okadaic-acid and maitotoxin

Topics: Bacterial Toxins; Ciguatoxins; Cyanobacteria Toxins; Ethers, Cyclic; Marine Toxins; Microcystins; Okadaic Acid; Oxocins; Peptides, Cyclic

Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid alpha-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-alpha (CaMPK-IIalpha) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified mu- and m-calpain produced fragmentation patterns for CaMPK-IIalpha and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-alpha are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIalpha and nNOS. The degradation of CaMPK-IIalpha, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Fetus; Kainic Acid; Kinetics; Marine Toxins; N-Methylaspartate; Neurons; Neurotoxins; Nitric Oxide Synthase; Okadaic Acid; Oxocins; Rats; Rats, Sprague-Dawley; Spectrin

Marine phycotoxins present a major public health problem due to their ability to contaminate seafoods. Toxic phytoplankton is not limited, however, to the coastal areas in Europe. There are more and more problems in freshwater reservoirs, in lakes, and in ponds from which grazing animals get their water. Okadaic acid (OA), a polyether toxin from the marine dinoflagellate, Prorocentrum lima, and microcystins (MCYSTs), a potent hepatotoxic cyclic heptapeptides produced by many strains of cyanobacteria have been shown to be a powerful tumor promotor. Thus, considerable effort has been undertaken to find sensitive and easy detection techniques for the determination of phycotoxins from various matrices (microalgae, freshwater, mussels, fish, ...). Different biological and chemical methods have been developed to identify these toxins in shellfish and fish. The mouse bioassay method has been widely used for the detection of toxins such as maitotoxin and ciguatoxin, which are not easily identified by chemical procedures. In this report we describe a capillary electrophoretic method that enables us to detect okadaic acid, microcystins (MCYST-YR, MCYST-LR, MCYST-RR) and maitotoxin in picogram range.

Topics: Animals; Cyanobacteria; Dinoflagellida; Electrophoresis, Capillary; Enzyme Inhibitors; Marine Toxins; Mice; Microcystins; Okadaic Acid; Oxocins; Peptides, Cyclic

Maitotoxin induces a concentration-dependent 45Ca uptake in primary cultures of rabbit tracheal epithelial cells. This response is insensitive to the calcium channel antagonists nifedipine, diltiazem and verapamil up to 20 microM. However, verapamil at 200 microM completely prevents 45Ca uptake. Measurements of indo-1 fluorescence show that MTX induces a very sustained (> or = 2 h) [Ca]i rise, which is completely inhibited by 200 microM of verapamil. Genistein (110 microM) (an inhibitor of tyrosine kinases) also strongly inhibits it. The inhibitory effect of 50 microM miconazole (an inhibitor of cytochrome P450) is only partial. Okadaic acid (inhibitor of protein-phosphatases) primarily delays the response to the toxin without decreasing its magnitude. MTX induces the formation of (1,4,5) inositol trisphosphate (IP3). The MTX response curve is biphasic. Stimulation is transient (< or = 10 min) and is not inhibited by chelation of intracellular Cai with BAPTA, nor by verapamil (200 microM) or U73122 (10 microM) (an inhibitor of activation of PLC beta 1 through a trimeric G protein). Results suggest that MTX independently activates a calcium transport process (which might imply phosphorylation on tyrosine residues) and a PLC not linked to a trimeric G protein.

Topics: Animals; Calcium; Calcium Radioisotopes; Cell Survival; Cells, Cultured; Cilia; Cytochrome P-450 Enzyme Inhibitors; Epithelial Cells; Epithelium; Ethers, Cyclic; Genistein; Isoflavones; Marine Toxins; Okadaic Acid; Oxocins; Phosphatidylinositols; Phosphoprotein Phosphatases; Protein-Tyrosine Kinases; Rabbits; Trachea

The cytotoxicity of maitotoxin (MTX) and okadaic acid (OA) was studied on three mammalian fibroblast cell lines. Neutral red uptake (NRU), which measures cell viability, and morphological alterations were selected as rapid suitable responses. NRU allowed a precise toxicity quantification while the observations of morphological damage revealed differences specific to MTX (cell blebbing) and OA (cell rounding). BHK21 C13 fibroblasts, although less sensitive to MTX than the other cell lines, were chosen since they gave stable information and a two-stage morphological response with OA ("square"-shaped cells, then round cells). When NRU and morphology alterations were studied with crude extracts of Gambierdiscus toxicus and Prorocentrum lima, responses were typical of the dominant toxins, MTX and OA or related toxins respectively. Applied to several dinoflagellate extracts, the two tests revealed no toxicity for Amphidinium carterae, Ostreopsis siamensis, O. ovata and Coolia monotis (from La Réunion) and toxicity for A. carterae and A. operculatum (from Saint Barthélémy). When toxic, A. carterae extracts showed blebbing similar to that caused by MTX. Morphology alterations caused by A. operculatum crude extracts, different from those corresponding to MTX or OA, were also observed.

Topics: 3T3 Cells; Animals; Cell Line; Cell Survival; Cricetinae; Dinoflagellida; Ethers, Cyclic; Fibroblasts; Kidney; Marine Toxins; Mice; Neutral Red; Okadaic Acid; Oxocins

A range of marine polyether toxins from dinoflagellates were analysed by ionspray mass spectrometry. Ciguatoxin-1 ([M+H]+ m/z = 1,111.8) purified from several fish species yielded singly charged ions corresponding to the parent ion, sodium and H2O adducts and ions for the loss of up to five H2O molecules. Ciguatoxin-1 was detected to 1 ng; however, interference from fish lipids precluded direct detection of ciguatoxin-1 in crude extracts from fish flesh spiked with ciguatoxin-1 at a level equivalent to 1.5 ng ciguatoxin-1/g of extracted flesh. Maitotoxin-2 yielded doubly and triply charged ions for sodium and potassium salts and likely possessed only one sulphate ester (M(r) = 3,298 for the mono-sodium salt). Maitotoxin-3, a recently isolated small maitotoxin, yielded singly charged ions including ions for the loss of one sulphate and up to four H2O molecules. Maitotoxin-3 is proposed to be a polyether compound possessing two sulphate esters (M(r) = 1,060.5 for the disodium salt). Brevetoxin-A ([M+H]+ m/z = 867.5) and brevetoxin-B ([M+H]+ m/z = 895.5) yielded singly charged ions corresponding to the parent ion, Na+ adducts and the loss of up to four H2O molecules. Okadaic acid ([M+H]+ m/z = 805.5) yielded singly charged ions corresponding to the parent ion and ions for the loss of up to three H2O molecules. A signal for M + 18 Da species that may represent [M+NH4]+ was observed for ciguatoxin-1, brevetoxin-A and -B, and okadaic acid. For all polyethers examined, the orifice potential influenced the relative intensity of the ions detected in a predictable manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinogens; Chromatography, High Pressure Liquid; Ciguatoxins; Dinoflagellida; Eels; Ethers, Cyclic; Fishes; Marine Toxins; Mass Spectrometry; Okadaic Acid; Oxocins; Spectrometry, Mass, Fast Atom Bombardment