Tissue distribution of amino acid- and lipid-brevetoxins after intravenous administration to C57BL/6 mice.

Brevetoxins produced during algal blooms of the dinoflagellate Karenia are metabolized by shellfish into reduction, oxidation, and conjugation products. Brevetoxin metabolites comprising amino acid- and lipid conjugates account for a large proportion of the toxicity associated with the consumption of toxic shellfish. However, the disposition of these brevetoxin metabolites has not been established. Using intravenous exposure to C57BL/6 mice, we investigated the disposition in the body of three radiolabeled brevetoxin metabolites. Amino acid-brevetoxin conjugates represented by S-desoxy-BTX-B2 (cysteine-BTX-B) and lipid-brevetoxin conjugates represented by N-palmitoyl-S-desoxy-BTX-B2 were compared to dihydro-BTX-B. Tissue concentration profiles were unique to each of the brevetoxin metabolites tested, with dihydro-BTX-B being widely distributed to all tissues, S-desoxy-BTX-B2 concentrated in kidney, and N-palmitoyl-S-desoxy-BTX-B2 having the highest concentrations in spleen, liver, and lung. Elimination patterns were also unique: dihydro-BTX-B had a greater fecal versus urinary elimination, whereas urine was a more important elimination route for S-desoxy-BTX-B2, and N-palmitoyl-S-desoxy-BTX-B2 persisted in tissues and was eliminated equally in both urine and feces. The structures particular to each brevetoxin metabolite resulting from the reduction, amino acid conjugation, or fatty acid addition of BTX-B were likely responsible for these tissue-specific distributions and unique elimination patterns. These observed differences provide further insight into the contribution each brevetoxin metabolite class has to the observed potencies.

Domoic acid epileptic disease.

Domoic acid epileptic disease is characterized by spontaneous recurrent seizures weeks to months after domoic acid exposure. The potential for this disease was first recognized in a human case study of temporal lobe epilepsy after the 1987 amnesic shellfish-poisoning event in Quebec, and was characterized as a chronic epileptic syndrome in California sea lions through investigation of a series of domoic acid poisoning cases between 1998 and 2006. The sea lion study provided a breadth of insight into clinical presentations, unusual behaviors, brain pathology, and epidemiology. A rat model that replicates key observations of the chronic epileptic syndrome in sea lions has been applied to identify the progression of the epileptic disease state, its relationship to behavioral manifestations, and to define the neural systems involved in these behavioral disorders. Here, we present the concept of domoic acid epileptic disease as a delayed manifestation of domoic acid poisoning and review the state of knowledge for this disease state in affected humans and sea lions. We discuss causative mechanisms and neural underpinnings of disease maturation revealed by the rat model to present the concept for olfactory origin of an epileptic disease; triggered in dendodendritic synapases of the olfactory bulb and maturing in the olfactory cortex. We conclude with updated information on populations at risk, medical diagnosis, treatment, and prognosis.

Semisynthesis of radiolabeled amino acid and lipid brevetoxin metabolites and their blood elimination kinetics in C57BL/6 mice.

Brevetoxin B (BTX-B), produced by dinoflagellates of the species Karenia, is a highly reactive molecule, due in part to an α,ß-unsaturated aldehyde group at the terminal side chain, leading to the production of metabolites in shellfish by reduction, oxidation, and conjugation. We have investigated in mice the blood elimination of three common bioactive brevetoxin metabolites found in shellfish, which have been semisynthesized from BTX-B in radioactive forms. BTX-B was reduced at C42 to yield [(3)H] dihydro-BTX-B. [(3)H] S-desoxy-BTX-B2 (cysteine brevetoxin B) was semisynthesized from BTX-B by the conjugation of cysteine at the C50 olefinic group then [(3)H] radiolabeled by C42 aldehyde reduction. [(14)C] N-Palmitoyl-S-desoxy-BTX-B2 was prepared using S-desoxy-BTX-B2 as the starting material with addition of the [(14)C] radiolabeled fatty acid via cysteine-amide linkage. The elimination of intravenously administered [(3)H] S-desoxy-BTX-B2, [(14)C] N-palmitoyl-S-desoxy-BTX-B2, or [(3)H] dihydro-BTX-B was measured in blood collected from C57BL/6 mice over a 48 h period. Each brevetoxin metabolite tested exhibited biexponential elimination kinetics and fit a two-compartment model of elimination that was applied to generate toxicokinetic parameters. The rate of transfer between the central compartment (i.e., blood) and the peripheral compartment (e.g., tissue) for each brevetoxin differed substantially, with dihydro-BTX-B exchanging rapidly with the peripheral compartment, S-desoxy-BTX-B2 eliminating rapidly from the central compartment, and N-palmitoyl-S-desoxy-BTX-B2 eliminating slowly from the central compartment. Toxicokinetic parameters were analyzed in the context of the unique structure of each brevetoxin metabolite resulting from a reduction, amino acid conjugation, or fatty acid addition to BTX-B.

