与酶联免疫分析法(ELISA)的测定结果。方法采用小鼠生物测定法与酶联免疫吸附法检测贝类中的麻痹性贝类毒素,并将2者的检测结果进行比较分析。结果 2种检测方法检测的麻痹性贝类毒素含量结果基本一致。5月份东极岛海域的厚壳贻贝中检出PSP((500±3.2)MU/100 g),超标率为5%;嵊泗枸杞海域贝类PSP含量较低,未超出安全食用标准。2个海域的紫贻贝PSP含量均未超出安全食用标准。结论小鼠生物法与ELISA方法的评价结果基本一致,其检测出的PSP结果可以为摄入PSP风险评估提供数据支撑。由于ELISA方法的检测成本较高,因此可采用小鼠生物法进行麻痹性贝类毒素风险监测。     

Analysis of paralytic shellfish toxins and their metabolites in shellfish from the North Yellow Sea of China

Samples of toxic scallop (Patinopecten yessoensis) and clam (Saxidomus purpuratus) collected on the northern coast of China from 2008 to 2009 were analysed. High-performance liquid chromatography with post-column oxidation and fluorescence detection was used to determine the profile of the main paralytic shellfish poisoning (PSP) toxins in these samples and their total toxicity. Hydrophilic interaction liquid ion chromatography with mass spectrometric detection confirmed the toxin profile and detected several metabolites in the shellfish. Results show that C1/2 toxins were the most dominant toxins in the scallop and clam samples. However, GTX1/4 and GTX2/3 were also present. M1 was the predominant metabolite in all the samples, but M3 and M5 were also identified, along with three previously unreported presumed metabolites, M6, M8 and M10. The results indicate that the biotransformation of toxins was species specific. It was concluded that the reductive enzyme in clams is more active than in scallops and that an enzyme in scallops is more apt to catalyse hydrolysis of both the sulfonate moiety at the N-sulfocabamoyl of C toxins and the 11-hydroxysulfate of C and GTX toxins to produce metabolites. This is the first report of new metabolites of PSP toxins in scallops and clams collected in China.     

Analysis of paralytic shellfish toxins and their metabolites in shellfish from the North Yellow Sea of China

Samples of toxic scallop (Patinopecten yessoensis) and clam (Saxidomus purpuratus) collected on the northern coast of China from 2008 to 2009 were analysed. High-performance liquid chromatography with post-column oxidation and fluorescence detection was used to determine the profile of the main paralytic shellfish poisoning (PSP) toxins in these samples and their total toxicity. Hydrophilic interaction liquid ion chromatography with mass spectrometric detection confirmed the toxin profile and detected several metabolites in the shellfish. Results show that C1/2 toxins were the most dominant toxins in the scallop and clam samples. However, GTX1/4 and GTX2/3 were also present. M1 was the predominant metabolite in all the samples, but M3 and M5 were also identified, along with three previously unreported presumed metabolites, M6, M8 and M10. The results indicate that the biotransformation of toxins was species specific. It was concluded that the reductive enzyme in clams is more active than in scallops and that an enzyme in scallops is more apt to catalyse hydrolysis of both the sulfonate moiety at the N-sulfocabamoyl of C toxins and the 11-hydroxysulfate of C and GTX toxins to produce metabolites. This is the first report of new metabolites of PSP toxins in scallops and clams collected in China.     

Assessment of the quantitative determination of paralytic shellfish poisoning toxins by pre-column derivatization and elimination of interfering compounds by solid-phase extraction

Monitoring programmes for paralytic shellfish poisoning toxins in bivalve molluscs still rely heavily on the use of mouse bioassays (MBA) for consumer protection. A high-performance liquid chromatography (HPLC) methodology (Lawrence method) was implemented in 1996 in the Portuguese monitoring programme as a complementary means of analysis. Comparison between MBA and HPLC was done at the time only by a qualitative approach due to the scarce number of positive samples tested. More quantitative data were obtained recently when studying toxin profiles in Moroccan shellfish, and the correlation found between these two methodologies is reported here for the first time. Two different matrices were studied: blue mussel and the giant cockle Acanthocardia tuberculatum. A good linear correlation was obtained for both matrices. However, a second-degree polynomial best fitted the data at both low and high extremes of toxicity. According to the HPLC quantitative results, 13% of false-negatives could be obtained by MBA due to an underestimation of toxicity near the limit of detection of the MBA. Difficulties on relying solely on HPLC for consumer protection have been aroused with uncommon matrices, such as imported clams or crustaceans, due to the presence of high concentrations of interfering compounds. The solid-phase extraction step of the Lawrence method was implemented to eliminate an unknown compound that could be mistaken for saxitoxin, and an 80% reduction of another common unknown compound eluting close to decarbamoylsaxitoxin. The implementation of the HPLC methodology achieved so far allows a high degree of consumer protection without the need to resource to animal sacrifice.     

