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At least 361 records · Page 20Linked to original sources

PubMed2000

Development of a method for the identification of azaspiracid in shellfish by liquid chromatography-tandem mass spectrometry.

Azaspiracid is the main toxin responsible for a number of recent human intoxications in Europe resulting from shellfish consumption. The first micro liquid chromatography-tandem mass spectrometry (micro-LC-MS-MS) method was developed for the determination of this novel shellfish poisoning toxin in mussels. The analyte was extracted from whole mussel meat with acetone and chromatographed on a C18 reversed-phase column (1.0 mm I.D.) by isocratic elution at 30 microl/min with acetonitrile-water (85:15, v/v), containing 0.03% trifluoroacetic acid. The toxin was ionised in an ionspray interface operating in the positive ion mode, where only the intact protonated molecule, [M+H]+, was generated at m/z 842. This served as precursor ion for collision-induced dissociation and three product ions, [M+H-nH2O]- with n=1-3, were identified for the unambiguous toxin confirmation by selected reaction monitoring LC-MS-MS analysis. A detection limit of 20 pg, based on a 3:1 signal-to-noise ratio, was achieved for the analyte. This LC-MS-MS method was successfully applied to determine azaspiracid in toxic cultivated shellfish from two regions of Ireland.

Animals↗

PubMed1997

A new method for determination of maitotoxin by capillary zone electrophoresis with ultraviolet detection.

Capillary zone electrophoresis with UV detection was applied to the rapid and efficient separation of an underivatized phycotoxin, maitotoxin, associated with ciguateric fish poisoning. Highly sensitive detection was obtained by UV absorption at 195 nm. A detection limit of 50 pg of maitotoxin was achieved at this wavelength. Analysis involved using a fused silica capillary coated with a hydrophobic phase, polyvinylalcohols. Confirmation of the electrophoretic peak of maitotoxin was further evaluated by cytotoxicity on a mammalian fibroblastic cell line, BHK 21 C13.

Animals↗

PubMed1999

Modulation of a calcium-activated chloride current by Maitotoxin.

The effect of Maitotoxin (MTX) on the calcium-activated chloride current (ICl-Ca) from Xenopus oocytes was studied, applying the two-electrode voltage clamp technique. MTX increased the current amplitude at all the voltages explored and reduced the time to reach the maximum current level (time to peak). At low toxin concentrations (15 pM), both effects were fully reversible. Activation of ICl-Ca by MTX was secondary to the increment in the intracellular Ca2+ concentration induced by this toxin, since incubation of the oocytes with the cell-permeant Ca2+ chelator BAPTA-AM, greatly reduced the effect of MTX on ICl-Ca. Furthermore, external chloride ions removal also diminished the MTX effect on the current, strongly suggesting that the main current activated by MTX is ICl-Ca. Subsequent applications of a fixed toxin concentration after toxin washout resulted in enhanced ICl-Ca, suggesting that the toxin effect potentiates.

Animals↗

PubMed2000

Neurotoxic shellfish poisoning and brevetoxin metabolites: a case study from Florida.

In June of 1996, three family members were diagnosed as suffering from neurotoxic shellfish poisoning (NSP) as a result of eating shellfish harvested from Sarasota Bay, Florida. Urine from two of these patients and extracts of shellfish collected from the same location were analyzed by radioimmunoassay (RIA) and by receptor binding assay. Activity consistent with brevetoxins was present in both urine and shellfish extracts. High performance liquid chromatographic (HPLC) analysis of shellfish extracts demonstrated multiple fractions recognized by specific anti-brevetoxin antibodies, suggesting metabolic conversion of parent brevetoxins. Affinity-purification of these extracts yielded four major peaks of activity. One peak was identified by HPLC-mass spectroscopy (HPLC-MS) to be PbTx-3, which was likely produced metabolically from the dominant parent toxin PbTx-2. No PbTx-2, however, was detected. Other peaks of activity were determined to consist of compounds of apparent masses of [M + H]+ of 1018, 1034, and 1005. These higher masses are suggestive of conjugated metabolites, but their structures have yet to be determined. The material associated with these latter three peaks were recognized by both RIA and receptor binding assay, but they quantitated differently. This finding suggests that these metabolites react differently in the two assays, and this result may have important implications for seafood safety and regulation. We suggest these metabolites to be the true cause of NSP, and they should be taken into account during regulatory testing.

