Academic literature on the topic 'Diarrhetic shellfish poisoning'

Abstract Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.

Pectenotoxins (PTXs) are a group of large cyclic polyether compounds associated with diarrhetic shellfish poisoning (DSP) as they are often found in combination with other DSPs such as okadaic acid (OA) and dinophysis toxins (DTXs) in shellfish. Although classified and regulated with the DSPs, there is debate over whether these toxins should be classified with DSP toxins. To date, ten different analogues of PTXs have been identified from shellfish and algae, and of these, the pectenotoxin-2 seco acids (PTX2-SAs) are of particular interest as they have previously been implicated in a shellfish poisoning incident in Australia, but relatively little was known of their toxicology. One such incident occurred in December 1997, when approximately 200 people were reported with severe diarrhoetic shellfish poisoning in Northern New South Wales (NSW). Analysis of the shellfish associated with this incident revealed relatively high PTX2-SA concentrations (approx. 300 micrograms/kg shellfish meat), with only trace amounts of pectenotoxin-2 (PTX2) and OA. Following this incident, PTX2-SAs were considered a health threat and guidelines were implemented in the absence of toxicological data, which has caused a great economic burden to shellfish industries around the globe, in particular to Australia, New Zealand and Ireland. Such regulation created in the absence of scientific data demonstrated the need to determine the toxicology of PTX2-SAs in commercial shellfish. Thus a comprehensive study on the toxicology and possible health implications of the PTX2-SAs in Australian shellfish was conducted. PTX2-SAs were isolated in different batches from shellfish (pipis, oysters and mussels) and from algal bloom samples of Dinophysis caudata. Toxin extraction was conducted with several purification stages and chemical analysis was performed with high-performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS). The chemical stability of the PTX2-SAs was investigated to ensure consistency of doses between toxicology experiments. Acute dosing studies with mice were then performed and included toxicopathology investigations with light microscopy and electron microscopy, in addition to toxin distribution studies and investigation of in vivo lipid peroxidation. In vitro studies with HepG2 cells included cytotoxicity assays, cell cycle investigations using flow cytometry and gene expression profiling of cells exposed to PTX2-SAs employing cDNA microarray technology. Acute pathology studies demonstrated that the PTX2-SAs do not cause the characteristic symptoms or lesions associated with DSP toxins. No diarrhoea was observed at any dose level in mice and no deaths occurred up to the maximum dosing level of 1.6mg/kg PTX2-SA. Only one batch of PTX2-SA extract produced toxic lesions characteristic of a DSP toxin (batch 1-pilot study) but after follow up studies, it was determined that this first batch of shellfish most likely contained an additional unidentified shellfish toxin or contaminant that co-extracted with PTX2-SAs during toxin isolation and purification procedures. This finding highlighted the importance of supporting the inclusion of the mice bioassay in procedures for shellfish toxin testing to enable detection of new toxins, and also highlighted the importance of toxin purification for toxicology studies. A significant rise in malondialdehyde excretion was observed within 24 hours of dosing mice, indicating that the PTX2-SAs may cause damage by lipid peroxidation in vivo. In vitro studies showed HepG2 cells to have cell cycle and gene expression changes within 24 hours of a dose of 800ng/mL PTX2-SAs. Cell cycle arrest was observed at the G2/M checkpoint and gene expression changes included alterations in genes involved in cell cycle control, lipid metabolism and transport, lipid genesis and trace metal transport. Many genes involved in DNA repair processes were moderated at the 24 hour point, but as no apoptosis was observed up to 72 hours post dosing it is a promising indication that any DNA damage that may have been caused by the administration of PTX2-SAs was not lethal, and was able to be repaired. In light of the information provided by toxicology investigations in this PhD, with particular reference to evidence of in vivo lipid peroxidation by raised levels of MDA in mouse urine, and changes in cell cycle distribution and gene expression in a cultured human cell line, it is concluded that there is potential for these toxins to induce biological changes in mammalian cells in vivo and in vitro, and hence potential for PTX2-SAs to cause health effects in humans. During the course of this three-year study, developments in techniques for shellfish toxin identification within our laboratories have revealed that the shellfish responsible for the 1997 NSW poisoning incident contained significant concentrations of okadaic acid acyl esters that were not detected at the time of the NSW incident. Although reportedly less toxic than okadaic acid itself, the OA ester concentrations present may have been sufficient to cause the observed symptoms. It is also theorized that these esters could be hydrolyzed in the human gastro-intestinal tract to release okadaic acid. In the light of this new evidence and with no pathology lesions or symptoms of diarrhoea being observed in PTX2-SA dosing studies with mice, we now believe these OA acyl esters to be the causative agent in the 1997 NSW DSP incident and not the PTX2-SAs. Nothing is currently known of the chronic toxicology of PTX2-SAs and thus their potential implications to public health in the long