RNA-Seq Analysis for Assessing the Early Response to DSP Toxins in Mytilus galloprovincialis Digestive Gland and Gill.

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.

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.

In Vitro Analysis of Early Genotoxic and Cytotoxic Effects of Okadaic Acid in Different Cell Types of the Mussel Mytilus galloprovincialis.

Okadaic acid (OA) is the predominant biotoxin responsible for diarrhetic shellfish poisoning (DSP) syndrome in humans. While its harmful effects have been extensively studied in mammalian cell lines, the impact on marine organisms routinely exposed to OA is still not fully known. Few investigations available on bivalve molluscs suggest less genotoxic and cytotoxic effects of OA at high concentrations during long exposure times. In contrast, no apparent information is available on how sublethal concentrations of OA affect these organisms over short exposure times. In order to fill this gap, this study addressed for the first time in vitro analysis of early genotoxic and cytotoxic effects attributed to OA in two cell types of the mussel Mytilus galloprovincialis. Accordingly, hemocytes and gill cells were exposed to low OA concentrations (10, 50, 100, 200, or 500 nM) for short periods of time (1 or 2 h). The resulting DNA damage, as apoptosis and necrosis, was subsequently quantified using the comet assay and flow cytometry, respectively. Data demonstrated that (1) mussel hemocytes seem to display a resistance mechanism against early genotoxic and cytotoxic OA-induced effects, (2) mussel gill cells display higher sensitivity to early OA-mediated genotoxicity than hemocytes, and (3) mussel gill cells constitute more suitable systems to evaluate the genotoxic effect of low OA concentrations in short exposure studies. Taken together, this investigation provides evidence supporting the more reliable suitability of mussel gill cells compared to hemocytes to evaluate the genotoxic effect of low short-duration exposure to OA.

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.

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.