Animals↗

PubMed2007

Sequential degradation of alphaII and betaII spectrin by calpain in glutamate or maitotoxin-stimulated cells.

Calpain-catalyzed proteolysis of II-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and nonapoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves II spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), betaII spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second II spectrin cleavage at G1230*S1231. BetaII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the betaII spectrin COOH-terminus. CaM promotes II spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant II spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds alphaII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both alphaII and betaII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates alphaII spectrin cleavage only at Y1176*G1177, while II spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of alphaII and then betaII spectrin, coinciding with the onset of nonapoptotic cell death. These results identify (i) novel calpain target sites in both alphaII and betaII spectrin; (ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and (iii) the preferential cleavage of alphaII spectrin vs betaII spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses: one that enhances skeletal plasticity without destroying the spectrin-actin skeleton, characterized by preservation of betaII spectrin; or an alternative response closely correlated with nonapoptotic cell death and characterized by proteolysis of betaII spectrin and complete dissolution of the spectrin skeleton.

Amino Acid Sequence↗

PubMed2006

Detection of marine toxins, brevetoxin-3 and saxitoxin, in seawater using neuronal networks.

There is a need for assay systems that can detect known and unanticipated neurotoxins associated with harmful algal blooms. The present work describes our attempt to monitor the presence of brevetoxin-3 (PbTx-3) and saxitoxin (STX) in a seawater matrix using the neuronal network biosensor (NNB). The NNB relies on cultured mammalian neurons grown over microelectrode arrays, where the inherent bioelectrical activity of the network manifested as extracellular action potentials can be monitored noninvasively. Spinal cord neuronal networks were prepared from embryonic mice and the mean spike rate across the network was analyzed before and during exposure to the toxins. Extracellular action potentials from the network are highly sensitive not only to purified STX and PbTx-3, but also when in combination with matrixes such as natural seawater and algal growth medium. Detection limits for STX and PbTx-3, respectively, are 0.031 and 0.33 nM in recording buffer and 0.076 and 0.48 nM in the presence of 25-fold-diluted seawater. Our results demonstrated that neuronal networks could be used for analysis of Alexandrium fundyense (STX-producer) and Karenia brevis (PbTx-producer) algal samples lysed directly in the seawater-based growth medium and appropriately diluted with HEPES-buffered recording medium. The cultured network responded by changes in mean spike rate to the presence of STX-or PbTx-producing algae but not to the samples of two non-STX and non-PbTx isolates of the same algal genera. This work provides evidence that the NNB has the capacity to rapidly detect toxins associated with cells of toxic algal species or as dissolved forms present in seawater and hasthe potential for monitoring toxin levels during harmful algal blooms.

Animals↗

PubMed2004

Detection of sodium channel activators by a rapid fluorimetric microplate assay.

Marine toxins such as brevetoxins and ciguatoxins are produced by dinoflagellates and can accumulate in seafood. These toxins affect humans through seafood consumption. Intoxication is mainly characterized by gastrointestinal and neurological disorders and, in most severe cases, by cardiovascular problems. To prevent the consumption of food contaminated with these toxins, shellfish have been tested by mouse bioassay. However, this method is expensive, time-consuming, and ethically questionable. The objective of this study was to use a recently developed fluorimetric microplate assay to rapidly detect brevetoxins and ciguatoxins. The method is based on the pharmacological effect of brevetoxins and ciguatoxins known to activate sodium channels and involves (i). the incubation of excitable cells in 96 well microtiter plates with the fluorescent dye bis-oxonol, whose distribution across the membrane is potential-dependent, and (ii). dose-dependent cell depolarization by the toxins. Our findings demonstrate that measuring changes in membrane potential induced by brevetoxins and ciguatoxins allowed their quantitation. Active toxins could be reliably detected at concentrations in the nanomolar range. The simplicity, sensitivity, and possibility of being automated provide the basis for development of a practical alternative to conventional testing for brevetoxins and ciguatoxins.

Animals↗

PubMed1997

Insulinotropic glucagon-like peptide-1-mediated activation of non-selective cation currents in insulinoma cells is mimicked by maitotoxin.