A cupric silver histochemical analysis of domoic acid damage to olfactory pathways following status epilepticus in a rat model for chronic recurrent spontaneous seizures and aggressive behavior.

The amnesic shellfish toxin, domoic acid, interferes with glutamatergic pathways leading to neuronal damage, most notably causing memory loss and seizures. In this study, the authors utilized a recently developed rat model for domoic acid-induced epilepsy, an emerging disease appearing in California sea lions weeks to months after poisoning, to identify structural damage that may lead to a permanent epileptic state. Sprague Dawley rats were kindled with several low hourly intraperitoneal doses of domoic acid until a state of status epilepticus (SE) appears. This kindling approach has previously been shown to induce a permanent state of epileptic disease in 96% animals within 6 months. Three animals were selected for neurohistology a week after the initial SE. An amino cupric silver staining method using neutral red counterstain was used on every eighth 40 µm coronal section from each brain to highlight neural degeneration from the olfactory bulb through the brain stem. The most extensive damage was found in the olfactory bulb and related olfactory pathways, including the anterior/medial olfactory cortices, endopiriform nucleus, and entorhinal cortex. These findings indicate that damage to olfactory pathways is prominent in a rat model for domoic acid-induced chronic recurrent spontaneous seizures and aggressive behavior.

Brevetoxin in blood, biological fluids, and tissues of sea turtles naturally exposed to Karenia brevis blooms in central west Florida.

In 2005 and 2006, the central west Florida coast experienced two intense Karenia brevis red tide events lasting from February 2005 through December 2005 and August 2006 through December 2006. Strandings of sea turtles were increased in the study area with 318 turtles (n = 174, 2005; n = 144, 2006) stranding between 1 January 2005 and 31 December 2006 compared to the 12-yr average of 43 +/- 23 turtles. Live turtles (n = 61) admitted for rehabilitation showed clinical signs including unresponsiveness, paresis, and circling. Testing of biological fluids and tissues for the presence of brevetoxin activity by enzyme-linked immunosorbent assay found toxin present in 93% (52 of 56) of live stranded sea turtles, and 98% (42 of 43) of dead stranded sea turtles tested. Serial plasma samples were taken from several live sea turtles during rehabilitation and toxin was cleared from the blood within 5-80 days postadmit depending upon the species tested. Among dead animals the highest brevetoxin levels were found in feces, stomach contents, and liver. The lack of significant pathological findings in the majority of animals necropsied supports toxin-related mortality.

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.

Analysis of interactions of brevetoxin-B and human serum albumin by liquid chromatography/mass spectrometry.

