brevetoxin and Neuroblastoma

Topics: Animals; Cell Line, Tumor; Ciguatoxins; Marine Toxins; Neuroblastoma; Ouabain; Oxocins; Veratridine

Topics: Animals; Biological Assay; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ciguatoxins; Dose-Response Relationship, Drug; Fishes; Limit of Detection; Marine Toxins; Mice; Neuroblastoma; Neurons; Ouabain; Oxocins; Reproducibility of Results; Saxitoxin; Time Factors; Veratridine; Voltage-Gated Sodium Channel Agonists; Voltage-Gated Sodium Channels

To develop a rapid and sensitive assay for detecting the common sea food toxins including paralytic shellfish poisoning toxin (PST), tetrodotoxin (TTX) and neurotoxic shellfish poisoning toxin (NST) based on their toxicological character.. Neuroblastoma cells were incubated with the fluorescent dye bis-oxonol, whose distribution across the membrane was potential-dependent. Changes in membrane potential of the cells induced by gonyautoxins (GTX2,3), brevetoxin (BTX) and TTX were observed respectively, using bis-oxonol.. Within 2 - 200 nmol/L of GTX2,3 or 20 - 600 nmol/L of TTX, veratridine-induced depolarization was shown to be inhibited by GTX2,3 or TTX in dose-dependent manner. Within 15 - 400 ng/ml, there was a dose-dependent relationship between the NSP-induced depolarization and toxin concentration.. It was likely to find a rapid, specific, and reliable method with bis-oxonol for detecting GTX2,3, TTX and BTX in sea food.

Topics: Animals; Cell Line, Tumor; Fluorescent Dyes; Humans; Marine Toxins; Membrane Potentials; Mollusca; Neuroblastoma; Oxocins; Saxitoxin; Shellfish; Staining and Labeling; Tetrodotoxin

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.

Topics: Animals; Binding Sites; Brain; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Cysteine; Dinoflagellida; Dose-Response Relationship, Drug; Heart; Humans; Kidney; Marine Toxins; Muscle, Skeletal; Myocardium; Neuroblastoma; Neurons; Neurotoxins; Oxocins; Sodium Channels; Spectrometry, Mass, Electrospray Ionization; Sulfoxides

Several novel brevetoxin derivatives were isolated and identified in Karenia brevis cultures and natural blooms by using solid phase extraction (SPE) and LC/MS(MS) techniques. These analogs were more polar compared with previously described brevetoxins, and were poorly extractable by conventional non-polar solvent (chloroform) partitioning. Brevetoxin analogs were structurally confirmed as hydrolyzed (open A-ring) forms of brevetoxins PbTx-1, PbTx-7, PbTx-2, and PbTx-3, and of oxidized PbTx-1 and PbTx-2. Some of these open A-ring derivatives were in greater abundance than their non-hydrolyzed counterparts. All were in much greater abundance in bloom water filtrate compared with cell-rich fractions. Open A-ring compounds were cytotoxic in mouse neuroblastoma (N2a) cell assay. In the K. brevis bloom-exposed Eastern oyster, brevetoxin metabolites with opened A rings were identified (e.g., open-ring cysteine-PbTx conjugates), contributing to their overall toxin burden.

Topics: Animals; Cell Line, Tumor; Chromatography, Liquid; Crassostrea; Dinoflagellida; Marine Toxins; Mass Spectrometry; Mice; Neuroblastoma; Oxocins

