Molecular forecasting of domoic acid during a pervasive toxic diatom bloom.

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis, this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis (dab) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters (sit1) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that coexpression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have colimited the diatom population along with low Si. Iron limitation represents an overlooked driver of both toxin production and ecological success of the low-iron-adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms.

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.

Identification of Candidate Protein Biomarkers Associated with Domoic Acid Toxicosis in Cerebrospinal Fluid of California Sea Lions (Zalophus californianus).

Since 1998, California sea lion (Zalophus californianus) stranding events associated with domoic acid toxicosis (DAT) have consistently increased. Outside of direct measurement of domoic acid in bodily fluids at the time of stranding, there are no practical nonlethal clinical tests for the diagnosis of DAT that can be utilized in a rehabilitation facility. Proteomics analysis was conducted to discover candidate protein markers of DAT using cerebrospinal fluid from stranded California sea lions with acute DAT (n = 8), chronic DAT (n = 19), or without DAT (n = 13). A total of 2005 protein families were identified experiment-wide. A total of 83 proteins were significantly different in abundance across the three groups (adj. p < 0.05). MDH1, PLD3, ADAM22, YWHAG, VGF, and CLSTN1 could discriminate California sea lions with or without DAT (AuROC > 0.75). IGKV2D-28, PTRPF, KNG1, F2, and SNCB were able to discriminate acute DAT from chronic DAT (AuROC > 0.75). Proteins involved in alpha synuclein deposition were over-represented as classifiers of DAT, and many of these proteins have been implicated in a variety of neurodegenerative diseases. These proteins should be considered potential markers for DAT in California sea lions and should be prioritized for future validation studies as biomarkers.

Astrocytic glutamate transport is essential for the memory-enhancing effects of 17ß-estradiol in ovariectomized mice.

Infusion of 17ß-estradiol (E2) into the dorsal hippocampus (DH) of ovariectomized (OVX) mice enhances memory consolidation, an effect that depends on rapid phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Astrocytic glutamate transporter 1 (GLT-1) modulates neurotransmission via glutamate uptake from the synaptic cleft. However, little is known about the contribution of DH astrocytes, and astrocytic glutamate transport, to the memory-enhancing effects of E2. This study was designed to test whether DH astrocytes contribute to estrogenic modulation of memory consolidation by determining the extent to which DH GLT-1 is necessary for E2 to enhance memory in object recognition and object placement tasks and trigger rapid phosphorylation events in DH astrocytes. OVX female mice were bilaterally cannulated into the DH or the DH and dorsal third ventricle (ICV). Post-training DH infusion of the GLT-1 inhibitor dihydrokainic acid (DHK) dose-dependently impaired memory consolidation in both tasks. Moreover, the memory-enhancing effects of ICV-infused E2 in each task were blocked by DH DHK infusion. E2 increased p42 ERK and Akt phosphorylation in DH astrocytes, and these effects were blocked by DHK. Results suggest the necessity of DH GLT-1 activity for object and spatial memory consolidation, and for E2 to enhance consolidation of these memories and to rapidly activate cell signaling in DH astrocytes. Findings indicate that astrocytic function in the DH of OVX females is necessary for memory formation and is regulated by E2, and suggest an essential role for DH astrocytic GLT-1 activity in the memory-enhancing effects of E2.

Spatiotemporal variations of domoic acid: New findings in the sedimentary environment of a typical nearshore mariculture bay, China.