Effects of atropine on plasma amino acid profiles and on the synthesis of individual milk proteins in dairy cows fed with pasture

Effects of atropine on blood plasma amino acid profile and on the yields and concentration of milk components were investigated in 12 Friesian cows in early lactation. Cows were housed indoors and fed with cut pasture ad libitum. Each cow received four treatments over 12 d during a replicated 4 x 4 Latin square experiment. Treatments were: control (saline); low dose (L; 30 microg atropine/kg body weight (BW)); medium dose (M; 40 microg atropine/kg BW); and 2 x L dose, 2 h apart (2 x L). On each of four treatment days, cows were milked at about 7.00, after which treatments were administered by subcutaneous injection. Cows were milked again at 2 h, 6 h and 10 h after injection. Milk samples were collected at each milking. Immediately after the 2 h milking, blood samples were drawn from each cow and the second injection was given for the 2 x L treatment. Atropine reduced hourly milk yield, and concentrations and hourly yields of total protein, casein, whey protein, alpha-casein, beta-casein, kappa-casein, beta-lactoglobulin and alpha-lactalbumin, but by differing amounts. Milk concentrations of bovine serum albumin and immunoglobulin G were increased by atropine, and overall yields of these proteins were mostly unchanged. Atropine lowered concentrations of most, but not all, amino acids in blood plasma, with essential amino acids reduced more than non-essential amino acids. Concentrations of alpha-amino N in whole blood, and glucose and insulin in blood plasma, fell after atropine injection. There was no difference between the L and M doses of atropine, but the 2 x L dose had greater effects on milk composition than the single doses. For yields of milk and milk components, the effect of the 2 x L dose was also more persistent. The results highlight the differential synthesis of individual milk proteins, and suggest that atropine might be useful for evaluating the mechanisms regulating milk protein composition.     

Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay

This study compared five methods of measuring paralytic shellfish toxins (PSTs) including the long-used mouse lethality bioassay, a commercially available cell culture test (MIST ® Quantification kit), HPLC analysis, and two newly developed radioreceptor assays utilizing mammalian sodium channels and saxiphilin. Methods were challenged with toxic shellfish extracts prepared according to the AOAC official method. The best correlations between predicted toxicity values being 0.9 or better, were those between HPLC analysis when compared with both radioreceptor assays and the mouse lethality bioassay, as well as that between the saxiphilin and the sodium channel radioreceptor assays. In all cases, statistically significant correlations existed between the toxicity measurements of the same extracts. The ratios between some methods were not unitary as measured by the slopes of the regression lines used for correlation analyses. HPLC analysis predicted more toxicity than all of the bioassays. The saxiphilin assay underestimated toxicity relative to the mouse bioassay, the MIST ® kit determinations and the sodium channel assay. The sodium channel assay predicted there to be less toxicity than the mouse bioassay and the MIST ® kit. Of all of the techniques used, the MIST ® kit correlation with the mouse bioassay was nearest to one. Each method possesses different virtues and it may be that a multi-method approach would harness the benefits of each method for various aspects of a shellfish testing regime.     

Lipophilic toxin profiles associated with diarrhetic shellfish poisoning in scallops, Patinopecten yessoensis, collected in Hokkaido and comparison of the quantitative results between LC/MS and mouse bioassay

Lipophilic toxins associated with diarrhetic shellfish poisoning (DSP) in scallops, Patinopecten yessoensis, collected in Hokkaido, Japan were quantified by liquid chromatography-mass spectrometry (LC/MS). Pectenotoxin-6 (PTX6) and yessotoxin (YTX) were the dominant toxins in the scallops, although the percentages of these toxins were different depending on the production area or the sampling period. The quantitative results obtained for the scallops in LC/MS and in mouse bioassay (MBA) were compared. Fifty of the 55 samples found to be exceeding the local quarantine level (0.025 MU/g whole meat) in Hokkaido by LC/MS were quantified by MBA as being below the quarantine level. It is suggested that this discrepancy is due to poor detection of YTX by MBA. These results indicate that LC/MS is a better method than MBA in terms of sensitivity and accuracy to quantify known lipophilic toxins, including YTX.     

Development and application of DNA markers to detect adulteration with Scopolia japonica in the medicinal herb Atractylodes lancea

Food Science and Biotechnology - Atractylodes lancea rhizomes are commonly consumed in east Asia as traditional medical herbs. However, in Korea, because of their morphological similarity, A....