Toxicokinetics and Physiologically Based Pharmacokinetic Modeling of the Shellfish Toxin Domoic Acid in Nonhuman Primates.

Domoic acid (DA), a neurotoxin, is produced by marine algae and has caused toxications worldwide in animals and humans. However, the toxicokinetics of DA have not been fully evaluated, and information is missing on the disposition of DA following oral exposures at doses that are considered safe for human consumption. In this study, toxicokinetics of DA were investigated in cynomolgus monkeys following single doses of 5 µg/kg DA intravenously, 0.075 mg/kg DA orally, and 0.15 mg/kg DA orally. After intravenous dosing, DA had a systemic clearance of 124 ± 71 (ml/h)/kg, volume of distribution at steady state of 131 ± 71 ml/kg and elimination half-life of 1.2 ± 1.1 hours. However, following oral dosing, the average terminal half-life of DA was 11.3 ± 2.4 hours, indicating that DA disposition follows flip-flop kinetics with slow, rate-limiting absorption. The absorption of DA was low after oral dosing with absolute bioavailability of 6% ± 4%. The renal clearance of DA was variable [21-152 (ml/h)/kg] with 42% ± 11% of the intravenous DA dose recovered in urine. A physiologically based pharmacokinetic model was developed for DA in monkeys and humans that replicated the flip-flop kinetics observed after oral administration and allowed simulation of urinary excretion and brain and kidney distribution of DA following intravenous and oral dosing. This study is the first to characterize DA disposition at exposure levels close to the current estimated tolerable daily intake and to mechanistically model DA disposition in a model species, providing important information of the toxicokinetics of DA for human safety assessment.

ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs.

The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate was 2.4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs.

Characterization of renal toxicity in mice administered the marine biotoxin domoic Acid.

Domoic acid (DA), an excitatory amino acid produced by diatoms belonging to the genus Pseudo-nitzschia, is a glutamate analog responsible for the neurologic condition referred to as amnesic shellfish poisoning. To date, the renal effects of DA have been underappreciated, although renal filtration is the primary route of systemic elimination and the kidney expresses ionotropic glutamate receptors. To characterize the renal effects of DA, we administered either a neurotoxic dose of DA or doses below the recognized limit of toxicity to adult Sv128/Black Swiss mice. DA preferentially accumulated in the kidney and elicited marked renal vascular and tubular damage consistent with acute tubular necrosis, apoptosis, and renal tubular cell desquamation, with toxic vacuolization and mitochondrial swelling as hallmarks of the cellular damage. Doses≥0.1 mg/kg DA elevated the renal injury biomarkers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, and doses≥0.005 mg/kg induced the early response genes c-fos and junb. Coadministration of DA with the broad spectrum excitatory amino acid antagonist kynurenic acid inhibited induction of c-fos, junb, and neutrophil gelatinase-associated lipocalin. These findings suggest that the kidney may be susceptible to excitotoxic agonists, and renal effects should be considered when examining glutamate receptor activation. Additionally, these results indicate that DA is a potent nephrotoxicant, and potential renal toxicity may require consideration when determining safe levels for human exposure.

Elimination kinetics of domoic acid from the brain and cerebrospinal fluid of the pregnant rat.

Domoic acid (DA) causes neurological effects in multiple species upon exposure, including status epilepticus in pregnant sea lions and an epileptic disease state that commonly develops in juveniles. This study aims to define brain toxicokinetic parameters in the pregnant rat in the larger context of maternal-fetal toxin transfer. Specifically, Sprague-Dawley rats were exposed to a low observable effect level of 1.0 mg DA/kg intravenously at gestational day 20, and plasma, brain, and cerebrospinal fluid (CSF) samples were taken at discrete time points over 24 h. Domoic acid concentrations were determined by a tandem LC/MS method recently optimized for brain tissue and CSF. Data showed that 6.6% of plasma DA reached the brain, 5.3% reached the CSF, and DA levels were nearly identical in both brain and CSF for 12 h, remaining above the threshold to activate isolated hippocampal neurons for 2 h. The calculated terminal half-life of CSF was 4 h, consistent with the time for complete CSF regeneration, suggesting that CSF acts as a mechanism to clear DA from the brain.

Toxicokinetics of domoic acid in the fetal rat.