term cannot determined. The toxicology investigations in this thesis were acute studies, and it has not been established if the observed changes could be repaired or returned within normal limits without the manifestation of illness or disease occurring. Utilizing the acute toxicology information in this thesis, a health risk assessment for consumption of PTX2-SA contaminated shellfish was performed. This risk assessment, employing numerous safety factors essential for an incomplete data set, produced guideline values that are lower than the current recommend concentrations. To date, there has been no solid evidence that PTX2-SAs cause illness in humans – all documented incidents involving the PTX2-SAs have also included other DSP contaminants that are known to cause human illness. Pathology has not unequivocally been demonstrated in animal studies and thus, in consideration of the epidemiological evidence, PTX2-SAs cannot be considered as high a risk to public health as was previously thought. For the reasons discussed above, and weighing up risk-benefit considerations of the economic burden the current guideline values are causing to shellfish industries around the globe, it is recommended that levels of PTX2-SAs be monitored in recognition of the precautionary principle, but no longer regulated as tightly with other DSPs until such a time that toxicological or epidemiological evidence can prove that the PTX2-SAs are a DSP and are a more considerable threat to human health than has been indicated by toxicology studies in this thesis. This study has produced a substantial amount of acute toxicology data and has provided a good basis for future chronic toxicology investigations with the PTX2-SAs for regulatory purposes.

Pectenotoxins (PTXs) are a group of large cyclic polyether compounds associated with diarrhetic shellfish poisoning (DSP) as they are often found in combination with other DSPs such as okadaic acid (OA) and dinophysis toxins (DTXs) in shellfish. Although classified and regulated with the DSPs, there is debate over whether these toxins should be classified with DSP toxins. To date, ten different analogues of PTXs have been identified from shellfish and algae, and of these, the pectenotoxin-2 seco acids (PTX2-SAs) are of particular interest as they have previously been implicated in a shellfish poisoning incident in Australia, but relatively little was known of their toxicology. One such incident occurred in December 1997, when approximately 200 people were reported with severe diarrhoetic shellfish poisoning in Northern New South Wales (NSW). Analysis of the shellfish associated with this incident revealed relatively high PTX2-SA concentrations (approx. 300 micrograms/kg shellfish meat), with only trace amounts of pectenotoxin-2 (PTX2) and OA. Following this incident, PTX2-SAs were considered a health threat and guidelines were implemented in the absence of toxicological data, which has caused a great economic burden to shellfish industries around the globe, in particular to Australia, New Zealand and Ireland. Such regulation created in the absence of scientific data demonstrated the need to determine the toxicology of PTX2-SAs in commercial shellfish. Thus a comprehensive study on the toxicology and possible health implications of the PTX2-SAs in Australian shellfish was conducted. PTX2-SAs were isolated in different batches from shellfish (pipis, oysters and mussels) and from algal bloom samples of Dinophysis caudata. Toxin extraction was conducted with several purification stages and chemical analysis was performed with high-performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS). The chemical stability of the PTX2-SAs was investigated to ensure consistency of doses between toxicology experiments. Acute dosing studies with mice were then performed and included toxicopathology investigations with light microscopy and electron microscopy, in addition to toxin distribution studies and investigation of in vivo lipid peroxidation. In vitro studies with HepG2 cells included cytotoxicity assays, cell cycle investigations using flow cytometry and gene expression profiling of cells exposed to PTX2-SAs employing cDNA microarray technology. Acute pathology studies demonstrated that the PTX2-SAs do not cause the characteristic symptoms or lesions associated with DSP toxins. No diarrhoea was observed at any dose level in mice and no deaths occurred up to the maximum dosing level of 1.6mg/kg PTX2-SA. Only one batch of PTX2-SA extract produced toxic lesions characteristic of a DSP toxin (batch 1-pilot study) but after follow up studies, it was determined that this first batch of shellfish most likely contained an additional unidentified shellfish toxin or contaminant that co-extracted with PTX2-SAs during toxin isolation and purification procedures. This finding highlighted the importance of supporting the inclusion of the mice bioassay in procedures for shellfish toxin testing to enable detection of new toxins, and also highlighted the importance of toxin purification for toxicology studies. A significant rise in malondialdehyde excretion was observed within 24 hours of dosing mice, indicating that the PTX2-SAs may cause damage by lipid peroxidation in vivo. In vitro studies showed HepG2 cells to have cell cycle and gene expression changes within 24 hours of a dose of 800ng/mL PTX2-SAs. Cell cycle arrest was observed at the G2/M checkpoint and gene expression changes included alterations in genes involved in cell cycle control, lipid metabolism and transport, lipid genesis and trace metal transport. Many genes involved in DNA repair processes were moderated at the 24 hour point, but as no apoptosis was observed up to 72 hours post