Brevetoxins are neurotoxins produced by marine dinoflagellates, primarily Karenia brevis, and can cause intoxication and even mortality of marine species, affect human health through the consumption of brevetoxin-contaminated shellfish, and effect respiratory irritation through aerosol exposure at coastal areas. Brevetoxin-A and brevetoxin-B, the major brevetoxins produced in algae, are metabolized to a series of amino acid and peptide-related derivatives in shellfish through the reactions of the amino acid residue cysteine with an α,ß-unsaturated aldehyde group. In this paper, covalent interactions between brevetoxin and proteins were investigated using brevetoxin-B and human serum albumin (HSA) as a model. It is demonstrated that both noncovalent and covalent interactions can occur between brevetoxin-B and HSA with in vitro experiments. Covalent adducts of brevetoxin-B and HSA were generated under physiological conditions and reduced with sodium borohydride based on the reaction conditions of single amino acid residues with brevetoxin-B. LC/MS analysis of toxin-treated HSA recognized the formation of the intact protein adducts with primarily one and two toxin molecules attached to one HSA molecule. HSA treated with/without brevetoxin-B was digested with trypsin, trypsin following chymotrypsin, and Pronase, respectively, for LC/MS analysis of adduction sites. Brevetoxin-B was found to react primarily with Cys(34) and His(3) and with His and Lys at other sites of HSA with variable reactivity and with Lys in general the least reactive.

Identification of ciguatoxins in Hawaiian monk seals Monachus schauinslandi from the Northwestern and Main Hawaiian Islands.

Ciguatoxins are potent algal neurotoxins that concentrate in fish preyed upon by the critically endangered Hawaiian monk seal (Monachus schauinslandi). The only report for Hawaiian monk seal exposure to ciguatoxins occurred during a 1978 mortality event when two seal liver extracts tested positive by mouse bioassay. Ciguatoxins were thus proposed as a potential threat to the Hawaiian monk seal population. To reinvestigate monk seal exposure to ciguatoxins we utilized more selective detection methods, the Neuro-2A cytotoxicity assay, to quantify ciguatoxin activity and an analytical method LC-MS/MS to confirm the molecular structure. Tissue analysis from dead stranded animals revealed ciguatoxin activity in brain, liver, and muscle, whereas analysis of blood samples from 55 free-ranging animals revealed detectable levels of ciguatoxin activity (0.43 to 5.49 pg/mL P-CTX-1 equiv) in 19% of the animals. Bioassay-guided LC fractionation of two monk seal liver extracts identified several ciguatoxin-like peaks of activity including a peak corresponding to the P-CTX-3C which was confirmed present by LC-MS/MS. In conclusion, this work provides first confirmation that Hawaiian monk seals are exposed to significant levels of ciguatoxins and first evidence of transfer of ciguatoxin to marine mammals. This threat could pose management challenges for this endangered marine mammal species.

Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response.

BACKGROUND: Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data. RESULTS: A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p < 0.0001) with microarray results. CONCLUSIONS: Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against neuroinflammation. Pathologic activity of the complement/coagulation cascade has been shown in patients suffering from a chronic form of ciguatera poisoning and is of particular interest in this model. Anti-inflammatory processes were at work not only in the brain but were also seen in whole blood and liver of these animals, creating a systemic anti-inflammatory environment to protect against the initial cellular damage caused by the toxin.

Sample preparation and liquid chromatography-tandem mass spectrometry for the analysis of selected Pacific ciguatoxins in blood samples.

Consumption of ciguatoxin-contaminated seafood can lead to ciguatera poisoning (CP). The diagnosis of CP in humans is based on the clinical symptoms after eating the fish from tropical or subtropical areas because no confirmatory clinical tests are available. One of the challenges for ciguatoxin analysis is their extremely low but toxicologically relevant concentration in biological samples. We previously reported a method using acetonitrile to precipitate proteins and extract the ciguatoxins simultaneously in whole blood samples from animals for toxin quantification by N2A cell-based assay. However, a test method for unambiguous confirmation of exposure of marine animals or humans to ciguatoxins is still needed. In the present study, we adopted the acetonitrile extraction method and added sample clean-up in the sample preparation for the determination of Pacific ciguatoxins CTX1B (aka P-CTX-1), 52-epi-54-deoxyCTX1B (aka P-CTX-2), and CTX3C (aka P-CTX-3C) in blood plasma by LC-MS/MS. We investigated sample clean-up, LC mobile phases, LC solvent programming, and settings of the two mass spectrometers (4000 Q TRAP and AB SCIEX Triple Quad 5500) in order to improve the ability to detect the Pacific ciguatoxins at ppt level. Rat blood plasma was used for the method development. Average recoveries of the three toxins in the rat plasma samples ranged from 90% to 116% with relative standard deviations of less than 15%. The method detection limits were still not low enough for the determination of the Pacific ciguatoxins in individual blood samples from Hawaiian monk seals with the two LC-MS systems. The methods were applied to a pooled sample of blood plasma collected from Hawaiian monk seals for confirmation of toxin exposure. This study will benefit monitoring of Pacific ciguatoxins in marine mammals and potentially humans by LC-MS/MS.