Previously, we analyzed Eastern oysters (Crassostrea virginica) naturally exposed to a Karenia brevis red tide and found that brevetoxins (PbTx) are rapidly accumulated and metabolized. Several metabolites were isolated and later identified, including a cysteine-PbTx conjugate (MH(+): m/z 1018) and its sulfoxide product (m/z 1034). In the present study, we confirm and extend those findings by examining PbTx metabolism and elimination in oysters exposed to pure toxins (PbTx-2 and -3) under controlled conditions. Waterborne PbTx-3 was rapidly accumulated, but not metabolized, in the oyster and was largely eliminated within 2 weeks after exposure. In contrast, PbTx-2 was accumulated and rapidly metabolized. Metabolites of PbTx-2 included the reduction product PbTx-3 (m/z 897), and the cysteine conjugates (m/z 1018 and 1034) isolated previously from the field samples. Levels of the metabolite PbTx-3 in PbTx-2-exposed oysters were highest immediately after exposure and declined at a rate similar to parent PbTx-3 in PbTx-3-exposed oysters. Cysteine-PbTx persisted for 8 weeks after exposure. The same metabolites were confirmed in oysters exposed to laboratory cultures of K. brevis. PbTx metabolites contribute to neurotoxic shellfish poisoning (NSP) and should be included in analytical protocols for monitoring shellfish toxicity after a K. brevis red tide event.

Topics: Animals; Brain Neoplasms; Cell Survival; Chromatography, Liquid; Dinoflagellida; Dose-Response Relationship, Drug; Marine Toxins; Mice; Neuroblastoma; Ostreidae; Oxocins; Spectrometry, Mass, Electrospray Ionization; Tumor Cells, Cultured

Ciguatoxins (CTXs) and brevetoxins (PbTxs) modify the activation and inactivation processes of voltage-sensitive sodium channels (VSSC). In this study, the specific binding to rat brain synaptosomes of two commercial PbTxs, five purified CTXs and their derivatives was evaluated in competition with various concentrations of radiolabelled brevetoxin ([3H]PbTx-3). The results indicate that all CTXs bind specifically and with high affinity to sodium channels. Statistical analysis of the calculated inhibition constants identified two classes of toxins: the PbTxs and the less polar CTXs, and a group of CTXs of very high affinity. Relatively small chemical differences between the CTXs gave rise to significant differences in their affinity to the rat brain sodium channels. Cytotoxic effects associated with sodium channel activation were evaluated for the two classes of toxins on murine neuroblastoma cells, and their acute toxicity was determined in mice. CTXs have shown high affinities to VSSC of rat brain membranes and strong cytotoxic effects on neuroblastoma cells which correlate with their very low LD50 in mice. For PbTxs, it is different. Although binding with high affinity to VSSC and giving rise to significant cytotoxic effects, they are known to be poorly toxic intraperitoneally to mice. Furthermore, within the CTXs family, even though the most toxic compound (CTX-1B) has the highest affinity and the less toxic one (CTX-4B) the lowest affinity, a detailed analysis of the data pointed out a complex situation: (i) high affinity and toxicity seem to be related to the hydroxylation of the molecule on the A-ring rather than to the backbone type, (ii) acute toxicity in mice does not follow exactly the sodium-dependent cytotoxicity on neuroblastoma cells. These data suggest that the high toxicity of CTXs is related to sodium-dependent disturbances of the excitable membranes but might also involve other cellular mechanisms.

Topics: Animals; Brain Neoplasms; Ciguatoxins; Female; In Vitro Techniques; Ion Channel Gating; Lethal Dose 50; Male; Marine Toxins; Mice; Neuroblastoma; Neurotoxins; Oxocins; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Structure-Activity Relationship; Synaptosomes

In October of 1996, a Gymnodinium breve bloom occurred in shellfish harvesting waters of Alabama, Mississippi and Louisiana, Gulf of Mexico, USA. Bloom densities reached 5.6x10(5) cells liter(-1) and bloom residence at shellfish sampling stations ranged from 3 to 28 days. Brevetoxin-2 dominated G. breve toxin profiles in bloom seawater extracts. Shellfish toxicity, assessed by mouse bioassay, exceeded the guidance level for up to 75 days after the bloom had dissipated. Cytotoxicity assays and mouse bioassays showed similar temporal patterns of shellfish toxicity, but the two methods differed in estimations of brevetoxin-3 equivalent toxicity by a factor of 93 to 1. LC-ESI-MS showed the temporal patterns in shellfish toxicity reflected metabolism of G. breve toxins. The molecular ions m/z 1004, 1017 and 1033 dominated LC-ESI-MS spectra of toxic chromatographic fractions from the extracts and were identified as brevetoxin metabolites on the basis of LC-APCI-MS-MS. The discrepancy between cytotoxicity and mouse bioassay estimates of brevetoxin-3 equivalent toxicity resulted from the difference in extraction efficiency of solvents used in the respective methods and the relative sensitivity of the assays to toxin metabolite mixtures present in the extracts. The normalized cytotoxicity assay showed 75% agreement with mouse bioassay positive test samples and 64% agreement with mouse bioassay negative test samples. Published in 1999 by John Wiley & Sons, Ltd.

