Academic literature on the topic 'Dinophysistoxin'

Abstract Increasing incidences of phytoplankton blooms with the potential danger of toxin release into the food chain have necessitated the search for new diagnostic methods that can detect toxins quickly and reliably. A competitive enzymelinked immunosorbent assay (ELISA) was developed to quantitate okadaic acid in shellfish and phytoplankton extracts. To determine the specificity of the assay, a number of toxins, such as calyculin A, brevetoxin-1, and dinophysistoxins-1, -2, and -3 were analyzed. Both dinophysistoxins-2 and -1 could be detected by the assay but in concentration ranges 10- and 20-fold higher than that for okadaic acid, respectively. Dinophysistoxin-3, calyculin A, or brevetoxin-1 could not be detected with this assay. To validate the accuracy of the method, 18 mussel and 7 phytoplankton extracts were analyzed in parallel for okadaic acid content by ELISA and liquid chromatography combined with either fluorescence or mass spectrometric detection. Very high correlation between the results was found.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-β-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1–5, the 24-O-β-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4–6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

For the first time, the aptamer of dinophysistoxin-1 was successfully obtained with high affinity and specificity by SELEX, and an aptasensor with a detection range from 40 to 600 nM was developed by biolayer interferometry.

Toxins produced by harmful algal blooms (HABs) are known to pose contamination risks to seafood products (e.g. fish and shellfish) consumed by human. In order to control contamination risks, monitoring regimes have to be performed rigorously. The effort to monitor the amount of toxins in consumable products has to rely on continuous supply to analytical standards. The current work presents the strategy in optimising the production of major diarrhetic shellfish poisoning (DSP) toxins, OA and DTX1, from Prorocentrum lima. The organism is also known to produce peridinin, a carotenoid pigment that has been found to have pharmaceutical potential. Results from this study showed that cultivation of P. lima CCAP 1136/11 was still, although not completely, reliant on supply of natural seawater. Characterisation of compounds produced by P. lima CCAP 1136/11 in batch culture identified three major bioactive compounds (OA, DTX1 and peridinin) and two minor OA-related compounds. Recovery of these major compounds was further optimised with two-stage extraction procedure. Several important considerations for the cultivation process include standardisation of inoculum age and initial cell density. These and several other growth parameters such as temperature, light and CO2 supplementation have been shown to affect the growth and production of DSP toxins and peridinin in the culture. One of the main highlights in this study revealed that providing culture with light and dark cycle at frequency of 0.5 hour benefit in the enhancement of OA, DTX1 and peridinin yield from P. lima CCAP 1136/11. As the last part of this study, a simple and scalable design of reactor has been proposed. Contrary to common observations for dinoflagellate culture, P. lima CCAP 1136/11 was found to be able to withstand increased sparging within the culture system, resulting in concomitant increased of growth and production of OA, DTX1 and peridinin. Future works have been suggested to focus on: (1) exploitation of different cultivation system, such as continuous or semicontinuous systems, and (2) exploration on genetic modification to enable commercial scale production of DSP toxins and peridinin.

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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Harmful algal blooms (HABs) are natural occurrences that can severely impact marine communities. Depending on the species, they can produce a wide array of toxins, which may elicit devastating effects on marine life. Diarrhetic Shellfish Poisoning (DSP) is a specific type of food poisoning, characterized by severe gastrointestinal illness due to the ingestion of filter feeding bivalves contaminated with a specific suite of toxins such as okadaic acid (OA) and dinophysistoxins (DTXs). OA and DTXs are lipophilic and heat-stable compounds that frequently accumulate in bivalve mollusks along European coasts. It is established that organisms such as bivalves accumulate these toxins in their tissues, mostly in the digestive gland, through their filter feeding behavior acting therefore as a vectors for other organisms such as planktivorous fish. Here, for the first time, the time course of accumulation and elimination of Diarrhetic shellfish toxins (DSTs) in juveniles of Zebra seabream (Diplodus cervinus) was assessed during an experimental period of 15 days. It was shown that fish present a high excretion rate and low toxin conversion, in fact no other compounds except for DTX-1 were found. Then fish aerobic metabolism and swimming performance were investigated, exposing individuals to OA group of toxins via dietary route during 3 days, after which fish were challenged with increasing swimming velocity trials using a swim tunnel respirometer. Results show that OA impaired on metabolic performance, assessed both during the standard metabolic rate (SMR) and during the maximum metabolic rate (MMR) as an oxygen consumption increment. Moreover, from critical swimming speed (Ucrit) analysis, significant differences among the two tested groups were observed. In conclusion, these studies show that fish can act temporary as a vector and, although a high elimination rate was found, at the same time toxins ingestion was able to impair their fitness.