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.

Brevetoxin forms covalent DNA adducts in rat lung following intratracheal exposure.

BACKGROUND: Human exposure to brevetoxins produced by the red tide organism, Karenia brevis, is an increasing public health concern. Using in vitro exposure of rat liver cells to brevetoxin B (PbTx-2), the primary toxin product of K. brevis, we previously showed that it formed C(27,28)-epoxy brevetoxin metabolites capable of covalently binding to nucleic acids, a common initiation step for carcinogenesis. OBJECTIVE: This study was undertaken to evaluate nucleic acid adduction in lung following in vitro and in vivo brevetoxin exposures. METHODS: To clarify reactions of brevetoxin epoxide with DNA, we analyzed reaction products of PbTx-6 (a C(27,28) epoxide metabolite of brevetoxin B) with nucleosides. We also analyzed adducts from nucleic acid hydrolysates of isolated rat lung cells treated with PbTx-2 or PbTx-6 in vitro and lung tissue from rats after intratracheal exposure to PbTx-2 or PbTx-6 at 45 microg toxin/kg body weight. RESULTS: Our results indicate that PbTx-2 forms DNA adducts with cytidine after treatment of isolated lung cells, and forms DNA adducts with adenosine and guanosine after intratracheal exposure. CONCLUSIONS: These results are consistent with metabolic activation of highly reactive brevetoxin intermediates that bind to nucleic acid. These findings provide a basis for monitoring exposure and assessing the hazard associated with depurination of brevetoxin-nucleotide adducts in lung tissue.

Repeat exposure to ciguatoxin leads to enhanced and sustained thermoregulatory, pain threshold and motor activity responses in mice: relationship to blood ciguatoxin concentrations.

Ciguatera is a common illness in tropical and subtropical regions that manifests in complex and long-lived symptoms which are more severe in subsequent exposures. This study measures central and peripheral neurologic signs, in parallel with blood toxin levels, in mice exposed once or twice (at 3 days interval) to a sublethal dose of ciguatoxin P-CTX-1 (0.26ng/g via i.p.). Mice were implanted with radiotransmitters to monitor motor activity and core temperature. A single exposure to ciguatoxin elicited an immediate and transient decrease in motor activity and temperature, and subsequent long-lasting thermoregulatory dysfunction resulting in stabilized body temperature around 36.0 degrees C with no observable circadian rhythm. The hypothermic response and the reduced activity were enhanced with a second exposure with 30% of the mice dying within 7h. Measurement of the peripheral nervous system by the tail flick assay revealed increased latency with a single ciguatoxin exposure, and a greater effect following the second exposure. Toxin was measurable in blood up to 3 days following the first exposure; at the 1h time point the concentrations were significantly elevated after a second exposure. These findings indicate an early response to ciguatoxin manifest in a central response to lower body temperature and reduce motor activity and a more persistent effect on the peripheral system leading to spinal heat antinociception and delayed fever-like response. The greater neurological response to a second ciguatoxin exposure was associated with elevated concentrations of ciguatoxin in the blood solely over the first hour of exposure. In conclusion, a single exposure to toxin exerts a significant neurological response which may be enhanced with subsequent exposure.

In utero domoic acid toxicity: a fetal basis to adult disease in the California sea lion (Zalophus californianus).

