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.

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.

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.

Domoic acid induced status epilepticus promotes aggressive behavior in rats.

Domoic acid (DA), a naturally occurring environmental toxin, has been observed to induce status epilepticus in humans, sea lions and pelicans. In a recent Sprague Dawley rat model, domoic acid dosing induced a state of status epilepticus which, after a symptom-free latent period without further dosing, progressed to recurrent spontaneous seizures, a hallmark of epilepsy. Certain individuals in this study also developed unusual behavioral changes, in particular an atypical aggression towards conspecifics. In this report we characterized the progression of aggressive behaviors after DA-induced status epilepticus and explored the relationship between aggressive behavior and recurrent spontaneous seizures. Experimental studies in this laboratory rat model are particularly relevant to California sea lions (Zapholus californianus), which show a spectrum of both epileptic and unusual behaviors, including aggression towards conspecifics in rehabilitation facilities, weeks to months after suspected exposure to domoic acid in the wild.

Domoic acid induced seizures progress to a chronic state of epilepsy in rats.

The emergence of an epilepsy syndrome in sea lions poisoned by domoic acid (DA) draws striking parallels to the single case study of temporal lobe epilepsy (TLE) that developed in an 84 yr old man one year after being poisoned by DA. To establish a basis for understanding this disease in sea lions and humans that appears to progress from DA poisoning, we have investigated the potential for a single incident of DA poisoning in rats to progress to spontaneous recurrent seizures (SRS), the hallmark of epilepsy. We have developed a DA administration protocol to induce a nonlethal status epilepticus (SE) and monitored the animals for SRS by 6 h/week of video recording. We demonstrate that a single episode of SE leads to SRS in 94% of rats (n = 23) in 6 months. These findings indicate that DA induced SE can efficiently translate to epileptic disease.

Brevetoxin B is a clastogen in rats, but lacks mutagenic potential in the SP-98/100 Ames test.

Brevetoxins are polyether toxins produced by the dinoflagellate Karenia brevis that are released into the air and are known to cause respiratory hemorrhage in manatees and irritation in humans. Brevetoxin has been previously reported to cause DNA breakage and chromosomal aberrations in in vitro cell assays. The toxin is subject to epoxidation reaction and the formation of nucleic acid adducts in cultured lung fibroblasts and in lung tissue after intratracheal administration to rats. We have exposed rats intratracheally to brevetoxin B (45 microg/kg) and analyzed liver cells for DNA fragmentation using a comet assay. Brevetoxin B (PbTx2) treated rats showed a two to three-fold increase in the amount of DNA in the comet tails, indicating that brevetoxin has in vivo clastogenic activity. We next tested brevetoxin B for mutagenic activity using the Ames 98/100 mutagenesis assay. Brevetoxin B at concentrations from 0.064 to 200 microg/mL failed to cause histidine revertants. Oxidative metabolism of brevetoxin B resulting from Aroclor 1259-induced rat liver microsomes also failed to cause histidine revertants. Finally, direct application of the brevetoxin B epoxide (PbTx6) in the Ames 98/100 assay at concentrations from 0.064 to 200 microg/mL failed to induce histidine revertants. These studies indicate that brevetoxin B retains clastogenic activity after intratracheal administration to the rat. Although brevetoxin B has been shown to form nucleic acid adducts in the lung, neither brevetoxin B nor its epoxide metabolite has mutagenic potential as assessed by the Ames 98/100 test.

Hepatic gene expression response to acute indomethacin exposure.

BACKGROUND: Rising morbidity and mortality related to the use of NSAIDs has led to the withdrawal of some of these agents and reconsideration of the adverse effects and usage paradigms of commonly available NSAIDs. Our objective in this study was to assay molecular indicators of acute hepatic injury associated with the administration of indomethacin, a prototypical NSAID, metabolized by the liver that undergoes enterohepatic circulation with associated gastrointestinal adverse effects. METHODS: Analysis of gene expression, using high-throughput, ADME (absorption, distribution, metabolism, excretion)-specific microarrays, was performed on RNA extracted from the livers of control or indomethacin treated rats, in parallel with serum enzyme tests and histological analysis of paraffin-embedded liver specimens. Male Sprague-Dawley rats (n = 45) were administered intraperitoneal injections of indomethacin for 3 days at the recommended normal dose (6.7 mg/kg), indomethacin at a high dose (20 mg/kg) or vehicle alone (controls). RESULTS: Upon termination of the study on day 4, serum gamma-glutamyl transferase activity and alkaline phosphatase/alanine aminotransferase ratios were significantly elevated in both high- and normal-dose cohorts compared with vehicle-treated animals. Diffuse microvascular steatosis was present in hepatic serial sections obtained from all animals subjected to the high-dosage regimen. High-resolution microarray analysis (six replicates/gene/animal) identified 256 genes, after outlier removal, in 17 functional classifications that were significantly altered by the high, but not by the normal dosage. These included depression of 10 of 11 cytochrome P450 genes (2B3, 2C70, 1A2-P2, 4F1, 2E1, 3A1, 2F1, 3AP7, 2C11, phenobarb-inducible P6) and 7 of 9 genes involved in the response to reactive oxygen species (e.g. glutathione reductase, glutathione transferase, and superoxide dismutase). Of 16 genes associated with toxin removal, nine exhibited significantly decreased transcripts. There was a marked shift away from lipid metabolism (decreased expression of eight genes) towards glucose utilization associated with steatosis. Despite the compromise of detoxification programs and a shift in metabolic substrate utilization, a compensatory remodeling response was activated, including genes for metalloproteases (ADAM10, MMP10, MMP11), integrins (integrin alpha-1 and alpha-E1), and extracellular matrix molecules (platelet/endothelial cell adhesion molecule-1 and heparan sulfate proteoglycan, perlecan), as well as transcripts associated with cell proliferation. The expression levels of only five genes were significantly altered among animals receiving the normal indomethacin dosage. CONCLUSION: These data confirmed that even brief exposure to indomethacin altered serum enzymatic activities and that high levels significantly altered gene expression in the liver and hepatic histology (by interfering with the clearance of toxins and xenobiotic substrates) and the regulation of basal metabolism.