Domoic acid (DA) is a neurotoxin produced by marine microalgae. It tends to accumulate in marine shellfish and fish, posing a threat to aquaculture and seafood consumers' health. In this study, DA in the surface and bottom seawater, sediment, and porewater of the Jiaozhou Bay, a typical mariculture bay in China, was systematically investigated for the first time over different seasons. Surprisingly, a high concentration of DA was discovered in the marine sediment porewater (maximum detected concentration: 289.49 ng/L) for the first time. DA was found to be extensively distributed in the water body and sedimentary environment of the Jiaozhou Bay. DA in the surface and bottom seawater of Jiaozhou Bay in spring was uniformly distributed, whereas DA showed obvious spatial variations in summer and winter. The high concentration areas of DA are located in the north of Jiaozhou Bay and decreased to the south areas. DA was also distributed in the sediment (spring mean: 316.57 ng/kg; summer mean: 10.22 ng/kg; winter mean: 237.08 ng/kg) and porewater (spring mean: 129.70 ng/L; summer mean: 53.54 ng/L; winter mean: 19.90 ng/L) of Jiaozhou Bay. The DA concentrations in the surface sediment and porewater were higher in the spring than in the winter and summer, contrary to the seasonal variation pattern observed in the surface and bottom seawater. The DA concentration in porewater was significantly higher than in the surface and bottom seawater, indicating that the risk of pollution contamination from DA to benthic fishery organisms may be underestimated. Overall, DA is widely distributed in the seawater and also in the benthic environment of Jiaozhou Bay and exhibited potential harm to fishery organisms varied greatly with seasons. It is an important discovery for marine algae toxins and has important guiding significance and important indicative role for the routine monitoring and management of DA pollution in water and benthic environment.

Molecular Dynamics Simulation Reveal the Structure-Activity Relationships of Kainoid Synthases.

Kainoid synthases are key enzymes in the biosynthesis of kainoids. Kainoids, as represented by DA and KA, are a class of naturally occurring non-protein amino acids with strong neurotransmitter activity in the mammalian central nervous system. Marine algae kainoid synthases include PnDabC from diatoms, which synthesizes domoic acid (DA), and DsKabC and GfKabC from red algae, which synthesize kainic acid (KA). Elucidation of the catalytic mechanism of kainoid synthases is of great significance for the rational design of better biocatalysts to promote the industrial production of kainoids for use in new drugs. Through modeling, molecular docking, and molecular dynamics simulations, we investigated the conformational dynamics of kainoid synthases. We found that the kainoid synthase complexes showed different stability in the simulation, and the binding and catalytic processes showed significant conformational transformations of kainoid synthase. The residues involved in specific interactions with the substrate contributed to the binding energy throughout the simulation process. Binding energy, the relaxed active pocket, electrostatic potential energy of the active pocket, the number and rotation of aromatic residues interacting with substrates during catalysis, and the number and frequency of hydrogen bonds between the individual functional groups revealed the structure-activity relationships and affected the degree of promiscuity of kainoid synthases. Our research enriches the understanding of the conformational dynamics of kainoid synthases and has potential guiding significance for their rational design.

Understanding the risks of co-exposures in a changing world: a case study of dual monitoring of the biotoxin domoic acid and Vibrio spp. in Pacific oyster.

Assessing the co-occurrence of multiple health risk factors in coastal ecosystems is challenging due to the complexity of multi-factor interactions and limited availability of simultaneously collected data. Understanding co-occurrence is particularly important for risk factors that may be associated with, or occur in similar environmental conditions. In marine ecosystems, the co-occurrence of harmful algal bloom toxins and bacterial pathogens within the genus Vibrio may impact both ecosystem and human health. This study examined the co-occurrence of Vibrio spp. and domoic acid (DA) produced by the harmful algae Pseudo-nitzschia by (1) analyzing existing California Department of Public Health monitoring data for V. parahaemolyticus and DA in oysters; and (2) conducting a 1-year seasonal monitoring of these risk factors across two Southern California embayments. Existing public health monitoring efforts in the state were robust for individual risk factors; however, it was difficult to evaluate the co-occurrence of these risk factors in oysters due to low number of co-monitoring instances between 2015 and 2020. Seasonal co-monitoring of DA and Vibrio spp. (V. vulnificus or V. parahaemolyticus) at two embayments revealed the co-occurrence of these health risk factors in 35% of sampled oysters in most seasons. Interestingly, both the overall detection frequency and co-occurrence of these risk factors were considerably less frequent in water samples. These findings may in part suggest the slow depuration of Vibrio spp. and DA in oysters as residual levels may be retained. This study expanded our understanding of the simultaneous presence of DA and Vibrio spp. in bivalves and demonstrates the feasibility of co-monitoring different risk factors from the same sample. Individual programs monitoring for different risk factors from the same sample matrix may consider combining efforts to reduce cost, streamline the process, and better understand the prevalence of co-occurring health risk factors.

Algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into an N-prenyltransferase.

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.

Potential endocrine correlation with exposure to domoic acid in Southern Right Whale (Eubalaena australis) at the Península Valdés breeding ground.

In waters off Península Valdés (PV), Argentina, southern right whales (SRW, Eubalaena australis) are occasionally exposed to domoic acid (DA), a neurotoxin produced by diatoms of the genus Pseudo-nitzschia. Domoic acid toxicity in marine mammals can cause gastrointestinal and neurological clinical signs, alterations in hematologic and endocrine variables, and can be fatal in extreme cases. In this study, we validated an enzyme immunoassay to quantify fecal glucocorticoid metabolites (fGCm) in 16 SRW fecal samples from live and dead stranded whales in PV from 2013 to 2018 and assessed fGCm levels associated with DA exposure. Overall, fGCm levels were significantly lower in SRWs with detectable fecal DA (n = 3) as compared to SRWs with undetectable fecal DA levels (n = 13). The highest fecal DA was observed in a live lactating female, which had low fGCm compared to the other lactating females studied. The highest fGCm was observed in a lactating female with undetectable DA; interestingly, at the time of sample collection, this female was sighted with two calves, an extremely unusual occurrence in this species. Though the sample size of these exceptionally rare breeding-season fecal samples was unavoidably small, our study provides evidence of potential adrenal alterations in whales exposed to an environmental neurotoxin such as DA.

Response mechanism of microbial community during anaerobic biotransformation of marine toxin domoic acid.

Domoic acid (DA) is a potent neurotoxin produced by toxigenic Pseudo-nitzschia blooms and quickly transfers to the benthic anaerobic environment by marine snow particles. DA anaerobic biotransformation is driven by microbial interactions, in which trace amounts of DA can cause physiological stress in marine microorganisms. However, the underlying response mechanisms of microbial community to DA stress remain unclear. In this study, we utilized an anaerobic marine DA-degrading consortium GLY (using glycine as co-substrate) to systematically investigate the global response mechanisms of microbial community during DA anaerobic biotransformation.16S rRNA gene sequencing and metatranscriptomic analyses were applied to measure microbial community structure, function and metabolic responses. Results showed that DA stress markedly changed the composition of main species, with increased levels of Firmicutes and decreased levels of Proteobacteria, Cyanobacteria, Bacteroidetes and Actinobacteria. Several genera of tolerated bacteria (Bacillus and Solibacillus) were increased, while, Stenotrophomonas, Sphingomonas and Acinetobacter were decreased. Metatranscriptomic analyses indicated that DA stimulated the expression of quorum sensing, extracellular polymeric substance (EPS) production, sporulation, membrane transporters, bacterial chemotaxis, flagellar assembly and ribosome protection in community, promoting bacterial adaptation ability under DA stress. Moreover, amino acid metabolism, carbohydrate metabolism and lipid metabolism were modulated during DA anaerobic biotransformation to reduce metabolic burden, increase metabolic demands for EPS production and DA degradation. This study provides the new insights into response of microbial community to DA stress and its potential impact on benthic microorganisms in marine environments.

Immuno-Enriched Microspheres - Magnetic Blade Spray-Tandem Mass Spectrometry for Domoic Acid in Mussels.

Paramagnetic microspheres can be used in planar array fluorescence immunoassays for single or multiplex screening of food contaminants. However, no confirmation of the molecular identity is obtained. Coated blade spray (CBS) is a direct ionization mass spectrometry (MS) technique, and when combined with triple quadrupole MS/MS, it allows for rapid confirmation of food contaminants. The lack of chromatography in CBS, though, compromises the specificity of the measurement for unequivocal identification of contaminants, based on the European Union (EU) regulation. Therefore, a rapid and easy-to-use immuno-magnetic blade spray (iMBS) method was developed in which immuno-enriched paramagnetic microspheres replace the coating of CBS. The iMBS-MS/MS method was fully optimized, validated in-house following the EU 2021/808 regulation, and benchmarked against a commercial lateral flow immunoassay (LFIA) for on-site screening of DA. The applicability of iMBS-MS/MS was further demonstrated by analyzing incurred mussel samples. The combination of immunorecognition and MS/MS detection in iMBS-MS/MS enhances the measurement's selectivity, which is demonstrated by the rapid differentiation between the marine toxin domoic acid (DA) and its structural analog kainic acid (KA), which cannot be achieved with the LFIA alone. Interestingly, this first-ever reported iMBS-MS/MS method is generic and can be adapted to include any other immuno-captured food contaminant, provided that monoclonal antibodies are available, thus offering a complementary confirmatory analysis approach to multiplex immunoassay screening methods. Moreover, thanks to its speed of analysis, iMBS-MS/MS can bridge the logistics gap between future large-scale on-site testings using LFIAs and classical time-consuming confirmatory MS analysis performed in official control laboratories.