Domoic acid (DA) is a potent neurotoxin that has both marine wildlife and human health impacts, including developmental effects during prenatal exposure in rodent models. However, little is known regarding DA toxicokinetics in the fetal unit during maternal-fetal transfer. Tissue distribution and toxicokinetics of DA were investigated in pregnant rats and their pups just prior to birth at gestational day 20. Pregnant Sprague Dawley rats were given an intravenous dose of 1.0 mg DA/kg and samples of maternal plasma, fetal plasma, placenta, amniotic fluid and fetal brain were taken at intervals over 24 h. Toxicokinetic parameters were determined using WinNonLin software analysis. Maternal plasma DA log concentration-time curves fit a two compartment pharmacokinetic profile, with alpha and beta half-lives of elimination of 26.9 and 297 min, respectively. Placenta had a C(max) of 752 ng/mL and a terminal half-life of 577 min. Maternal-fetal transfer between the plasma compartments was 31% with a fetal plasma C(max) of 86 ng/mL at 60 min and terminal half-life of 553 min. Amniotic fluid and fetal brain had overall averages of 27±12 ng/mL and 8.12 ng/g, respectively, and did not show evidence of elimination over 24 h. The longer fetal retention of DA, particularly in amniotic fluid, indicates that the fetus may be continually re-exposed during gestation, which could potentially lead to a disease state even at small exposure dose. This has implications for the California sea lions (Zalophus californianus), which exhibit an epilepsy-like disease that arises months after DA producing blooms.

Endangered North Atlantic right whales (Eubalaena glacialis) experience repeated, concurrent exposure to multiple environmental neurotoxins produced by marine algae.

The western North Atlantic population of right whales (Eubalaena glacialis) is one of the most critically endangered of any whale population in the world. Among the factors considered to have potentially adverse effects on the health and reproduction of E. glacialis are biotoxins produced by certain microalgae responsible for causing harmful algal blooms. The worldwide incidence of these events has continued to increase dramatically over the past several decades and is expected to remain problematic under predicted climate change scenarios. Previous investigations have demonstrated that N. Atlantic right whales are being exposed to at least two classes of algal-produced environmental neurotoxins-paralytic shellfish toxins (PSTs) and domoic acid (DA). Our primary aims during this six-year study (2001-2006) were to assess whether the whales' exposure to these algal biotoxins occurred annually over multiple years, and to what extent individual whales were exposed repeatedly and/or concurrently to one or both toxin classes. Approximately 140 right whale fecal samples obtained across multiple habitats in the western N. Atlantic were analyzed for PSTs and DA. About 40% of these samples were attributed to individual whales in the North Atlantic Right Whale Catalog, permitting analysis of biotoxin exposure according to sex, age class, and reproductive status/history. Our findings demonstrate clearly that right whales are being exposed to both of these algal biotoxins on virtually an annual basis in multiple habitats for periods of up to six months (April through September), with similar exposure rates for females and males (PSTs: ∼70-80%; DA: ∼25-30%). Notably, only one of 14 lactating females sampled did not contain either PSTs or DA, suggesting the potential for maternal toxin transfer and possible effects on neonatal animals. Moreover, 22% of the fecal samples tested for PSTs and DA showed concurrent exposure to both neurotoxins, leading to questions of interactive effects. Targeted studies employing both in vivo and in vitro model systems represent the next logical step in assessing how and to what extent these algal biotoxins might compromise the health and reproduction of this endangered population.

Transcellular transport of domoic acid across intestinal Caco-2 cell monolayers.

The intestinal absorption mechanism of domoic acid (DA) was investigated using Caco-2 cells. DA is a tricarboxylic amino acid that contains a glutamic acid moiety, and causes deficits in short-term memory by binding to glutamate receptors as an agonist of glutamic acid. Caco-2 cell monolayers cultured on permeable membranes were incubated with 100 µM DA on either the apical or basolateral side, and the transcellular transport of DA was measured. The transcellular transport of DA from the apical to basolateral side was about twofold that in the opposite direction. The transcellular transport of DA from the apical side was optimal at a neutral pH, and was temperature- and Cl(-)-dependent, but was Na(+)-independent. Coincubation of DA with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, significantly decreased the apical-to-basolateral transport of DA by 48%, and coincubation with probenecid (a non-specific anion transport inhibitor) significantly decreased the transport of DA by 31%. In contrast, coincubation with glutamic acid, succinic acid (a dicarboxylic acid), or citric acid (a tricarboxylic acid) did not decrease the transport of DA. These results suggest that the apical-to-basolateral transport of DA across the Caco-2 cell monolayers is mediated by DIDS-sensitive anion transporters.