Maitotoxin (MTX) activates a Ca2+-dependent non-selective cation current (ICa-NS) in insulinoma cells whose time course is identical to non-selective cation currents activated by incretin hormones such as glucagon-like peptide-1 (GLP-1), which stimulate glucose-dependent insulin secretion by activating cAMP signaling pathways. We investigated the mechanism of activation of ICa-NS in insulinoma cells using specific pharmacological reagents, and these studies further support an identity between MTX- and GLP-1-activated currents. ICa-NS is inhibited by extracellular application of genistein, econazole, and SKF 96365. This inhibition by genistein suggests that tyrosine phophorylation may play a role in the activation of ICa-NS. ICa-NS is not inhibited by incubation of cells in glucose-free solution, by extracellular tetrodotoxin, nimodipine, or tetraethylammonium, or by intracellular dialysis with 4-aminopyridine, ATP, ryanodine, or heparin. ICa-NS is also not significantly inhibited by staurosporine, which does, however, partially inhibit the MTX-induced rise of intracellular Ca2+ concentration. These effects of staurosporine suggest that protein kinase C may not be involved in the activation of ICa-NS but that it may regulate intracellular Ca2+ release. Alternatively, ICa-NS may have a small component that is carried through separate divalent cation-selective channels that are inhibited by staurosporine. ICa-NS is neither activated nor inhibited by dialysis with KF, KF + AlF3 or GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), suggesting that GTP-binding proteins do not play a major role in the activation of this current.

Animals↗

PubMed1986

Palytoxin is a non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter in two-stage mouse skin carcinogenesis.

Palytoxon, which is a toxin with a molecular weight of 2681 daltons isolated from a marine coelenterate, is a potent skin irritant. However, it did not induce ornithine decarboxylase in mouse skin, or adhesion of human promyelocytic leukemia cells (HL-60). Moreover, it did not inhibit the specific binding of [3H]12-O-tetradecanoylphorbol-13-acetate (TPA) to a mouse skin particulate fraction or activate protein kinase C isolated from mouse brain in vitro. Since palytoxin showed strong irritation on mouse ear in one short-term screening test for a promoter, it was examined in a two-stage carcinogenesis experiment. The incidence of tumors in a group of mice treated with 7,12-dimethylbenz[a]anthracene plus palytoxin was 62.5% in week 25. These tumors were identified histologically as seven papillomas and one carcinoma. This paper reports the potent tumor-promoting activity of palytoxin, which is classified as a non-TPA-type tumor promoter.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2005

Identification of a rapid detoxification mechanism for brevetoxin in rats.

We examined detoxification of brevetoxin in rats through metabolic activities and key elimination routes by analyzing samples from individual rats exposed to two brevetoxin congeners (PbTx-2 and PbTx-3). Brevetoxins were detected by radioimmunoassay in methanolic extracts of blood within 1 h post intraperitoneal (ip) administration. The toxin assay response was about three times higher in PbTx-2-treated rats versus the same dose (180 microg/kg) of PbTx-3. This difference persisted for up to 8 h postexposure. When the blood samples were reextracted with 20% methanol to enhance recovery of potential polar brevetoxin metabolites, 25-fold higher assay activity was present in the PbTx-2-treated rats. Analysis of urine from the same animals identified 7-fold more activity in the PbTx-2-treated rats that accumulated over the course of 24 h. Radioimmunoassay-guided high performance liquid chromatographic analysis of urine from PbTx-2-treated rats yielded three major peaks of activity. The first peak was attributed to the two cysteine adducts, cysteine-PbTx sulfoxide and cysteine-PbTx (MH(+): m/z 1034 and 1018). The second peak was attributed to the oxidized form of PbTx-2 (MH(+): m/z 911) and its reduction product PbTx-3. The third peak remains unidentified. Brevetoxin cysteine conjugate and its sulfoxide product contributed nearly three-quarters of the brevetoxin immunoactivity. Our findings indicate the most commonly occurring PbTx-2 is rapidly transformed to a polar metabolite of a reduced biological activity that appears in blood and remains for up to 8 h, yet is cleared mostly to the urine within 24 h.

Animals↗

PubMed2006

Azaspiracid-1 alters the E-cadherin pool in epithelial cells.