Behavioural effects in mice orally exposed to domoic acid or ibotenic acid are influenced by developmental stages and sex differences.

The structure of the brain is dramatically altered during the critical period. Physiological substances (neurotransmitters, hormones, etc.) in the body fluctuate significantly before and after sexual maturation. Therefore, the effect of chemical exposure on the central nervous system often differs depending on the developmental stage and sex. We aimed to compare the behavioural effects that emerged from the administration of chemicals to mice of different life stages (immature or mature) and different sex (male or female). We administered mice with domoic acid (DA), a marine poison, and ibotenic acid (IA), found in poisonous mushrooms. These excitatory amino acids act as agonists for glutamate and are potent neurotoxins. Interestingly, the behavioural effects of these chemicals were completely different. Following DA administration, we observed memory deficits only in groups of male mice treated at maturity. Following IA administration, we observed deviations in emotional behaviour in groups of male mice treated at both immaturity and maturity. In contrast, few characteristic changes were detected in all groups of females. Our results support the theory that the behavioural effects of chemical administration vary considerably with developmental stages and sex. In conclusion, our findings promote better understanding of individual differences in excitatory chemical-induced neurotoxicity and provide evidence for future risk strategies and treatments.

Preparation of domoic acid analogues using a bioconversion system, and their toxicity in mice.

Domoic acid (1, DA), a member of the natural kainoid family, is a potent agonist of ionotropic glutamate receptors in the central nervous system. The chemical synthesis of DA and its derivatives requires considerable effort to establish a pyrrolidine ring containing three contiguous stereocenters. Recently, a biosynthetic cyclase for DA, DabC, was identified. This enzyme cyclizes the linear precursor of isodomoic acid A (IA) to IA, a bioactive DA analogue. In this study, we developed a bioconversion system to obtain DA analogues from linear substrates prepared by simple chemical synthesis using DabC expressed in Escherichia coli, in vivo. Three IA analogues with various substitutions at the C7'-geranyl terminus were prepared using this system: two minor natural analogues, 7'-methyl-IA (5) and 7'-hydroxy-IA (6), and one new unnatural analogue, 7'-amide-IA (7). In addition, the toxicity of these DA analogues in mice was examined by intracerebroventricular injection. Most of the mice injected with 5 (3 nmol) and 6 (3 nmol) did not show any adverse symptoms, whereas the mice injected with 7 (3 nmol) showed typical symptoms induced by DA (1, 0.7 nmol) and IA (2, 3 nmol). These results suggest that the 7'-carbonyl group in the side chain of IA (2) is crucial for its toxicity. The docking studies of DA, IA (2), 5, 6, and 7 to GluK1 supported these results.

A mussel tissue certified reference material for multiple phycotoxins. Part 5: profiling by liquid chromatography-high-resolution mass spectrometry.

