Transcriptome Analysis Reveals MAPK/AMPK as a Key Regulator of the Inflammatory Response in PST Detoxification in Mytilus galloprovincialis and Argopecten irradians.

Paralytic shellfish toxins (PSTs) are an increasingly important source of pollution. Bivalves, as the main transmission medium, accumulate and metabolize PSTs while protecting themselves from damage. At present, the resistance mechanism of bivalves to PSTs is unclear. In this study, Mytilus galloprovincialis and Argopecten irradians were used as experimental shellfish species for in situ monitoring. We compared the inflammatory-related gene responses of the two shellfish during PSTs exposure by using transcriptomes. The results showed that the accumulation and metabolism rate of PSTs in M. galloprovincialis was five-fold higher than that in A. irradians. The inflammatory balance mechanism of M. galloprovincialis involved the co-regulation of the MAPK-based and AMPK-based anti-inflammatory pathways. A. irradians bore a higher risk of death because it did not have the balance system, and the regulation of apoptosis-related pathways such as the PI3K-AKT signaling pathway were upregulated. Taken together, the regulation of the inflammatory balance coincides with the ability of bivalves to cope with PSTs. Inflammation is an important factor that affects the metabolic pattern of PSTs in bivalves. This study provides new evidence to support the studies on the resistance mechanism of bivalves to PSTs.

Domoic acid biosynthesis in the red alga Chondria armata suggests a complex evolutionary history for toxin production.

Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages.

Performance of different extraction methods for paralytic shellfish toxins and toxin stability in shellfish during storage.

Accurate analysis of paralytic shellfish toxins (PSTs) in shellfish is important to protect seafood safety and human health. In this study, the performance of different extraction protocols for PSTs from scallop tissues is compared and discussed, including regular extraction solvents hydrochloric acid (HCl) and acetic acid (AcOH) followed by heating and solid-phase extraction (SPE) purification, and a novel technique of matrix solid-phase dispersion (MSPD) without heating. The possible conversion of C1/2 and GTX2/3 standards after heating, and the stability of PSTs in wet scallop tissues stored at -20 °C for a 6-month period are also explored. Results showed that the MSPD technique could effectively mitigate matrix interference, but its recoveries of PSTs were significantly lower than those of the HCl and AcOH extraction methods followed by carbon SPE purification. The molar concentrations of M-toxins obtained by the MSPD method were generally lower than those analyzed by the HCl and AcOH extraction methods, which demonstrated a weak chemical conversion of C1/2 and GTX2/3 due to the heating process. Most of the PSTs were relatively stable in scallop tissues during 1-month storage at -20 °C, while the concentrations of PSTs in scallop tissues obviously changed after 6 months due to the degradation and transformation of PSTs during long-term storage at -20 °C. This work helps improve our understanding of the performance of different extraction methods and the stability of PSTs in scallop tissues stored at -20 °C.

Cinnamaldehyde Could Reduce the Accumulation of Diarrhetic Shellfish Toxins in the Digestive Gland of the Mussel Perna viridis under Laboratory Conditions.

Diarrhetic shellfish toxins (DSTs), some of the most important phycotoxins, are distributed almost all over the world, posing a great threat to human health through the food chain. Therefore, it is of great significance to find effective methods to reduce toxin accumulation in shellfish. In this paper, we observed the effects of four phytochemicals including cinnamaldehyde (CA), quercetin, oridonin and allicin on the accumulation of DSTs in the digestive gland of Perna viridis after exposure to the DSTs-producing Prorocentrum lima. We found that, among the four phytochemicals, CA could effectively decrease the accumulation of DSTs (okadaic acid-eq) in the digestive gland of P. viridis. Further evidence demonstrated that CA could reduce the histological alterations of the digestive gland of a mussel caused by DSTs. RT-qPCR showed that CA could suppress the CYP3A4 induction by DSTs, suggesting that the DSTs' decrease induced by CA might be related to the inhibition of CYP3A4 transcription induction. However, further studies on the underlying mechanism, optimal treatment time, ecological safety and cost should be addressed before cinnamaldehyde is used to decrease the accumulation of DSTs in field.

Differences in Toxic Response Induced by Three Variants of the Diarrheic Shellfish Poisoning Phycotoxins in Human Intestinal Epithelial Caco-2 Cells.

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Limnological Differences in a Two-Basin Lake Help to Explain the Occurrence of Anatoxin-a, Paralytic Shellfish Poisoning Toxins, and Microcystins.