dosing it is a promising indication that any DNA damage that may have been caused by the administration of PTX2-SAs was not lethal, and was able to be repaired. In light of the information provided by toxicology investigations in this PhD, with particular reference to evidence of in vivo lipid peroxidation by raised levels of MDA in mouse urine, and changes in cell cycle distribution and gene expression in a cultured human cell line, it is concluded that there is potential for these toxins to induce biological changes in mammalian cells in vivo and in vitro, and hence potential for PTX2-SAs to cause health effects in humans. During the course of this three-year study, developments in techniques for shellfish toxin identification within our laboratories have revealed that the shellfish responsible for the 1997 NSW poisoning incident contained significant concentrations of okadaic acid acyl esters that were not detected at the time of the NSW incident. Although reportedly less toxic than okadaic acid itself, the OA ester concentrations present may have been sufficient to cause the observed symptoms. It is also theorized that these esters could be hydrolyzed in the human gastro-intestinal tract to release okadaic acid. In the light of this new evidence and with no pathology lesions or symptoms of diarrhoea being observed in PTX2-SA dosing studies with mice, we now believe these OA acyl esters to be the causative agent in the 1997 NSW DSP incident and not the PTX2-SAs. Nothing is currently known of the chronic toxicology of PTX2-SAs and thus their potential implications to public health in the long term cannot determined. The toxicology investigations in this thesis were acute studies, and it has not been established if the observed changes could be repaired or returned within normal limits without the manifestation of illness or disease occurring. Utilizing the acute toxicology information in this thesis, a health risk assessment for consumption of PTX2-SA contaminated shellfish was performed. This risk assessment, employing numerous safety factors essential for an incomplete data set, produced guideline values that are lower than the current recommend concentrations. To date, there has been no solid evidence that PTX2-SAs cause illness in humans – all documented incidents involving the PTX2-SAs have also included other DSP contaminants that are known to cause human illness. Pathology has not unequivocally been demonstrated in animal studies and thus, in consideration of the epidemiological evidence, PTX2-SAs cannot be considered as high a risk to public health as was previously thought. For the reasons discussed above, and weighing up risk-benefit considerations of the economic burden the current guideline values are causing to shellfish industries around the globe, it is recommended that levels of PTX2-SAs be monitored in recognition of the precautionary principle, but no longer regulated as tightly with other DSPs until such a time that toxicological or epidemiological evidence can prove that the PTX2-SAs are a DSP and are a more considerable threat to human health than has been indicated by toxicology studies in this thesis. This study has produced a substantial amount of acute toxicology data and has provided a good basis for future chronic toxicology investigations with the PTX2-SAs for regulatory purposes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Public Health
Faculty of Health Sciences
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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
The okadaic acid phycotoxin AO is produced by a group of micro seaweed known as Dinoflagellated. The mussels when feeding themselves from this micro seaweed accumulate in their hepatopancreas, this toxin, trigging in the human being the Syndrome or Diarrhetic Shellfish Poisoning (DSP). The symptoms appear at around 30 minutes after the consumption of the contaminated clam; the symptoms vary among abdominal nauseas, pains, vomits and diarrhea. When the toxin ingestion happens in amounts lower than 48 μg.g-1, the above described symptoms do not develop, however, its continued consumption favors the appearing of tumors in the gastrointestinal tract due to the high carcinogenic power of AO. This study intended to detect and quantify the diarrheic toxin AO in Perna perna mussels collected between the months of May and December of the 2006 and verification of the potentially toxic micro seaweed presence, in the seasons Spring/Summer (from September until December of 2006). Since May until december of 2006, mussels (Perna perna) were collected and analyzed regarding to the presence of the Phycotoxin Ao .In order to collect the micro seaweed were used a plankton net (20μm of mesh) and Bomb Rule 2000. The identification of the dinoflagellates ones was carried through in inverted biological microscope. The detection of AO in the mussels was carried through by High Efficiency of Liquid Chromatography with Fluorimetric Detection (HPLC-FC). The chromatographic results had indicated the presence of AO toxin in all the gotten mussel samples from May until October of 2006 in low concentrations. In the analyses of phytoplankton, the diatoms were the most representative group compared with the Dinoflagellated. The species Prorocentrum micans and P. gracile examined were not pointed as toxin producers until the moment. Among the Dino-flagellated potentially toxic were found the species : Dinophysis acuminata, D. tripos, D. rotundata and D. fortii. The results indicate the necessity of elaboration and effective application of a hygienic-sanitary controlling program of the clams as well as monitoring the environment, aiming above everything the public health Safety.