Bivalves play a marked role in coastal marine ecosystems, impacting primary productivity with a strong top-down control on phytoplankton communities. In addition, they are an important food source for higher trophic levels, including humans. However, bivalves can be impaired by several external factors, either biotic or abiotic, natural or anthropogenic. Exposure to these stressors, coupled with intensive farming, raised the need for adequate management of the wild populations and farmed production. To improve the difficult balance between ecological and socio-economic interests, it must be investigated how bivalves cope with external stressors. For this reason, the effects caused by the following stressors were studied in the present thesis: (i) harmful algal blooms (HABs) and (ii) climate change drivers in the seawater, such as warming (W) and acidification (A). HABs are increasing in frequency, intensity, persistence and geographic distribution, but are ambiguous regarding their ecological impacts and physiological effects on bivalves. A strong relationship is emerging between HABs and climate change drivers, what may affect both shellfisheries and wild populations. Hence, the objectives of this work are: 1) To determine whether native and invasive species cope with exposure to HABs differently, providing new insights on species-specificities and ecosystem functioning fragilities in the presence of invasive species and HABs; 2) To evaluate how commercially valuable bivalve species cope with simultaneous exposure to several climate change drives and HABs, providing new insights on how environmental changes affects toxicokinetics, physiological and genotoxic bivalves responses, under HABs; 3) To assess, marine biotoxins bioaccessibility, contributing for new information relevant for risk assessment. Different approaches were used to achieve these goals. Complementary field and laboratory works were performed to assess toxicokinetics patterns of marine biotoxins in native vs. invasive bivalve species. Bivalves were exposed to toxic blooming algae species under climate change scenarios in laboratory controlled conditions to assess toxicokinetics as well as physiological and genotoxic responses. Naturally contaminated bivalves were used to investigate their role as vectors of marine biotoxins to humans through in vitro digestion methodology. Relevant new data were obtained regarding the effects of several marine biotoxins in bivalves. Assessing accumulation of regulated and non-regulated biotoxins in native (Ruditapes decussatus) and invasive (R. philippinarum) clam species from Aveiro Lagoon revealed that higher toxin content, particularly regarding OA-group, the most abundant and frequent toxins in the Portuguese coast, is reached by native species. Accumulation of lower toxin levels by invasive clam may then favour farmers interest for their production. The kinetics and genotoxicity study in native and invasive clams reinforced the existence of species-specific behaviours. Exposure to the dinoflagellate Prorocentrum lima, an OA and dinophysistoxin-1 producer, increases the pressure over native clams, with higher toxin accumulation and genetic damage, as well as early and increased induction of DNA repair activity. Invasive clams, on the other hand, are better adapted to cope with these challenges. Investigation of the effects of combined exposure to multiple stressors revealed that W, A and HABs alter the accumulation/elimination dynamics of Paralytic Shellfish Poisoning (PSP) toxins in mussels (Mytilus galloprovincialis). Lower accumulation levels and slower elimination rates were observed. The predicted climate change scenarios and exposure to HABs may then lead to lower contamination levels but to longer harvesting closures. Simultaneous exposure to altered environmental conditions and HABs also had significant impacts in the antioxidant system and DNA integrity, resulting in an organ-specific modulation of the antioxidant response, increasing genetic damage and preventing/retarding DNA damage repair. However, the DNA damage observed seems to be non-oxidative. While the investigation of factors enhancing the elimination of toxins was not a main objective of this study, it was shown that acidification might promote PSP toxins elimination in mussels. Finally, the bioaccessibility studies revealed a significant reduction in the OA group content available after in vitro digestion, suggesting an overestimation of exposure to these biotoxins and an over-conservative approach in safety levels definition and risk assessment studies. This was the first study assessing bioaccessibility of OA-group toxins, pointing to in vitro digestion as a promising tool to obtain accurate data regarding toxin ingestion from bivalves to the consumer. Overall, the complexity of the relation between HABs, bivalves and the environmental factors to consider in marine biotoxins management is increasing, and only continuous and extensive monitoring of environmental, biological and anthropogenic conditions may allow for a healthier balance between environmental and socio-economic interests.