Not all desensitizations are created equal: physiological evidence that AMPA receptor desensitization differs for kainate and glutamate.

AMPA receptor-mediated responses to the agonist kainate differ from those of glutamate in two important respects. Glutamate is a full agonist that elicits strongly desensitizing responses, whereas kainate is a partial agonist with responses that are often described as weakly desensitizing or non-desensitizing. The efficacy of kainate relative to glutamate has previously been shown to be increased by mutations in the AMPA receptor ligand-binding cleft (Mano et al., 1996) and by coexpression with the auxiliary subunit stargazin (Tomita et al., 2005; Turetsky et al., 2005), but much less is known about factors that affect kainate desensitization. We therefore designed experiments to compare kainate and glutamate desensitization and efficacy in wild-type and mutant AMPA receptors expressed with and without stargazin in HEK293 cells. Desensitization to the two agonists was differentially affected by mutations in the helices participating in bonds between two subunits in the active state of the receptor (Sun et al., 2002), indicating that the protein interactions maintaining the stability of the dimer interface differ depending on which agonist is bound. Kainate efficacy was affected by factors distinct from ligand-binding cleft closure, including mutations in the dimer interface and channel vestibule as well as receptor composition. The increase in kainate responses for AMPA receptors coexpressed with stargazin was the result of both reduced kainate desensitization and increased kainate efficacy. These results provide critical new insights into the agonist dependence of both AMPA receptor activation and desensitization and the mechanism of the effects of stargazin on responses of partial agonists.

Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1.

Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.

Decreased glutamine synthetase, increased citrulline–nitric oxide cycle activities, and oxidative stress in different regions of brain in epilepsy rat model

No disponible

To understand their role in epilepsy, the nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite (NOx), thiobarbituric acid reactive substances (TBARS), and total antioxidant status (TAS), were estimated in different regions of brain in rats subjected to experimental epilepsy induced by subcutaneous administration of kainic acid (KA). The short-term (acute) group animals were killed after 2 h and the long term (chronic) group animals were killed after 5 days of single injection of KA (15 mg/kg body weight). After decapitation of rats, the brain regions were separated and in their homogenates, the concentration of NOx, TBARS and TAS and the activities of NOS, AS, AL, arginase and glutamine synthetase were assayed by colorimetric methods. The results of the study demonstrated the increased activity of NOS and formation of NO in acute and chronic groups epilepsy. The activities of AS and AL were increased and indicate the effective recycling of citrulline to arginine. The activity of glutamine synthetase was decreased in acute and chronic groups of epilepsy compared to control group and indicate the modulation of its activity by NO in epilepsy. The activity of arginase was not changed in acute group; however it was decreased in chronic group and may favor increased production of NO in this condition. The concentration TBARS were increased and TAS decreased in acute and chronic groups of epilepsy and supports the oxidative stress in epilepsy (AU)

In vivo seizure induction and affinity studies of domoic acid and isodomoic acids-D, -E and -F.

Domoic acid and its isomers are produced via algal blooms and are found in high concentrations in shellfish. Here, we assessed the acute seizurogenic potencies of isomers-D, -E and -F and their binding affinities at heterogeneous populations of KA receptors from rat cerebrum. In addition, binding affinities of all six isomers (Iso-A through -F) were assessed at AMPA receptors. Radioligand displacement studies indicated that the seizurogenic potency of Iso-F (E-configuration) closely correlates with its affinities at both KA and AMPA receptors, whereas isomers-D (Z) and -E (E), which exhibit distinctly lower seizurogenic potencies, are quite weak displacers. Previously observed functional potencies for isomers-A, -B and -C (Sawant et al., 2008) correlated with AMPA receptor affinities observed here. Taken together, these findings call into question previous structure-activity rules. Significantly, in our hands, Iso-D was ten-fold less potent than Iso-F. To further explain observed links between structural conformation and functional potency, molecular modeling was employed. Modeling results closely matched the rank order of potency and binding data observed. We further assessed the efficacy of isomers-D, -E and -F as pharmacological preconditioning agents. Acute preconditioning with low-dose Iso-D, -E or -F, before high-dose DA failed to impart behavioural tolerance. This study has shed new light on structural conformations affecting non-NMDA ionotropic glutamate receptor binding and functional potency, and provides a foundation for future work in areas of AMPA and KA receptor modeling.

Feeding mechanics as the basis for differential uptake of the neurotoxin domoic acid by oysters, Crassostrea virginica, and mussels, Mytilus edulis.