A ficotoxina ?cido ocadaico (AO) ? produzida por um grupo de microalgas conhecidas como dinoflagelados. Os mexilh?es ao se alimentarem destas microalgas acumulam em seu hepatop?ncreas, esta toxina, desencadeando no ser humano a S?ndrome ou Envenenamento Diarr?ico por Moluscos - EDM. Os sintomas se apresentam em torno de 30 minutos ap?s o consumo do molusco contaminado; variando entre n?useas, dores abdominais, v?mitos e diarr?ia. Quando a ingest?o da toxina acontece em quantidades inferiores a 48 μg.g-1, os sintomas acima descritos n?o se desenvolvem, por?m, seu consumo continuado favorece o surgimento de tumores no trato gastrointestinal devido ao poder carcinog?nico do AO. Este estudo pretendeu detectar e quantificar a toxina diarr?ica AO em mexilh?es Perna perna coletados entre os meses de maio e dezembro de 2006, e a verifica??o da presen?a de microalgas potencialmente t?xicas, nas esta??es primavera/ver?o (setembro a dezembro de 2006). De maio a dezembro de 2006, mexilh?es (Perna perna) foram coletados e analisados quanto ? presen?a da ficotoxina AO. As microalgas foram coletadas entre setembro e dezembro de 2006, com aux?lio de uma rede de pl?ncton (de 20μm de malha) e Bomba Rule 2000. A identifica??o dos dinoflagelados foi realizada em microsc?pio biol?gico invertido. A detec??o do AO nos mexilh?es foi realizada por Cromatografia L?quida de Alta Efici?ncia com Detec??o Fluorim?trica (CLAE-DF). Os resultados cromatogr?ficos indicaram a presen?a da toxina AO em todas as amostras obtidas de mexilh?es, de maio a outubro de 2006, em baixas concentra??es. Nas an?lises do fitopl?ncton, as diatom?ceas foram o grupo mais representativo comparado aos dinoflagelados. As esp?cies Prorocentrum micans e P. gracile observadas n?o foram apontadas como produtoras de toxinas at? o momento. Dentre os dinoflagelados potencialmente t?xicos foram encontradas as esp?cies: Dinophysis acuminata, D. tripos, D. rotundata e D. fortii. Os resultados indicam a necessidade da elabora??o e aplica??o efetiva de um programa de controle higi?nico-sanit?rio dos moluscos assim como monitoramento do ambiente, objetivando acima de tudo a seguran?a ? sa?de p?blica.

Dissertação de Mestrado em Segurança Alimentar
As biotoxinas marinhas mais frequentes e abundantes em Portugal são as toxinas lipofílicas, nomeadamente as toxinas do grupo do ácido ocadaico (AO), que inclui as dinofisistoxinas (DTX1 e DTX2) e os seus derivados (DTX3), responsáveis pela intoxicação diarreica (DSP diarrhetic shellfish poisoning). A investigação da presença, variabilidade e transformação de biotoxinas marinhas em moluscos bivalves é de elevada importância não só para os consumidores e produtores de moluscos bivalves, mas também para as entidades reguladoras em segurança alimentar. Até à data, a quantidade de toxinas ingeridas nos alimentos tem sido considerada igual à quantidade de toxinas disponível para a absorção pelo corpo humano após o processo digestivo. Neste estudo, avaliou-se através de um modelo de digestão in vitro estático, a fração de AO, DTX2 e das suas formas esterificadas (DTX3), libertada do alimento para os fluidos digestivos (bioacessibilidade) em amostras naturalmente contaminadas de mexilhão, berbigão, conquilha e lingueirão. As amostras de mexilhão e conquilha apresentam regularmente um perfil de toxinas composto por AO e DTX2 nas suas formas livres, enquanto que o berbigão e o lingueirão têm uma elevada capacidade de biotransformação, o que conduz a que o seu perfil de toxinas seja constituído maioritariamente por formas esterificadas (DTX3). Amostras colhidas na costa Portuguesa foram utilizadas para avaliar a bioacessibilidade destes compostos, quer na matriz crua, como após cozedura a vapor. A Bioacessibilidade do conteúdo total de toxinas variou entre as espécies analisadas. A maior percentagem de toxinas bioacessiveis foi encontrada no mexilhão (86 ± 4 %), seguido da conquilha (74 ± 10 %), do lingueirão (71 ± 10 %) e, por último, do berbigão (59 ± 10 %). As toxinas que não se libertaram da matriz alimentar foram detetadas na fração nãobioacessivel, correspondente à fração excretada. Nas amostras de mexilhão e conquilha determinou-se mais AO na fração bioacessivel do que o presente na matriz alimentar antes da digestão. Não podendo haver mais AO do que aquele que foi ingerido, e