California sea lions have been a repeated subject of investigation for early life toxicity, which has been documented to occur with increasing frequency from late February through mid-May in association with organochlorine (PCB and DDT) poisoning and infectious disease in the 1970's and domoic acid poisoning in the last decade. The mass early life mortality events result from the concentrated breeding grounds and synchronization of reproduction over a 28 day post partum estrus cycle and 11 month in utero phase. This physiological synchronization is triggered by a decreasing photoperiod of 11.48 h/day that occurs approximately 90 days after conception at the major California breeding grounds. The photoperiod trigger activates implantation of embryos to proceed with development for the next 242 days until birth. Embryonic diapause is a selectable trait thought to optimize timing for food utilization and male migratory patterns; yet from the toxicological perspective presented here also serves to synchronize developmental toxicity of pulsed environmental events such as domoic acid poisoning. Research studies in laboratory animals have defined age-dependent neurotoxic effects during development and windows of susceptibility to domoic acid exposure. This review will evaluate experimental domoic acid neurotoxicity in developing rodents and, aided by comparative allometric projections, will analyze potential prenatal toxicity and exposure susceptibility in the California sea lion. This analysis should provide a useful tool to forecast fetal toxicity and understand the impact of fetal toxicity on adult disease of the California sea lion.

Embryonic exposure to domoic Acid increases the susceptibility of zebrafish larvae to the chemical convulsant pentylenetetrazole.

BACKGROUND: Domoic acid (DA) is a neurotoxin produced by diatoms of the genus Pseudo-nitzschia that targets the limbic system to induce tonic-clonic seizures and memory impairment. In utero DA exposure of mice leads to a reduction in seizure threshold to subsequent DA exposures in mid-postnatal life, and similar studies have shown neurotoxic effects in rats that were delayed until adolescence. OBJECTIVE: We used in ovo microinjection of zebrafish (Danio rerio) to characterize the effect of embryonic exposure of DA on seizure-inducing agents later in life as an alternative species model to screen environmental contaminants that might induce a fetal-originating adult disease. METHODS: Embryos were microinjected within hours of fertilization to DA concentrations ranging from 0.12 to 1.26 ng/mg egg weight. Seven days later, the larval animals were characterized for sensitivity to the chemical convulsant pentylenetetrazole (PTZ), an agent that is well-defined in laboratory rodents and, more recently, in zebrafish. RESULTS: In ovo DA exposure, most significantly at 0.4 ng/mg, reduces the latency time until first PTZ seizure in larval fish and increases the severity of seizures as determined by seizure stage and movement parameters. The interaction between in ovo DA exposure and PTZ caused seizure behaviors to individually asymptomatic doses of PTZ (1.0 and 1.25 mM) and DA (0.13 and 0.22 ng/mg). CONCLUSION: These studies demonstrate that in ovo exposure to DA reduces the threshold to chemically induced seizures in larval fish and increases the severity of seizure behavior in a manner that is consistent with in utero studies of laboratory rodents.

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.

Distribution of Brevetoxin to Lipoproteins in human plasma.

To better understand the distribution of brevetoxins in lipoproteins, including their role in tissue delivery and toxin elimination in humans, we examined the interaction of brevetoxin congener PbTx-3 with human lipoproteins. In a scintillation proximity assay (SPA) and microtiter equilibrium dialysis, brevetoxin bound linearly to purified human high density, low density, and very low density lipoproteins (HDL, LDL, and VLDL). Both methods demonstrated higher binding capacity per weight for HDL over the other lipoproteins; approximately 50% higher with SPA and 100% higher with equilibrium dialysis. The preferential binding of brevetoxin to HDL particles is consistent with the higher surface to volume ratio of these particles and the association of the toxin with the surface phospholipid/cholesterol domain of the lipoprotein particle. Lipoprotein components were next separated from a well-characterized human plasma sample to determine the mass distribution of brevetoxin within plasma. Equilibrium dialysis of the fractionated and recombined lipoproteins and plasma proteins determined that brevetoxin distributed predominately (>80%) to lipoproteins associating with each lipoprotein class. These results provide useful information to consider human susceptibility differences, such as those based on dyslipidemia, to the transport and elimination of polyether toxins.

Intrinsic potency of synthetically prepared brevetoxin cysteine metabolites BTX-B2 and desoxyBTX-B2.