A freeze-dried mussel tissue-certified reference material (CRM-FDMT1) was prepared containing the marine algal toxin classes azaspiracids, okadaic acid and dinophysistoxins, yessotoxins, pectenotoxins, cyclic imines, and domoic acid. Thus far, only a limited number of analogues in CRM-FDMT1 have been assigned certified values; however, the complete toxin profile is significantly more complex. Liquid chromatography-high-resolution mass spectrometry was used to profile CRM-FDMT1. Full-scan data was searched against a list of previously reported toxin analogues, and characteristic product ions extracted from all-ion-fragmentation data were used to guide the extent of toxin profiling. A series of targeted and untargeted acquisition MS/MS experiments were then used to collect spectra for analogues. A number of toxins previously reported in the literature but not readily available as standards were tentatively identified including dihydroxy and carboxyhydroxyyessotoxin, azaspiracids-33 and -39, sulfonated pectenotoxin analogues, spirolide variants, and fatty acid acyl esters of okadaic acid and pectenotoxins. Previously unreported toxins were also observed including compounds from the pectenotoxin, azaspiracid, yessotoxin, and spirolide classes. More than one hundred toxin analogues present in CRM-FDMT1 are summarized along with a demonstration of the major acyl ester conjugates of several toxins. Retention index values were assigned for all confirmed or tentatively identified analogues to help with qualitative identification of the broad range of lipophilic toxins present in the material.

A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca2+-signalling.

Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.

Stability of Domoic Acid in 50% Methanol Extracts and Raw Fecal Material from Bowhead Whales (Balaena mysticetus).

Domoic acid (DA), the toxin causing amnesic shellfish poisoning (ASP), is produced globally by some diatoms in the genus Pseudo-nitzschia. DA has been detected in several marine mammal species in the Alaskan Arctic, raising health concerns for marine mammals and subsistence communities dependent upon them. Gastrointestinal matrices are routinely used to detect Harmful Algal Bloom (HAB) toxin presence in marine mammals, yet DA stability has only been studied extensively in shellfish-related matrices. To address this knowledge gap, we quantified DA in bowhead whale fecal samples at multiple time points for two groups: (1) 50% methanol extracts from feces, and (2) raw feces stored in several conditions. DA concentrations decreased to 70 ± 7.1% of time zero (T0) in the 50% methanol extracts after 2 weeks, but remained steady until the final time point at 5 weeks (66 ± 5.7% T0). In contrast, DA concentrations were stable or increased in raw fecal material after 8 weeks of freezer storage (-20 °C), at room temperature (RT) in the dark, or refrigerated at 1 °C. DA concentrations in raw feces stored in an incubator (37 °C) or at RT in the light decreased to 77 ± 2.8% and 90 ± 15.0% T0 at 8 weeks, respectively. Evaporation during storage of raw fecal material is a likely cause of the increased DA concentrations observed over time with the highest increase to 126 ± 7.6% T0 after 3.2 years of frozen storage. These results provide valuable information for developing appropriate sample storage procedures for marine mammal fecal samples.

Capacity of the potentially toxic diatoms Pseudo-nitzschia mannii and Pseudo-nitzschia hasleana to tolerate polycyclic aromatic hydrocarbons.

This study investigates the effects of polycyclic aromatic hydrocarbons (PAHs) on two potentially toxic Pseudo-nitzschia hasleana and P. mannii, isolated from a PAH contaminated marine environment. Both species, maintained in non-axenic cultures, have been exposed during 144 h to increasing concentrations of a 15 PAHs mixture. Analysis of the domoic acid, showed very low concentrations. Dose-response curves for growth and photosynthesis inhibition were determined. Both species have maintained their growth until the end of incubation even at the highest concentration tested (120 µg l-1), Nevertheless, P mannii showed faster growth and seemed to be more tolerant than P. hasleana. To reduce PAH toxicity, both species have enhanced their biovolume, with a higher increase for P. mannii relative to P hasleana. Both species were also capable of bio-concentrating PAHs and were able to degrade them probably in synergy with their associated bacteria. The highest biodegradation was observed for P. mannii, which could harbored more efficient hydrocarbon-degrading bacteria. This study provides the first evidence that PAHs can control the growth and physiology of potentially toxic diatoms. Future studies should investigate the bacterial community associated with Pseudo-nitzschia species, as responses to pollutants or to other environmental stressors could be strongly influence by associated bacteria.

Occurence and Variability of Domoic Acid in Mussel (Mytilus galloprovincialis) Samples from the Golden Horn Estuary, Sea of Marmara (Turkey).