Chautauqua Lake, New York, is a two-basin lake with a deeper, cooler, and less nutrient-rich Northern Basin, and a warmer, shallower, nutrient-replete Southern Basin. The lake is populated by a complex mixture of cyanobacteria, with toxigenic strains that produce microcystins, anatoxins, and paralytic shellfish poisoning toxins (PSTs). Samples collected from 24 sites were analyzed for these three toxin classes over four years spanning 2014-2017. Concentrations of the three toxin groups varied widely both within and between years. During the study, the mean and median concentrations of microcystins, anatoxin-a, and PSTs were 91 and 4.0 µg/L, 0.62 and 0.33 µg/L, and 32 and 16 µg/L, respectively. Dihydro-anatoxin was only detected once in Chautauqua Lake, while homo-anatoxin was never detected. The Northern Basin had larger basin-wide higher biomass blooms with higher concentrations of toxins relative to the more eutrophied Southern Basin, however blooms in the North Basin were infrequent. Chlorophyll concentrations and toxins in the two basins were correlated with different sets of environmental and physical parameters, suggesting that implementing controls to reduce toxin loads may require applications focused on more than reductions in cyanobacterial bloom density (e.g., reduction of phosphorus inputs), and that lake limnological factors and morphology are important determinants in the selection of an appropriate management strategy. Chautauqua Lake is a drinking water source and is also heavily used for recreation. Drinking water from Chautauqua Lake is unlikely to be a significant source of exposure to cyanotoxins due to the location of the intakes in the deeper North Basin, where there were generally low concentrations of toxins in open water; however, toxin levels in many blooms exceeded the US Environmental Protection Agency's recreational guidelines for exposure to cyanotoxins. Current cyanotoxin monitoring in Chautauqua Lake is focused on microcystins. However, the occurrence of blooms containing neurotoxic cyanotoxins in the absence of the microcystins indicates this restricted monitoring may not be sufficient when aiming to protect against exposure to cyanotoxins. The lake has a large number of tourist visitors; thus, special care should be taken to prevent recreational exposure within this group.

A Mediterranean Alexandrium taylorii (Dinophyceae) Strain Produces Goniodomin A and Lytic Compounds but Not Paralytic Shellfish Toxins.

Species of the dinophyte genus Alexandrium are widely distributed and are notorious bloom formers and producers of various potent phycotoxins. The species Alexandrium taylorii is known to form recurrent and dense blooms in the Mediterranean, but its toxin production potential is poorly studied. Here we investigated toxin production potential of a Mediterranean A. taylorii clonal strain by combining state-of-the-art screening for various toxins known to be produced within Alexandrium with a sound morphological and molecular designation of the studied strain. As shown by a detailed thecal plate analysis, morphology of the A. taylorii strain AY7T from the Adriatic Sea conformed with the original species description. Moreover, newly obtained Large Subunit (LSU) and Internal Transcribed Spacers (ITS) rDNA sequences perfectly matched with the majority of other Mediterranean A. taylorii strains from the databases. Based on both ion pair chromatography coupled to post-column derivatization and fluorescence detection (LC-FLD) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis it is shown that A. taylorii AY7T does not produce paralytic shellfish toxins (PST) above a detection limit of ca. 1 fg cell-1, and also lacks any traces of spirolides and gymnodimines. The strain caused cell lysis of protistan species due to poorly characterized lytic compounds, with a density of 185 cells mL-1 causing 50% cell lysis of cryptophyte bioassay target cells (EC50). As shown here for the first time A. taylorii AY7T produced goniodomin A (GDA) at a cellular level of 11.7 pg cell-1. This first report of goniodomin (GD) production of A. taylorii supports the close evolutionary relationship of A. taylorii to other identified GD-producing Alexandrium species. As GD have been causatively linked to fish kills, future studies of Mediterranean A. taylorii blooms should include analysis of GD and should draw attention to potential links to fish kills or other environmental damage.

OMICs Approaches in Diarrhetic Shellfish Toxins Research.