Azaspiracids cause severe damages in the epithelium of several organs. In this study we have investigated the effects of azaspiracid-1 (AZA-1) on two epithelial cell lines. Nanomolar concentrations of AZA-1 reduced MCF-7 cell proliferation and impaired cell-cell adhesion. AZA-1 altered the cellular pool of the adhesion molecule E-cadherin by inducing a dose- and time-dependent accumulation of an E-cadherin fragment (E-cadherin-related antigen [ECRA(100)]), with a concentration inducing the half-maximal effect (EC(50)) of 0.47nM. The immunological characterization of ECRA(100) revealed that it consists of an E-cadherin molecule lacking the intracellular domain, and these data showed that the effect induced by AZA-1 in MCF-7 cells is undistinguishable from that induced by yessotoxin (YTX) in the same experimental system. A comparison of toxin effects in MCF-7 and Caco 2 cells confirmed that the effects induced by AZA-1 and YTX are undistinguishable in these cells. Treatment of fibroblasts with AZA-1 did not affect the cellular pool of N-cadherin showing that the toxin effect is cadherin-specific. A comparison of the effects induced by AZA-1, YTX, and okadaic acid on F-actin and E-cadherin in MCF-7 and Caco 2 cells showed that 1nM AZA-1 did not cause significant changes in F-actin and that accumulation of ECRA(100) did not correlate with decreased levels of F-actin under our experimental conditions. Matching our results with those available in literature, we notice that, when molecular effects induced by AZA-1 and YTX have been studied in the same in vitro systems, experimental data show that they are undistinguishable in terms of sensitive cellular parameters, effective doses, and kinetics of responses in several cell lines. The possibility that azaspiracids and YTXs might share their molecular mechanism(s) of action in defined biological settings should be considered.

Actins↗

PubMed1988

Maitotoxin triggers the cortical reaction and phosphatidylinositol-4,5-bisphosphate breakdown in amphibian oocytes.

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.

Animals↗

PubMed1999

Maitotoxin activates a nonselective cation channel and a P2Z/P2X(7)-like cytolytic pore in human skin fibroblasts.

Maitotoxin (MTX), a potent cytolytic agent, activates Ca(2+) entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts was examined. MTX produced a time- and concentration-dependent increase in cytosolic free Ca(2+) concentration that depended on extracellular Ca(2+) and was relatively insensitive to blockade by extracellular lanthanides. MTX also produced a time- and concentration-dependent increase in plasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da), 2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately, 5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation, N-methyl-D-glucamine (167 Da). Thus MTX initially activates Ca(2+)-permeable cation channels and later induces the formation of large pores. These effects of MTX on plasmalemmal permeability are similar to those seen on activation of P2Z/P2X(7) receptors in a variety of cell types, raising the intriguing possibility that MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore.

Adenosine Triphosphate↗

PubMed1986

Dopamine inhibits maitotoxin-stimulated pituitary 45Ca2+ efflux and prolactin release.

We examined the hypothesis that dopaminergic inhibition of prolactin release is coupled to modulation of cellular calcium flux. Dispersed female rat pituitary cells were prelabeled in 45Ca2+ and perifused to determine simultaneously fractional calcium efflux and prolactin release, as stimulated by maitotoxin, a calcium channel activator. The integrated response of each parameter to 5 ng/ml maitotoxin was obtained in individual perifusion columns in the absence or presence of various concentrations of dopamine. Maitotoxin-stimulated calcium efflux was suppressed by dopamine concentrations of 0.01 microM and greater (P less than 0.01) and achieved a maximal effect at approximately 0.1 microM, at which calcium efflux was reduced by 50%. Maitotoxin-stimulated prolactin release was inhibited by 0.03 microM dopamine and greater concentrations (P less than 0.01), and at a concentration of approximately 10.0 microM dopamine the effect became maximal at approximately 85% suppression. Haloperidol (0.1 microM) blocked the effects of 0.1 microM dopamine on both parameters. Simultaneous suppression of maitotoxin-stimulated calcium efflux and prolactin release by concentrations of dopamine within the nanomolar range suggests that dopamine receptor activation is negatively coupled to modulation of calcium flux in the physiological regulation of prolactin secretion.

Animals↗

PubMed1990

Interactions of dopamine and neurotensin on calcium fluxes and prolactin release in normal rat pituitary cells.