Os bivalves desempenham um papel fundamental nos ecossistemas marinhos costeiros, limitando a produtividade primária através de um forte mecanismo de controlo descendente (top-down) sobre as comunidades fitoplanctónicas e constituindo uma importante fonte de alimento para níveis tróficos superiores, incluindo o Homem. No entanto, os bivalves são afetados negativamente por diversos fatores externos, sejam estes bióticos ou abióticos, naturais ou antropogénicos. A exposição a estes fatores, juntamente com a exploração intensiva, leva a que seja necessária a implementação de uma gestão adequada, quer das populações selvagens, quer da produção em viveiros. Para melhorar esta gestão e alcançar o difícil equilíbro entre os interesses ecológicos e socioeconómicos é então necessário entender como os bivalves lidam com estes fatores. Na presente tese foram estudados os efeitos provocados por (i) blooms de algas tóxicas (HABs do inglês harmful algal blooms) e (ii) alterações das condições climáticas da água do mar, nomeadamente o aumento da temperatura (W) e acidificação (A). Os HABs são fenómenos que estão a aumentar em frequência, intensidade, persistência e abrangência geográfica, mas cujos impactos ecológicos e efeitos fisiológicos em bivalves são ainda ambíguos. Concomitantemente com a pressão causada pelo aumento da procura de bivalves como alimento para as populações humanas, fortes indícios apontam para uma relação entre a ocorrência de HABs e os parâmetros de alterações climáticas, o que pode afectar quer a indústria, quer as populações de bivalves selvagens. Assim, os objetivos deste trabalho são: 1) Determinar se as espécies nativas e invasoras lidam com a exposição a HABs de maneira diferente, dando particular atenção às respostas específicas de cada espécie e às fragilidades do ecossistema em relação à presença de espécies invasoras e ocorrência dos referidos blooms; 2) Avaliar como espécies de bivalves de elevado valor comercial lidam com a exposição simultânea a alterações climáticas e HABs, focando o efeito destas modificações ambientais na toxicocinética, respostas fisiológicas e genotoxicidade exibida pelos bivalves; 3) Avaliar a bioacessibilidade das biotoxinas marinhas, focando a obtenção de informação relevante para estudos de avaliação de risco. Foram realizados trabalhos complementares de campo e laboratório, de modo a avaliar os padrões de toxicocinética de biotoxinas marinhas em espécies nativas e invasoras. Em laboratório, foram expostos bivalves a algas tóxicas em condições alteradas, simulando a ocorrência de HABs sob cenários previstos de alterações climáticas, de modo a avaliar toxicocinética, respostas fisiológicas e genotoxicidade. Exemplares naturalmente contaminados foram utilizados para investigar o papel dos bivalves como vectores de biotoxinas marinhas para humanos, através da simulação da digestão in vitro. O estudo da acumulação de biotoxinas marinhas regulamentadas e não regulamentadas em espécies de amêijoa nativa (Ruditapes decussatus) e invasora (R. philippinarum) na Ria de Aveiro revelou que a espécie nativa acumula, recorrentemente, concentrações de toxinas mais elevadas, particularmente no que toca a toxinas grupo do ácido ocadaico (OA do inglês Okadaic acid), sendo estas as toxinas mais abundantes e frequentes na costa Portuguesa. A acumulação de menores concentrações por parte da amêijoa invasora pode promover o interesse dos produtores na sua exploração. A avaliação da cinética e da genotoxicidade em amêijoas nativas e invasoras reforçou a existência de comportamentos específicos para cada espécie. A exposição a Prorocentrum lima, dinoflagelado produtor de OA e dinofisistoxina 1, aumenta a pressão sobre a espécie nativa, uma vez que esta apresenta as concentrações de toxina mais elevadas, os maiores níveis de dano genético e ainda uma indução mais precoce e intensa dos mecanismos de reparação de dano no ADN. A espécie invasora, por outro lado, está melhor adaptada para lidar com estes desafios. O estudo dos efeitos de fatores múltiplos combinados revelou que a exposição a W, A e HABs altera a dinâmica de acumulação/eliminação de toxinas paralisantes (PSP) em mexilhões (Mytilus galloprovincialis). Os cenários previstos de alterações climáticas e a exposição a HABs sugerem níveis de contaminação mais baixos, mas períodos de interdição de apanha mais prolongados. A exposição simultânea a condições ambientais alteradas e HABs também teve um impacto significativo no sistema antioxidante e na integridade do ADN, resultando numa modulação da resposta antioxidante especifica para cada órgão, aumentando o dano genético e prevenindo/atrasando a sua reparação. O dano encontrado no ADN parece, no entanto, não ser oxidativo. Embora a investigação de fatores que favoreçam a rápida eliminação de toxinas não constasse dos objetivos deste estudo, foi demonstrado que a acidificação pode promover a eliminação de toxinas PSP em mexilhões. Por último, os estudos de bioacessibilidade revelaram uma redução na quantidade de toxinas do grupo-OA disponível após a digestão in vitro, sugerindo uma sobrestimação da exposição a estas biotoxinas e recomendando uma abordagem conservadora na definição dos níveis de segurança e na subsequente avaliação do risco. Este foi o primeiro estudo a determinar a bioacessibilidade das toxinas do grupo-OA e sugere a técnica de digestão in vitro como uma ferramenta promissora na obtenção de dados rigorosos no que diz respeito à ingestão de toxinas através de bivalves e a acessibilidade destes compostos no organismo humano. De um modo geral, a complexidade da relação entre HABs, bivalves e os fatores ambientais a serem considerados na gestão de biotoxinas marinhas está a aumentar, e apenas uma contínua e extensiva monitorização das condições ambientais, biológicas e antropogénicas pode permitir um melhor equilíbrio entre os interesses ambientais e socio-económicos.
Programa Doutoral em Ciência, Tecnologia e Gestão do Mar