In mammals and shellfish, brevetoxins produced by the dinoflagellate Karenia brevis are rapidly metabolized to cysteine conjugates. These metabolites identified by mass spectrometry are produced in abundance in mammals and are potentially major bioactive products for intoxication. They are also abundant metabolites in shellfish where they are, in contrast to mammals, retained for prolonged periods, posing a potential threat to shellfish consumers. In this work, we analyze the intrinsic potency of the semi-synthetic cysteine brevetoxin sulfoxide (BTX-B2) and the cysteine brevetoxin (desoxyBTX-B2), each confirmed for purity by LC-MS and NMR techniques, on receptor site 5 of the voltage-gated sodium channels (VGSCs) in brain, heart and skeletal muscle. We show that both brevetoxin conjugates compete with the tritiated reduced parent brevetoxin ([(3)H]PbTx-3) in rat brain membrane preparations and in HEK cells expressing skeletal muscle or cardiac VGSC, albeit, with 8-16-fold lower affinity than the PbTx-3. On neuroblastoma cell assays we show a 3-fold reduction in cytotoxic potency for BTX-B2 relative to PbTx-3, and an 8-fold reduction for desoxyBTX-B2. In conclusion, the major transformation product of brevetoxin observed in diverse species through cysteine adduction and oxidation leads to metabolites with reduced potency on brain, skeletal muscle and heart cells.

Optimization of ciguatoxin extraction method from blood for Pacific ciguatoxin (P-CTX-1).

Ciguatera diagnosis relies on clinical observations associated with a recent consumption of fish. Although needed, direct confirmation of exposure in subjects showing ciguatera disease symptoms is currently unavailable. We previously reported that ciguatoxins were measurable in the blood of mice exposed to extracts of Pacific ciguatoxins isolated from Gambierdiscus polynesiensis, and of Indian Ocean or Caribbean Sea ciguatoxins, isolated from fish. Although highly efficient for extracting spiked purified Caribbean-CTX-1, the methanolic extraction method previously described is found here to yield only 6% recovery of spiked Pacific-CTX-1 (P-CTX-1). We report in this short communication a substantially modified method for ciguatoxin extraction from both dried and fresh blood. With this method, toxin measurement is directly accomplished in acetonitrile deproteinated whole fresh blood or phosphate buffer solution (PBS) eluted dried blood using the N2A cell-based assay. Spike studies using increasing concentrations of purified ciguatoxins reveal linear (r2 above 0.87 for all toxins) and overall efficient toxin recoveries (62%, 96%, and 96% from fresh blood and 75%, 90%, and 74% from dried blood, for C-CTX-1, P-CTX-3C, and P-CTX-1, respectively). Comparative blood matrix analysis for P-CTX-1 recovery shows increased recovery of ciguatoxin activity from whole fresh blood than from dried blood, greater by 20% in P-CTX-1 spiked mice blood and by over 85% in P-CTX-1 exposed mouse blood. In conclusion, both Caribbean and Pacific ciguatoxins can be readily extracted from blood using this modified method; however, in the case of P-CTX-1 we find that fresh blood is optimal.

Transcriptional profiling of whole blood and serum protein analysis of mice exposed to the neurotoxin Pacific Ciguatoxin-1.

Ciguatoxins (CTX) are a suite of cyclic polyether toxins produced by the marine dinoflagellate Gambierdiscus sp., are potent activators of voltage-gated sodium channels and a leading cause of human poisoning from food fish. This report characterizes the genomic and proteomic response in whole blood of adult male mice exposed i.p. to 264 ng/kg of the Pacific congener of CTX (P-CTX-1) at 1, 4 and 24h. Whole genome microarray expression data were filtered by tightness of fit between replicates, fold change (1.8) and p-value (10(-5)), resulting in 183 annotated genes used for trending analysis, K-means clustering and ontology classification. Genes involved with cytokine signaling, proteasome complex and ribosomal function were dominant. qPCR performed on 19 genes of interest had a correlation of 0.95 to array results by Pearson's correlation coefficient. Serum protein analysis showed small but significant changes in 6 of 60 proteins assayed: Ccl2, Ccl12, CD40, IL-10, leptin and M-CSF. In large part, the gene expression was consistent with a Th2 immune response with interesting similarities to expression seen in asthmatic models.