Diarrhetic shellfish toxins (DSTs) are among the most prevalent marine toxins in Europe's and in other temperate coastal regions. These toxins are produced by several dinoflagellate species; however, the contamination of the marine trophic chain is often attributed to species of the genus Dinophysis. This group of toxins, constituted by okadaic acid (OA) and analogous molecules (dinophysistoxins, DTXs), are highly harmful to humans, causing severe poisoning symptoms caused by the ingestion of contaminated seafood. Knowledge on the mode of action and toxicology of OA and the chemical characterization and accumulation of DSTs in seafood species (bivalves, gastropods and crustaceans) has significantly contributed to understand the impacts of these toxins in humans. Considerable information is however missing, particularly at the molecular and metabolic levels involving toxin uptake, distribution, compartmentalization and biotransformation and the interaction of DSTs with aquatic organisms. Recent contributions to the knowledge of DSTs arise from transcriptomics and proteomics research. Indeed, OMICs constitute a research field dedicated to the systematic analysis on the organisms' metabolisms. The methodologies used in OMICs are also highly effective to identify critical metabolic pathways affecting the physiology of the organisms. In this review, we analyze the main contributions provided so far by OMICs to DSTs research and discuss the prospects of OMICs with regard to the DSTs toxicology and the significance of these toxins to public health, food safety and aquaculture.

Detoxification of paralytic shellfish poisoning toxins in naturally contaminated mussels, clams and scallops by an industrial procedure.

Paralytic shellfish poisoning (PSP) episodes cause important economic impacts due to closure of shellfish production areas in order to protect human health. These closures, if are frequent and persistent, can seriously affect shellfish producers and the seafood industry, among others. In this study, we have developed an alternative processing method for bivalves with PSP content above the legal limit, which allows reducing toxicity to acceptable levels. A modification of the PSP detoxifying procedure stablished by Decision 96/77/EC of the European Union in Acanthocardia tuberculata, was developed and implemented for PSP elimination in other bivalves species. The procedure was applied to 6 batches of mussels, 2 batches of clams and 2 batches of scallops, achieving detoxification rates of around 85%. A viable industrial protocol which allows the transformation of a product at risk into a safe product was developed. Although a significant reduction was obtained, in a sample circa 9000 µg STX diHCl equiv/kg, the final toxin level in these highly toxic mussels did not fall below the European limit. The processing protocol described may be applied efficiently to mussels, clams and scallops and it may be a major solution to counteract the closure of shellfish harvesting areas, especially if persistent.

Experimental uptake and depuration of paralytic shellfish toxins in Southern Rock Lobster, Jasus edwardsii.

In October 2012, paralytic shellfish toxins (PST) were detected in the hepatopancreas of Southern Rock Lobsters (Jasus edwardsii) collected from the east coast of Tasmania, Australia. This resulted in the first commercial closure in Australia for this species. Questions were raised on how the toxins were transferred to the lobsters, how long the toxins would persist, whether PST-contaminated hepatopancreas posed a risk to human health, and what management strategies could be applied. The aim of this study was to investigate whether PST-contaminated mussels are a potential vector enabling toxin accumulation in J. edwardsii and to collect information on toxin uptake, distribution and depuration rates and toxin profiles under controlled experimental settings. Lobsters were fed mussels naturally contaminated with PST for a period of 28 days in an experimental setting; following this, lobsters were allocated to either fed or starved treatment groups. PST were not detected in the tail tissue of lobsters at any stage of the experiment. Lobster hepatopancreas contained mean levels of 2.4 mg STX.2HCl eq/kg after 28 days of uptake, although substantial variability in total toxicity was observed. The PST profile of the hepatopancreas was similar to that of the contaminated mussels used as feed. Significant differences were noted in the PST depuration rates between fed and starved treatment groups. The daily depuration rate for total PST was estimated to be 0.019 and 0.013 mg STX.2HCl eq/kg for the fed and starved treatment groups respectively using a constant-rate decay model. After 42 days of depuration, total PST (STX equivalents) levels in the hepatopancreas of all lobsters were below 0.8 mg STX.2HCl eq/kg, which represents the regulatory level applied to bivalves. This result indicates that long-term holding to depurate PST may potentially be used as a risk management tool.

Proposed Biotransformation Pathways for New Metabolites of Paralytic Shellfish Toxins Based on Field and Experimental Mussel Samples.