tendo sido observado paralelamente um valor muito reduzido de AO esterificado na fração bioacessivel, sugere-se uma conversão dos compostos esterificados no composto parental durante o processo digestivo. A conversão no trato digestivo dos compostos menos tóxicos (DTX3) nos seus compostos parentais mais potentes, vem justificar os casos de intoxicação após o consumo de bivalves contendo um perfil de toxinas dominado por DTX3. No entanto, nas amostras de berbigão e lingueirão não foi observada a conversão das formas esterificadas de AO, sugerindo um perfil de formas esterificadas diferente do mexilhão e conquilha. O tratamento a vapor foi avaliado nas amostras de mexilhão e berbigão, tendo-se observado um aumento da concentração das toxinas lipofílicas, mas uma redução da bioacessibilidade dos compostos face às matrizes cruas, sendo de destacar que a bioacessibilidade estimada para as amostras de berbigão foi interior a 50%. Este estudo fornece novos dados relevantes que podem melhorar e levar a estudos de avaliação de risco em segurança alimentar mais precisos sobre estas toxinas. A avaliação de risco com base exclusivamente na ocorrência das toxinas DSP em moluscos bivalves pode conduzir a uma sobrestimação da exposição e levar a medidas regulamentares mais conservadoras do que tendo em conta a quantidade de toxinas que podem ser absorvidas pelos epitélios intestinais.
ABSTRACT - In vitro bioaccessibility of the marine biotoxins okadaic acid, dinophysistoxin-2 and their derivatives in raw and steamed shellfish - The most common and abundant biotoxins in Portugal are lipophilic toxins, including okadaic acid group (OA) toxins, which includes the dinophysistoxins (DTX1 and DTX2) and its derivatives (DTX3), responsible for the human food borne illness diarrhetic shellfish poisoning (DSP). Investigating the presence, variability and processing of marine biotoxins in bivalve molluscs is highly important not only for consumers and shellfish producers but also for governmental agencies with responsibilities on food safety. To date, the amount of toxins ingested in the food has been considered equal to the amount of toxins available for uptake by the human body after the digestive process. In this study, we evaluated through a static in vitro digestion model, the fraction of OA, DTX2 and DTX3 released from the food to the digestive fluids (bioaccessibility) in naturally contaminated mussels, donax clams, cockles, and razor clams. Bioaccessibility was assessed in both raw and steamed shellfish matrices. Higher bioaccessibilty was estimated for mussels (86 ± 4 %), followed by donax clams (74 ± 10 %), razor clams (71 ± 10 %) and finally cockles (59 ± 10 %). The amount of toxins not released into the digestive juices were detected in the non-bioaccessible fraction. In mussels and donax clams higher levels of OA were determined in the digestive juices than in the food matrix. Parallel to higher amounts of OA in bioaccessible fraction was observed a reduction of its esterified forms (DTX3), suggesting that DTX3 are converted into the parental compounds during the digestion process. This gastrointestinal conversion of less potent toxins into their more toxic parental compounds can be pointed out as a reason for human food poisonings after ingestion of contaminated shellfish containing mostly DTX3, as was previously observed in Portugal. However, conversion of DTX3 into OA was not observed in cockles and razor clams, suggesting that the suite of esterified compounds in these species is different from mussels and donax clams. Steaming of shellfish matrices, simulating a cooking treatment, lead to an increase of toxins concentration but significantly reduced their bioaccessibility, which was particular evident for cockles having the release of toxins been estimated by this in vitro model to levels below 50%. This study provides relevant new data that can improve and lead to more accurate food safety risk assessment studies concerning these toxins. Risk assessment based solely on DSP toxins occurrence in seafood can conduct to an overestimation of the exposure and lead to regulatory measures more conservative than taking into account the amount of toxins that can be absorbed by the intestinal epithelia.
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