The neurotoxin domoic acid (DA), produced by diatoms Pseudo-nitzschia spp., is transferred to humans via consumption of contaminated bivalves. This study examines feeding mechanisms, namely reduced filtration, pre-ingestive rejection and poor absorption, that might explain the comparatively low DA levels commonly found in oysters during toxic Pseudo-nitzschia blooms. Clearance rate (CR), absorption efficiency (AE) of organic matter and selective rejection in pseudofeces of oysters (Crassostrea virginica) and mussels (Mytilus edulis) were investigated in relation to the DA levels accumulated during 2-wk, simultaneous exposure to toxic Pseudo-nitzschia multiseries. Effects of temperature and P. multiseries cell size were also tested to identify conditions, if any, under which oysters can accumulate unsafe DA levels. Oysters accumulated 3.0-7.5x less DA than mussels from a short-celled P. multiseries clone (length=24microm) at 12 degrees C. This was related to the 7.4-8.5x lower CRs determined for oysters relative to mussels at this temperature. Exposure to a longer-celled P. multiseries clone (81microm) resulted in up to 70x lower toxin levels in oysters compared to mussels, which was attributed to differential feeding selectivity. Mussels were unable to discriminate between long- and short-celled P. multiseries clones from a mixed suspension, whereas oysters were previously shown to preferentially reject long cells (>70microm) in pseudofeces. Both bivalves selectively rejected P. multiseries cells from mixed suspensions containing a flagellate but not another diatom. AE of organics from P. multiseries cells by oysters and mussels was comparably low (42 and 39%, respectively) and thus unlikely to explain their differential DA accumulation. CR and DA uptake by oysters were negligible at

Distribution and linkage of domoic acid (amnesic shellfish poisoning toxins) in subcellular fractions of the digestive gland of the scallop Pecten maximus.

The king scallop Pecten maximus accumulates domoic acid, the main amnesic shellfish poisoning toxin, in the digestive gland for a long time. To try to find if the cause of this characteristic is the binding of the toxin to some cellular component, the subcellular distribution of domoic acid in the cells of the digestive gland was studied, by means of serial centrifugation, ultrafiltration and size exclusion chromatography (SEC). Domoic acid was found to be present mostly in soluble form in the cytosol, as more than 90% was found in the supernatant after a centrifugation of 1h at 45,000 x g, and passed a 10 kDa ultrafilter. The retention time of the peak with an absorption maximum of 242 nm--the one characteristic of domoic acid--observed in the SEC chromatograms of the scallop samples was found identical to be one of a reference solution of the toxin, indicating therefore, that domoic acid is free in the cytosol of the digestive gland of Pecten maximus. This finding turns the focus from binding to the lack of membrane transporters in this species of the scallop as the cause of the long retention time of domoic acid in this species.

Pharmacological activity of C10-substituted analogs of the high-affinity kainate receptor agonist dysiherbaine.

Kainate receptor antagonists have potential as therapeutic agents in a number of neuropathologies. Synthetic modification of the convulsant marine toxin neodysiherbaine A (NDH) previously yielded molecules with a diverse set of pharmacological actions on kainate receptors. Here we characterize three new synthetic analogs of NDH that contain substituents at the C10 position in the pyran ring of the marine toxin. The analogs exhibited high-affinity binding to the GluK1 (GluR5) subunit and lower affinity binding to GluK2 (GluR6) and GluK3 (GluR7) subunits in radioligand displacement assays with recombinant kainate and AMPA receptors. As well, the natural toxin NDH exhibited approximately 100-fold selectivity for GluK2 over GluK3 subunits, which was attributable to the C8 hydroxyl group in NDH. We used molecular dynamic simulations to determine the specific interactions between NDH and residues within the ligand-binding domains of these two kainate receptor subunits that contribute to the divergent apparent affinities for the compound. These data demonstrate that interactions with the GluK1 subunit are preserved in analogs with substitutions at C10 in NDH and further reveal the determinants of selectivity and pharmacological activity of molecules acting on kainate receptor subunits, which could aid in design of additional compounds that target these receptors.

DDT exposure of zebrafish embryos enhances seizure susceptibility: relationship to fetal p,p'-DDE burden and domoic acid exposure of California sea lions.