A seafood poisoning event occurred in Qinhuangdao, China, in April 2016. Subsequently, the causative mussels (Mytilus galloprovincialis) were harvested and analyzed to reveal a high concentration [∼10 758 µg of saxitoxin (STX) equiv kg-1] of paralytic shellfish toxins (PSTs), including gonyautoxin (GTX)1/4 and GTX2/3, as well as new metabolites 11-hydroxy-STX (M2), 11,11-dihydroxy-STX (M4), open-ring 11,11-dihydroxy-STX (M6), 11-hydroxy-neosaxitoxin (NEO) (M8), and 11,11-dihydroxy-NEO (M10). To understand the origin and biotransformation pathways of these new metabolites, uncontaminated mussels (M. galloprovincialis) were fed with either of two Alexandrium tamarense strains (ATHK and TIO108) under laboratory conditions. Similar PST metabolites were also detected in mussels from both feeding experiments. Results supposed that 11-hydroxy-C2 toxin (M1) and 11,11-dihydroxy-C2 (M3) are transformed from C2, while 11-hydroxy-C4 toxin (M7) and 11,11-dihydroxy-C4 (M9) are converted from C4. In addition, the metabolites M2, M4, and M6 appear to be products of GTX2/3, and the metabolites M8 and M10 are likely derived from GTX1/4.

Analysis of diarrhetic shellfish poisoning toxins and pectenotoxin-2 in the bottlenose dolphin (Tursiops truncatus) by liquid chromatography-tandem mass spectrometry.

Toxins produced by harmful algae are associated with detrimental health effects and mass mortalities of marine mammals. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is generally used to confirm the presence of algal toxins in marine mammals. Sample preparation and LC-MS/MS methods for the determination of three diarrhetic shellfish poisoning (DSP) toxins (okadaic acid, OA; dinophysistoxin-1, DTX1; dinophysistoxin-2, DTX2) and pectenotoxin-2 (PTX2) in bottlenose dolphin (Tursiops truncatus) urine and tissue samples were evaluated using spike-and-recovery tests. Sample clean-up with either reversed-phase silica or polymeric solid-phase extraction (SPE) reduced interference of sample matrices and improved toxin recoveries, with polymeric SPE showing higher sample loading capacity. LC separation on Xbridge C18 columns using acetonitrile/water gradient elutions with ammonia as the additive was chosen for its high detectivity and sensitivity in the MS detection of DSP toxins in negative ion mode. The retention times of OA, DTX1, and DTX2, separated as negative ions, increased with LC column temperature while the retention time of PTX2, separated as the neutral molecule, was weakly affected. At the same column temperature, retention times of OA, DTX1, and DTX2 gradually increased as the mobile phases aged while the retention time of PTX2 remained unchanged; higher column temperatures resulted in a greater increase in the retention time of each DSP toxin with mobile phase aging. Average recoveries of the 4 toxins in bottlenose dolphin samples ranged from 80% to 130% with relative standard deviations of less than 15% using the LC mobile phases prepared within one week at a column temperature of 30°C or 40°C. The preferred column temperature was 30°C, as the retention times of DSP toxins were less affected by mobile phase aging at this temperature. The limit of detection of each toxin analyzed in bottlenose dolphin samples was 2.8 ng/g or less in tissue samples and 0.7 ng/ml or less in urine.

A tyrosine-containing analog of mu-conotoxin GIIIA as ligand in the receptor binding assay for paralytic shellfish poisons.

Development of novel analytical tools to detect marine biotoxins has been warranted in view of the apparent global pervasiveness of algal-derived shellfish poisoning, and the limitations of existing methods. Here, we describe the initial phase in the development and evaluation of a tyrosine-containing analog of µ-conotoxin (µ-CTX) GIIIA as an alternative to saxitoxin (STX) in a receptor binding assay (RBA) for paralytic shellfish poisons. The peptide analog was synthesized and characterized for structure and bioactivity. The major product of oxidation elicited paralytic symptoms in mice at a minimum dose of 1.31 mg kg(-1) (i.p.). Mass spectrometry analysis of the bioactive peptide gave a molecular mass of 2637.52 Da that was close to the predicted value. Iodination via chloramine-T produced non-, mono- and di-iodinated peptides (respectively, NIP, MIP and DIP). Competition assays against (3)H-STX revealed higher Ki and EC50 (P < 0.0001, ANOVA) indicating reduced affinity for the receptor, and limited displacement of receptor-bound STX. However, subsequent use of MIP may extend the application of RBA to detect small changes in toxin levels owing to its likely enhanced displacement by STX. This may be useful in analyzing samples with toxicities near the regulatory limit, or in establishing baseline values in high risk environments.

Cultivation of the benthic microalga Prorocentrum lima for the production of diarrhetic shellfish poisoning toxins in a vertical flat photobioreactor.