Topics: Animals; Biological Assay; Brain Neoplasms; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Dinoflagellida; Marine Toxins; Mass Spectrometry; Mice; Neuroblastoma; Ostreidae; Oxocins; Seawater; Sodium Channels; Time Factors

We have modified the cell-based directed cytotoxicity assay for sodium channel and calcium channel active phycotoxins using a c-fos-luciferase reporter gene construct. In this report we describe the conceptual basis to the development of reporter gene assays for algal-derived toxins and summarize both published and unpublished data using this method. N2A mouse neuroblastoma cells, which express voltage-dependent sodium channels, were stably transfected with the reporter gene c-fos-luc, which contains the firefly luciferase gene under the transcriptional regulation of the human c-fos response element. The characteristics of the N2A reporter gene assay were determined by dose response with brevetoxin and ciguatoxin. Brevetoxin-1 and ciguatoxin-1 induced c-fos-luc with an EC50 of 4.6 and 3.0 ng ml(-1), respectively. Saxitoxin caused a concentration-dependent inhibition of brevetoxin-1 induction of c-fos-luc with an EC50 of 3.5 ng ml(-1). GH4C1 rat pituitary cells, which lack voltage-dependent sodium channels but express voltage-dependent calcium channels, were also stably transfected with the c-fos-luc. GH4C1 cells expressing c-fos-luciferase were responsive to maitotoxin (1 ng ml(-1)) and a putative toxin produced by Pfiesteria piscicida. Although reporter gene assays are not designed to replace existing detection methods used to measure toxin activity in seafood, they do provide a valuable means to screen algal cultures for toxin activity, to conduct assay-guided fractionation and to characterize pharmacologic properties of algal toxins.

Topics: Animals; Biological Assay; Ciguatoxins; Dose-Response Relationship, Drug; Genes, fos; Genes, Reporter; Luciferases; Marine Toxins; Mice; Neuroblastoma; Neurotoxins; Oxocins; Pfiesteria piscicida; Pituitary Gland; Proto-Oncogene Proteins c-fos; Rats; RNA, Messenger; Saxitoxin; Transfection; Tumor Cells, Cultured

In the present study we have developed an assay for the detection of sodium channel-specific marine toxins based upon mitochondrial dehydrogenase activity in the presence of veratridine and ouabain. This cell bioassay allows detection of either sodium channel enhancers, such as the brevetoxins and the ciguatoxins, or sodium channel blocking agents, such as the saxitoxins. The assay responds in a dose dependent manner and differentiates the toxic activity as either sodium channel blocking or enhancing. In addition, the assay is highly sensitive, with present detection limits of 2 ng/ml for either saxitoxins or brevetoxins (PbTx-1 and PbTx-3). Assay response to a ciguatoxic extract and to brevetoxins is rapid, allowing dose dependent detection within 4 to 6 h. The method is simple, utilizes readily available reagents, uses substantially less sample than required for mouse bioassay, and is well within the scope of even modest tissue culture facilities. This cell-based protocol has the potential to serve as an alternate and complementary method to the standard mouse bioassay.

Topics: Animals; Biological Assay; Cell Line; Cell Survival; Ciguatoxins; Marine Toxins; Mice; Neuroblastoma; Neurotoxins; Oxocins; Saxitoxin; Sodium Channels; Tetrazolium Salts; Tumor Cells, Cultured