BACKGROUND: California sea lions have a large body burden of organochlorine pesticides, and over the last decade they have also been subject to domoic acid poisoning. Domoic acid poisoning, previously recognized in adult animals, is now viewed as a major cause of prenatal mortality. The appearance of a chronic juvenile domoic acid disease in the sea lions, characterized by behavioral abnormalities and epilepsy, is consistent with early life poisoning and may be potentiated by organochlorine burden. OBJECTIVE: We investigated the interactive effect of DDT (dichlorodiphenyltrichloroethane) on neurodevelopment using a zebrafish (Danio rerio) model for seizure behavior to examine the susceptibility to domoic acid-induced seizures after completion of neurodevelopment. METHODS: Embryos were exposed (6-30 hr postfertilization) to either o,p'-DDT or p,p'-DDE (dichlorodiphenyldichloroethylene) during neurodevelopment via a 0.1% dimethyl sulfoxide solution. These larval (7 days postfertilization) fish were then exposed to either the seizure-inducing drug pentylenetetrazol (PTZ) or domoic acid; resulting seizure behavior was monitored and analyzed for changes using cameras and behavioral tracking software. RESULTS: Embryonic exposure to DDTs enhanced PTZ seizures and caused distinct and increased seizure behaviors to domoic acid, most notably a type of head-shaking behavior. CONCLUSION: These studies demonstrate that embryonic exposure to DDTs leads to asymptomatic animals at completion of neurodevelopment with greater sensitivity to domoic acid-induced seizures. The body burden levels of p,p'-DDE are close to the range recently found in fetal California sea lions and suggest a potential interactive effect of p,p'-DDE embryonic poisoning and domoic acid toxicity.

Maternal-fetal transfer of domoic acid in rats at two gestational time points.

BACKGROUND AND OBJECTIVES: Prenatal exposure to asymptomatic doses of domoic acid (DA) causes learning and memory deficits later in life; therefore, we sought to measure distribution of DA in maternal plasma and brain, prenatal brain, and amniotic fluid 1 hr after exposure, a time frame that normally encompasses acute seizure behavior. METHODS: Pregnant rats were given a single intravenous dose of DA (0.6 or 1.6 mg/kg body weight) at either gestational day (GD) 13 or GD20, which correspond to the beginning of rat embryo neurogenesis and the last day of gestation, respectively. Using a direct ELISA, dose-dependent levels of DA were detected in each sample matrix tested. RESULTS: An average of 6.6 and 14 ng DA/g brain tissue was found in GD13 and GD20 prenatal rats, respectively. Brain concentrations of DA in the GD13 prenates were identical to amniotic fluid levels, consistent with no restriction for DA to enter the GD13 prenatal brain. At GD20 the prenatal brain contained half the concentration of DA in the amniotic fluid, and was approximately half that found in the brain of the dams. After 1 hr, fetal brain and amniotic fluid contained between 1 and 5% of DA found in the maternal circulation. The amniotic fluid levels of DA in this study were also within the same range measured in stranded California sea lions that showed reproductive failure. CONCLUSIONS: DA crosses the placenta, enters brain tissue of prenates, and accumulates in the amniotic fluid. Amniotic fluid appears to be a useful fluid to monitor DA exposure.

Changes of metabolite profile in kainic acid induced hippocampal injury in rats measured by HRMAS NMR.

The solid-state high resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) technique was applied in this work to characterize and quantify the neurochemical changes in the rat hippocampus (CA1 or CA3) after local administration of kainic acid (KA). Intact tissue samples obtained from the KA treated and control brain samples were analyzed using HRMAS NMR. Metabolite profiles from NMR spectra of KA treated and control samples revealed the statistical significant decrease of N-acetylaspartate (NAA) and an increase of choline derivatives in the CA1 and CA3 directly receiving KA injection. Less extensive KA-induced metabolic changes were found in the hippocampi sample from the area contralateral to the site receiving KA administration. These results provided quantitative metabolic information on KA-induced neuronal loss and cell breakdown. In addition, the present study also revealed increased level of gamma-aminobutyric acid (GABA) and glutamate after KA treatment, suggesting that the cellular release of inhibitory and excitatory amino acids can be quantified using this method. KA induced microglia activation was evidenced by increased level of myo-insitol (myo-I). This study demonstrates that ex vivo HRMAS NMR is a useful tool for analyzing and quantifying changes of neurochemistry and cerebral metabolism in the intact brain.

Uptake, tissue distribution and excretion of domoic acid after oral exposure in coho salmon (Oncorhynchus kisutch).