We studied the role of calcium in dopaminergic control of the neuroendocrine effects of neurotensin. In primary cultures of dispersed normal female rat anterior pituitary cells the interactions of dopamine and neurotensin were examined with reference to the rate of PRL release, the magnitude of 45Ca2+ uptake, the rate of fractional 45Ca2+ efflux, and the dynamic response of the intracellular calcium concentration (Cai) monitored with the fluorescent dye, Indo-1. Neurotensin stimulated calcium uptake and also mobilized a pool of intracellular calcium to increase Cai in a sustained plateau-like pattern. The response of PRL release and fractional efflux to neurotensin, however, each displayed typical spike and plateau profiles. In the presence of dopamine the stimulation of PRL release and calcium uptake due to neurotensin were abolished, and the rise in Cai was barely detectable, but neurotensin-stimulated fractional efflux persisted almost unchanged. These data suggest that dopamine may modulate Cai by inhibiting calcium uptake and possibly also by enhancing cellular calcium extrusion under stimulated conditions. Further, the increased inositol trisphosphate production reportedly stimulated by neurotensin apparently does not generate a spike-like response of intracellular calcium, and stimulated hormone release may display a spike and plateau pattern solely with a plateau Cai profile.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1989

Calcium-dependent effects of maitotoxin on phosphoinositide breakdown and on cyclic AMP accumulation in PC12 and NCB-20 cells.

The marine dinoflagellate toxin maitotoxin (MTX) stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells and in neuroblastoma hybrid NCB-20 cells. In both cell lines, the stimulation of phosphoinositide breakdown by MTX is dependent on extracellular calcium, but it is not reduced by organic or inorganic calcium channel blockers. In PC12 cells, the maximal stimulation of phosphoinositide breakdown occurs at 1.5 mM [Ca2+]o, whereas in NCB-20 cells the maximal stimulation is observed at 2.5-4.5 mM [Ca2+]o. Phosphoinositide breakdown is known to lead to formation of both inositol phosphates and diacylglycerols. The latter, through stimulation of protein kinase C, would, like phorbol esters, be expected to augment cyclic AMP accumulation in PC12 cells and to inhibit receptor-mediated cyclic AMP accumulation in NCB-20 cells. MTX does potentiate forskolin-induced accumulation of cyclic AMP in PC12 cells and does inhibit prostaglandin E2-induced accumulation of cyclic AMP in NCB-20 cells. The effects of MTX on accumulation of cyclic AMP are calcium dependent and the concentrations of calcium required for maximal responses are the same as the ones required for maximal stimulation of phosphoinositide breakdown. MTX increases intracellular calcium in both cell lines, as measured by calcium-quin2 fluorescence. But the effects of MTX on forskolin- and prostaglandin E2-mediated cyclic AMP accumulation are not mimicked by a calcium ionophore and are not blocked by nifedipine, a calcium channel blocker. Translocation of protein kinase C occurs after treatment with MTX in both cell lines; the protein kinase C activity and content are reduced in the cytosol and increased in membranes after exposure to either MTX or a phorbol ester. The results confirm previous studies on the heterogeneous input of protein kinase C to cyclic AMP-generating systems performed with phorbol esters and demonstrate the utility of MTX as a unique tool for studies of systems that involve second messengers generated through stimulation of phosphoinositide breakdown.

Adrenal Gland Neoplasms↗

PubMed1980

Mechanism of the excitatory action of palytoxin and N-acetylpalytoxin in the isolated guinea-pig vas deferens.

Palytoxin (PTX), one of the most well known potent toxic substances among marine toxins, caused a slow phasic contraction of the isolated guinea-pig vas deferens (second component) followed by the first rapid phasic contraction (first component) at concentrations above 3 x 10(-9) M. N-acetylpalytoxin, one of its derivatives, also produced similar actions but its potency was about 1/100 of that of PTX. The second component of PTX-induced contraction, but not the first component, was markedly inhibited by treatments with phentolamine, reserpine and 6-hydroxydopamine, but remained unaffected by atropine and mecamylamine pretreatment. Tetrodotoxin partially inhibited the second component, whereas the second component was markedly inhibited by solutions low in Na+ (85.2 mM) or containing verapamil (10(-6) M). Both components were completely abolished by high Mg++ or Ca++-free medium. It is concluded that the first component was the result of a direct action of PTX on smooth muscle sites, whereas the second phase was the result of an indirect action mediated through the norephinephrine release from the adrenergic nerve terminals.

Acrylamides↗

PubMed1999

Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts.

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.

Animals↗