The occurrence and variability of domoic acid (DA) levels in wild Mytilus galloprovincialis samples, compared with the Pseudo-nitzschia spp. abundance and particulate DA (pDA) concentrations in relation to the environmental changes in the Golden Horn Estuary, Turkey from October 2018 to September 2019. Biotoxin analysis were performed by high-performance liquid chromatography with diode-array detection (HPLC-DAD). DA concentrations in particulate matter (pDA) and mussel samples were found between 0.090-0.685 µg L-1 and 0.905-2.413 µg g-1, respectively. Accumulation of DA in wild mussel samples could be the result of the increasing tendency of P.nitzschia spp. abundances between April and May. Maximum DA levels were detected in particulate matter when the salinity was measured as the lowest in May. Thus, it can be said that the DA production was driven by the significant salinity decrease in the GHE. This is the first attempt regarding the presence of DA in M. galloprovincialis samples collected from Turkish coasts.

PHARMACOKINETICS OF SUBCUTANEOUS ALPHA LIPOIC ACID, A PROPOSED THERAPEUTIC AID FOR DOMOIC ACID INTOXICATION IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

Domoic acid (DA) is a potent neurotoxin produced by certain marine algae that can cause neurologic and cardiac dysfunction by activating glutamate receptors. Glutamate receptor overexcitation results in continuous cellular activation, oxidative damage, and cell death. DA toxicosis causes disorientation and seizures, and antiseizure medications are the primary treatment. Alpha lipoic acid (ALA), a powerful antioxidant and glutathione precursor widely used in humans and dogs, can cross the blood-brain barrier to provide antioxidant availability to brain tissue. Hundreds of stranded California sea lions (CSL; Zalophus californianus) are diagnosed annually with DA toxicosis and thus are an appropriate animal in which to establish ALA dosing recommendations for treatment. The objective of this study was to determine the population pharmacokinetics of a single 10- or 20-mg/kg dose of ALA administered subcutaneously into the interscapular region to healthy rehabilitated CSL. Blood was collected at two time points between 15 min and 24 h after administration. Serum ALA concentrations were measured by liquid chromatography-mass spectrometry, and parameters were evaluated using a nonlinear mixed effects model. ALA was rapidly absorbed for each dose, peaking within 20 to 30 minutes, and t1/2 of 40 and 32 min (10 and 20 mg/kg, respectively), followed by an initial steep distribution phase and prolonged elimination phase. Peak concentration (CMAX) was 1,243 ng/ml (10-mg/ml dose) and 5,010 ng/ml (20-mg/ml dose). Serum from 13 CSLd with DA toxicosis treated with 10 mg/kg ALA for 1 to 9 d had measurable levels, and ALA was also measurable in cerebrospinal fluid from two treated CSLs. Therapeutic effects are noted with a CMAX of 4,000 to 5,000 ng/ml in humans; thus in CSLs, 20 mg/kg administered subcutaneously once daily may be sufficient to achieve a therapeutic level in this species. Determination of efficacy and optimal dosing interval and duration require additional investigation.

[Determination of domoic acid in shellfish by liquid chromatography- tandem mass spectrometry coupled with dispersive solid phase extraction].

OBJECTIVE: A method utilizing liquid chromatography-tandem mass spectrometry(LC-MS/MS) coupled with dispersive solid phase extraction for quantitative analysis of domoic acid in four kinds of shellfish was established. METHODS: The sample of 0. 1 g was extracted with 25% methanol aqueous solution, the extract was purified by dispersive solid phase extraction with 50 mg HLB and 5 mg GCB, and then filtered through a PTFE membrane. The analytes were separated on a C_(18) column(100 mm×2. 1 mm, 1. 9 µm), and detected in selected reaction monitoring(SRM) mode via positive electrospray ionization. The matrix matching and external standard method was used for quantitation. RESULTS: Domoic acid showed good linearity in the concentration range between 1. 0 ng/mL and 50. 0 ng/mL with correlation coefficients higher than 0. 9994. The detection limits of domoic acid in shellfish was 5 µg/kg. The inter-and intra-day recoveries were 91. 6%-109. 2% and 90. 9%-109. 3%, respectively. The inter-and intra-day ralitive standard deviations(RSDs) were lower than 8. 2% at spiked concentrations of 20, 50 and 100 µg/kg. CONCLUSION: The method is accurate, fast, easy to operate, which can satisfy the requirements of public health emergency testing or routine testing.