Domoic acid (DA) is a potent neurotoxin naturally produced by some pennate diatom species of the genus Pseudo-nitzschia. It is well known that during harmful algal blooms fish can accumulate DA in the gastrointestinal (GI) tract and act as vectors of the toxin to higher trophic level piscivores, often with severe neurotoxic consequences to the predators. Although neurotoxicity and mass mortality have been observed in vertebrates (i.e. marine mammals and sea birds) feeding on contaminated fish, to date there has been no evidence of neurobehavioral toxicity in the fish vectors themselves. It has been hypothesized that fish may not absorb DA from the digestive tract, thus making them insensitive to dietary consumption of DA. To test this hypothesis, we performed oral gavage exposures followed by a time series of tissue dissections to characterize uptake, depuration, and tissue distribution of DA in fish. Intracoelomic (IC) injection exposures (which bypass the GI tract) were also performed to determine if coho neurons are neurologically susceptible to DA. Excitotoxic symptoms were observed in fish via IC injection at similar toxin levels that have been reported to induce excitotoxic symptoms in intraperitoneal (IP) exposures with mammalian models such as mice, suggesting that fish neurons have a similar sensitivity to DA as other vertebrates. Surprisingly, after oral gavage with ecologically relevant doses of DA, the toxin was detected in plasma collected from the dorsal aorta via a permanent intraarterial catheter within 15 min, yet excitotoxic symptoms were not observed. Additionally, DA was detected in liver, heart, spleen, kidney, muscle, brain and bile. These data indicate that although DA is absorbed from the gut, fish do not exhibit neuroexcitatory effects at maximum ecologically relevant oral doses of DA. Tissue distribution and DA uptake and depuration patterns suggest that a majority of the absorbed toxin is excreted via the kidneys and bile, thereby preventing toxic levels of DA from reaching sensitive nervous tissue. Additionally, greater than 20% of total IC administered DA doses were sequestered in bile within 1h of injection in five symptomatic fish, providing evidence for biliary sequestration of the toxin from blood. Here, we comprehensively describe the uptake, depuration, and tissue distribution patterns of DA and propose that renal and biliary processes may serve as primary routes of toxin clearance in fish.

Exposure to domoic acid affects larval development of king scallop Pecten maximus (Linnaeus, 1758).

Domoic acid (DA) is a highly toxic phycotoxin produced by bloom forming marine diatoms Pseudo-nitzschia spp. Bivalves can accumulate this toxin to a high level through their feeding activities, and thus illness or death in can occur in consumers of bivalves. In this study, king scallop, Pecten maximus, larvae were exposed to dissolved domoic acid (DA) for 25d, and the toxin accumulation and effects of harbouring this toxin were investigated. Scallop larvae incorporated DA continuously during the larval culture period and accumulated a maximum DA level of 5.21pgind(-1) when exposed to a solution of 50ngml(-1) dissolved DA. As a result of the DA treatment, larval growth, measured in terms of shell length and the appearance of the eye-spot, and larval survival were significantly compromised. This is the first study on DA incorporation dynamics in P. maximus larvae, signifying the potential of using shellfish larvae for the study on mechanisms of phycotoxin accumulation. The negative effect of DA exposure suggests that this toxin could possibly influence natural recruitment in P. maximus, and it may be necessary to protect hatchery-cultured scallop larvae from DA during toxic Pseudo-nitzschia blooms.

Tissue distribution and effects of heat treatments on the content of domoic acid in blue mussels, Mytilus edulis.

The effect of heat treatment on domoic acid (DA) content in soft tissues of mussels Mytilus edulis was investigated using high performance liquid chromatography. DA concentrations in whole flesh, hepatopancreas and tissue remainder were measured in fresh, steamed and autoclaved mussel flesh. Relative decreases in DA and tissue fluid following heat treatments of whole flesh were similar resulting in approximately equal concentrations of DA pre- and post-treatment. DA concentration decreased in the hepatopancreas and increased in tissue remainder suggesting some organ disruption of mussels during heat treatment. These findings suggest that heat treatments using either conventional steaming or autoclaving at 121 degrees C are not appropriate techniques to reduce DA in mussels during commercial processing. We also conclude that sample pre-treatment has a minimal effect on the result of a DA analysis on whole mussel tissues. The stability of DA at different temperatures within a shellfish matrix was separately tested. Reductions in DA concentration (ca. 3-7%) compensate for some of the discrepancies between what was found in the cooking fluids in the initial study and what was expected based on the whole flesh concentration of the uncooked material.