Assessment of the early effects of the Diarrhetic Shellfish Toxins in the mussel Mytilus galloprovincialis using cellular and molecular biomarkers

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01[Abstract] Diarrhetic shellfish poisoning (DSP) toxins constitute a group of marine toxins displaying wide geographic distribution and involved in frequent toxic episodes (Harmful algal blooms, HABs) in coastal areas. Okadaic acid (OA) and its derivatives the dinophysistoxins (DTXs) are the main toxic compounds of this group. Seafood contamination by DSP toxins, especially bivalve molluscs, constitutes the principal cause of diarrhetic shellfish poisoning syndrome (DSP) in human consumers. In order to reduce its prevalence, efficient pollution control programs are currently conducted in shellfish farming industries, causing collateral economic losses. Despite the toxicity caused by DSP toxins in human and mammalian cell lines, bivalve molluscs show an apparent resistance to these compounds. This thesis evaluates, for the first time, the early effects of DSP toxins in the mussel Mytilus galloprovincialis using cellular and molecular biomarkers. Accordingly, the genotoxic and cytotoxic effects of OA were evaluated in hemolymph and gill cells in vitro. This approach was supplemented with the in vivo assessment of the effects of DSP-toxin-producing dinoflagellate Prorocentrum lima. Additionally, the transcriptional and biochemical responses of several antioxidant enzymes were determined in mussels exposed in vivo to P. lima. The results presented in this thesis increase the general knowledge pertaining the genotoxic and cytotoxic potential of DSP toxins in marine organisms and, more especifically, in the case bivalve molluscs. In addition, this work helps elucidate the connection between the exposure to these toxins and the induction of oxidative stress. Similarly, the results obtained suggest that the resistance of these organisms to DSP toxins is associated with the ability of their antioxidant system to respond immediately to damage. In conclusion, this thesis lays the foundation for the future development of efficient monitoring programs.[Resumen] Las toxinas diarreicas (Diarrhetic shellfish poisoning, DSP) constituyen un grupo de toxinas marinas de amplia distribución geográfica responsable de frecuentes episodios tóxicos de contaminación en áreas costeras. Los principales compuestos tóxicos que conforman este grupo son el ácido ocadaico (Okadaic acid, OA) y sus derivados las dinofisitoxinas (Dinophysistoxins, DTXs). La contaminación por toxinas DSP en mariscos, especialmente en moluscos bivalvos, causa intoxicación diarreica (síndrome DSP) en consumidores humanos. Para reducir su prevalencia se llevan a cabo programas de control eficientes, pero que provocan pérdidas económicas colaterales en el cultivo de moluscos. A pesar de la toxicidad causada por las toxinas DSP en líneas celulares de humanos y mamíferos, los moluscos bivalvos muestran aparente resistencia a estos compuestos. En esta tesis se han evaluado, por primera vez, los efectos tempranos de las toxinas DSP en el mejillón Mytilus galloprovincialis mediante el uso de biomarcadores celulares y moleculares. Para ello se llevó a cabo una primera aproximación mediante el estudio in vitro de los efectos genotóxicos y citotóxicos del OA en células de hemolinfa y branquia de mejillón. Esta aproximación se complementó con el estudio in vivo de los efectos del dinoflagelado productor de toxinas DSP Prorocentrum lima. Además, se determinó la respuesta transcripcional y bioquímica de varias enzimas antioxidantes de mejillón a la exposición in vivo a P. lima. Los resultados obtenidos en la presente tesis incrementan el conocimiento del potencial genotóxico y citotóxico de las toxinas DSP en los moluscos bivalvos y lo relacionan con el estrés oxidativo. Del mismo modo, la resistencia de estos organismos a las toxinas DSP se asocia con la capacidad del sistema antioxidante para responder de forma inmediata a los primeros daños. En conclusión, la presente tesis sienta las bases para el desarrollo futuro de mecanismos eficientes de monitorización.[Resumo] As toxinas diarreicas (Diarrhetic shellfish poisoning, DSP) constitúen un grupo de toxinas mariñas de ampla distribución xeográfica responsable de frecuentes episodios tóxicos de contaminación en áreas costeiras. Os principais compostos tóxicos que conforman este grupo son o ácido ocadaico (Okadaic acid, OA) e os seus derivados as dinofisitoxinas (Dinophysistoxins, DTXs). A contaminación por toxinas DSP en mariscos, especialmente en moluscos bivalvos, causa intoxicación diarreica (síndrome DSP) en consumidores humanos. Para reducir a súa prevalencia lévanse a cabo programas de control eficientes, pero que provocan perdas económicas colaterais no cultivo de moluscos. A pesar da toxicidade causada polas toxinas DSP en liñas celulares de humanos e mamíferos, os moluscos bivalvos mostran aparente resistencia a estes compostos. Nesta tese avaliáronse, por primeira vez, os efectos temperáns das toxinas DSP no mexillón Mytilus galloprovincialis mediante o uso de biomarcadores celulares e moleculares. Para iso levouse a cabo unha primeira aproximación mediante o estudo in vitro dos efectos xenotóxicos e citotóxicos do OA en células de hemolinfa e branquia de mexillón. Esta aproximación complementouse co estudo in vivo dos efectos do dinoflaxelado produtor de toxinas DSP Prorocentrum lima. Ademais, determinouse a resposta transcricional e bioquímica de varias enzimas antioxidantes de mexillón á exposición in vivo a P. lima. Os resultados obtidos na presente tese incrementan o coñecemento do potencial xenotóxico e citotóxico das toxinas DSP nos moluscos bivalvos e relaciónano co estrés oxidativo. Do mesmo xeito, a resistencia destes organismos ás toxinas DSP asóciase coa capacidade do sistema antioxidante para responder de forma inmediata aos primeiros danos. En xeral, nesta tese séntanse as bases para o desenvolvemento futuro de mecanismos eficientes de monitoraxe