In this study, the cultivation conditions of Prorocentrum lima, including temperature, nutrient concentration, photoperiod, and salinity were observed, and then an effective method for the large-volume cultivation of P. lima using a vertical flat photobioreactor was developed for the first time. The maximum cell concentrations and toxin contents of P. lima cultured in the photobioreactor were reached after a 35 days cultivation. Moreover, a step-wise double-sedimentation centrifugation method was used to harvest the microalgae cells, with the harvest rate of 89%. Toxin analysis of dry microalgal powder indicated that OA and DTX1 contents were 15.2 and 21.6 mg g(-1), respectively. These results verify that the culture method using the proposed photobioreactor is effective to massively produce DSP toxin-containing P. lima. This study may serve as a guide for the large-scale production of toxin-producing red-tide benthic microalgae.

RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium minutum.

BACKGROUND: The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation. RESULTS: The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens. CONCLUSION: The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic.

SxtA and sxtG gene expression and toxin production in the Mediterranean Alexandrium minutum (Dinophyceae).

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: comparison of detection methods.

Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre- or postcolumn oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on-site monitoring. In this study, CE methods were developed for C(4) D, FLD, UV absorption detection, and MS-making this first report of C(4) D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE-UV and CZE-C(4) D methods provide better resolution, selectivity, and separation efficiency compared to CZE-MS and MEKC-FLD. The sensitivity of the CZE-C(4) D and MEKC-FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE-C(4) D and 60.9 to 104 ng/mL for MEKC-FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C(4) D and MEKC-FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE-C(4) D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was successfully used for PST screening of a periodate-oxidized mussel sample, with results confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in shellfish samples.

Exposure to the neurotoxic dinoflagellate, Alexandrium catenella, induces apoptosis of the hemocytes of the oyster, Crassostrea gigas.

This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.

Sex differences in effects of low level domoic acid exposure.

Consumption of seafood containing the phytoplankton-derived toxin domoic acid (DOM) causes neurotoxicity in humans and in animals. It has been reported that DOM-induced symptoms may be more severe in men than women, but to date the effect of sex on DOM-induced effects in adults is not known. We investigated sex differences in DOM-induced effects in adult rats. Since low level exposure is of greatest relevance to human health (due to DOM regulatory limit), we examined the effects of low level exposure. Adult male and female Sprague Dawley rats were administered a single intraperitoneal injection of DOM (0, 1.0, 1.8 mg/kg). Behaviour was monitored for 3h and immunohistochemistry in the dorsal hippocampus and olfactory bulb was also examined. DOM increased locomotor and grooming activity, compared to vehicle group. DOM exposure also significantly increased stereotypic behaviours and decreased phosphorylated cAMP response element-binding protein immunoreactivity (pCREB-IR). There was no effect of sex on the magnitude of the behavioural responses, but the onset of DOM-induced locomotor activity and ear scratches was quicker in females than in males. Mixed effect modelling revealed the predicted peak in locomotor activity in response to DOM was also quicker in females than in males. Severe toxicity was evident in 2/7 male rats and 0/8 female rats dosed with 1.8 mg/kg DOM. These data suggest that males exposed to low level DOM may be more susceptible to severe neurotoxicity, whereas females are affected more quickly. Understanding sex differences in DOM-induced neurotoxicity may contribute to future protective strategies and treatments.

Alterations in metabolism-related genes induced in SHSY5Y cells by okadaic acid exposure.

Okadaic acid (OA) is a widely distributed marine toxin produced by several phytoplanktonic species and responsible for diarrheic shellfish poisoning in humans. At the molecular level OA is a specific inhibitor of several types of serine/threonine protein phosphatases. Due to this enzymic inhibition, OA was reported to induce numerous alterations in relevant cellular physiological processes, including several metabolic pathways such as glucose uptake, lipolysis and glycolysis, heme metabolism, and glycogen and protein synthesis. In order to further understand the underlying mechanisms involved in OA-induced effects on cellular metabolism, the expression levels of six genes related to different catabolic and anabolic metabolism-related processes were analyzed by real-time polymerase chain reaction. Specifically, the expression patterns of GAPDH, TOMM5, SLC25A4, COII, QARS, and RGS5 genes were determined in SHSY5Y human neuroblastoma cells exposed to OA for 3, 24, or 48 h. All these genes showed alterations in their expression levels after at least one of the OA treatments tested. These alterations provide a basis to understand the mechanisms underlying the previously described OA-induced effects on different metabolic processes, mainly regarding glucose and mitochondrial metabolism. However, other OA-induced affected genes can not be ruled out, and further studies are required to more comprehensively characterize in the mechanisms of OA-induced interaction on cell metabolism.