RNA-Seq analysis for assessing the early response to DSP toxins in Mytilus galloprovincialis digestive gland and gill

[Abstract] The harmful effects of diarrhetic shellfish poisoning (DSP) toxins on mammalian cell lines have been widely assessed. Studies in bivalves suggest that mussels display a resistance to the cytogenotoxic effects of DSP toxins. Further, it seems that the bigger the exposure, the more resistant mussels become. To elucidate the early genetic response of mussels against these toxins, the digestive gland and the gill transcriptomes of Mytilus galloprovincialis after Prorocentrum lima exposure (100,000 cells/L, 48 h) were de novo assembled based on the sequencing of 8 cDNA libraries obtained using an Illumina HiSeq 2000 platform. The assembly provided 95,702 contigs. A total of 2286 and 4523 differentially expressed transcripts were obtained in the digestive gland and the gill, respectively, indicating tissue-specific transcriptome responses. These transcripts were annotated and functionally enriched, showing 44 and 60 significant Pfam families in the digestive gland and the gill, respectively. Quantitative PCR (qPCR) was performed to validate the differential expression patterns of several genes related to lipid and carbohydrate metabolism, energy production, genome integrity and defense, suggesting their participation in the protective mechanism. This work provides knowledge of the early response against DSP toxins in the mussel M. galloprovincialis and useful information for further research on the molecular mechanisms of the bivalve resistance to these toxins.Ministerio de Economía y Competitividad; AGL2012-3089

Okadaic Acid Meet and Greet: An Insight into Detection Methods, Response Strategies and Genotoxic Effects in Marine Invertebrates

Harmful Algal Blooms (HABs) constitute one of the most important sources of contamination in the oceans, producing high concentrations of potentially harmful biotoxins that are accumulated across the food chains. One such biotoxin, Okadaic Acid (OA), is produced by marine dinoflagellates and subsequently accumulated within the tissues of filtering marine organisms feeding on HABs, rapidly spreading to their predators in the food chain and eventually reaching human consumers causing Diarrhetic Shellfish Poisoning (DSP) syndrome. While numerous studies have thoroughly evaluated the effects of OA in mammals, the attention drawn to marine organisms in this regard has been scarce, even though they constitute primary targets for this biotoxin. With this in mind, the present work aimed to provide a timely and comprehensive insight into the current literature on the effect of OA in marine invertebrates, along with the strategies developed by these organisms to respond to its toxic effect together with the most important methods and techniques used for OA detection and evaluation

Okadaic Acid: More than a Diarrheic Toxin

Okadaic acid (OA) is one of the most frequent and worldwide distributed marine toxins. It is easily accumulated by shellfish, mainly bivalve mollusks and fish, and, subsequently, can be consumed by humans causing alimentary intoxications. OA is the main representative diarrheic shellfish poisoning (DSP) toxin and its ingestion induces gastrointestinal symptoms, although it is not considered lethal. At the molecular level, OA is a specific inhibitor of several types of serine/threonine protein phosphatases and a tumor promoter in animal carcinogenesis experiments. In the last few decades, the potential toxic effects of OA, beyond its role as a DSP toxin, have been investigated in a number of studies. Alterations in DNA and cellular components, as well as effects on immune and nervous system, and even on embryonic development, have been increasingly reported. In this manuscript, results from all these studies are compiled and reviewed to clarify the role of this toxin not only as a DSP inductor but also as cause of alterations at the cellular and molecular levels, and to highlight the relevance of biomonitoring its effects on human health. Despite further investigations are required to elucidate OA mechanisms of action, toxicokinetics, and harmful effects, there are enough evidences illustrating its toxicity, not related to DSP induction, and, consequently, supporting a revision of the current regulation on OA levels in food

Early Genotoxic and Cytotoxic Effects of the Toxic Dinoflagellate Prorocentrum lima in the Mussel Mytilus galloprovincialis

Okadaic acid (OA) and dinophysistoxins (DTXs) are the main toxins responsible for diarrhetic shellfish poisoning (DSP) intoxications during harmful algal blooms (HABs). Although the genotoxic and cytotoxic responses to OA have been evaluated in vitro, the in vivo effects of these toxins have not yet been fully explored. The present work fills this gap by evaluating the in vivo effects of the exposure to the DSP-toxin-producing dinoflagellate Prorocentrum lima during the simulation of an early HAB episode in the mussel Mytilus galloprovincialis. The obtained results revealed that in vivo exposure to this toxic microalgae induced early genotoxicity in hemocytes, as a consequence of oxidative DNA damage. In addition, the DNA damage observed in gill cells seems to be mainly influenced by exposure time and P. lima concentration, similarly to the case of the oxidative damage found in hemocytes exposed in vitro to OA. In both cell types, the absence of DNA damage at low toxin concentrations is consistent with the notion suggesting that this level of toxicity does not disturb the antioxidant balance. Lastly, in vivo exposure to growing P. lima cell densities increased apoptosis but not necrosis, probably due to the presence of a high number of protein apoptosis inhibitors in molluscs. Overall, this work sheds light into the in vivo genotoxic and cytotoxic effects of P. lima. In doing so, it also demonstrates for the first time the potential of the modified (OGG1) comet assay for assessing oxidative DNA damage caused by marine toxins in marine invertebrates