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The fractionation of an aqueous extract of yam Dioscorea antaly from Madagascar led to the isolation of terpenoids and flavonoids. Compounds were identified on the basis of modern mass spectrometry and two-dimensional nuclear magnetic resonance (2D-NMR). Toxicological effects of the most abundant isolated compound, 8-epidiosbulbin E were studied on medaka Oryzias latipes embryo-larval development. The lethal concentration (killing 50%; LC(50) ) to embryos treated 24 h before hatching and for 3 days after hatching was estimated to be 0·56 mg ml(-1) (P< 0·05). No mortality was observed with O. latipes larvae exposed after hatching until day 4. Anatomo-pathological studies of embryos exposed to 0·56 mg ml(-1) showed development anomalies of the central nervous system, liver, muscle and intestine. The present data thus extend the model of O. latipes embryos as a useful animal model to analyse the effects of food toxins.

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Although aluminum (Al), a known environmental toxin, has been implicated in a variety of neurological disorders, the molecular mechanism responsible for these conditions is not fully understood. In this report, we demonstrate the ability of Al to trigger mitochondrial dysfunction and ineffective adenosine triphosphate (ATP) production. This situation severely affected cytoskeletal dynamics. Whereas the control cells had well-defined structures, the Al-exposed astrocytoma cells appeared as globular structures. Creatine kinase (CK) and profilin-2, two critical modulators of cellular morphology, were markedly diminished in the astrocytoma cells treated with Al. Antioxidants such as alpha-ketoglutarate and N-acetylcysteine mitigated the occurrence of the globular-shaped cells promoted by Al toxicity. Taken together, these data reveal an intricate link between ATP metabolism and astrocytic dysfunction and provide molecular insights into the pathogenesis of Al-induced neurological diseases.

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Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

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This article reports the results of investigations into the process of cell death induced in the Chinese hamster ovary cell K1 subclone (CHO K1) by okadaic acid (OA), a hydrophobic polyether produced by marine dinoflagellates. The IC50 was about 13 nM OA after 24 h of treatment, as determined using neutral red. With the MTT assay, the IC50 was 25 nM, although in this case 25% of the initial staining was still observed at 100 nM. Hoechst staining showed that mitotic figures accumulated at 12 nM OA after a 24- or 48-h treatment. In experiments limited to a 3-day treatment without changing the medium, CHO K1 cells were engaged in the death process at 50 nM OA after about 20 h and at 10 nM OA after 48 h. In many cells nuclear fragmentation that resulted in the apparent appearance of vesicles correlated with increasing cellular volume. But additional cell fragmentation was not observed with any treatment, and the chromatin material seemed to progressively disappear inside the cells. DNA fragmentation was analyzed by electrophoresis and with the TUNEL technique. With both techniques, the DNA was fragmented by 48 h in both 25 and 50 nM OA. Electrophoresis showed that both adherent and nonadherent cells were affected. Annexin-positive/ propidium iodide (PI)-negative cells were rarely observed after OA treatment. Some were seen under the scanning cytometer after 20 h at 50 nM OA or after 48 h at 10 nM OA, but they were never detected by flow cytometry. Most of the time scanning cytometry showed either unstained cells or PI-positive (annexin-positive or -negative) cells (48 h, 50 nM, or 72 h, 10 nM). Flow cytometry cytograms showed two cell subpopulations: one composed of a majority of smaller cells, the other of larger cells. The larger cells markedly decreased with time and OA treatment (50 and 100 nM). Stained-cell counting showed that all cells that stained were both annexin- and PI positive and that most PI-positive cells were smaller. Ki67 antigen labeling showed the proliferative activity of CHO K1 cultures but also demonstrated the loss of this activity in smaller cells treated with 50 nM OA for 48 h. We concluded that in our culture conditions the main OA target within CHO K1 cultures was dividing cells. Our results suggest that cells with disturbed metaphase-anaphase enter apoptosis, leading to necrotic daughter cells.

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Pseudomonas fluorescens was grown in millimolar amounts of yttrium. The tolerance to yttrium appeared to be mediated by the ability of the organism to accumulate the trivalent metal predominantly in the outer membrane component(s). At the stationary phase of growth, 65 to 70% of the metal was associated with the constituent(s) of the outer membrane. Treatment with 2 mM (EDTA) did not release the metal. Incubation of the outer membrane fraction with yttrium led to further accumulation of the metal. The outer membrane equivalent to 1 mg of protein was shown to immobilize 175 microg of yttrium. There was no significant variation in uronic acid and the lipid contents of the control and yttrium-stressed cells as monitored by colorimetric assays. The protein profiles of the outer and inner membrane components obtained from the control and metal-stressed cells showed marked variations as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis.

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Eight clones of the toxic dinoflagellate Prorocentrum lima (Ehrenberg) Dodge from four sites (two clones per site) on the coral reef of La Réunion, Mayotte, Europa, and Mauritius Islands in the SW Indian Ocean were isolated and cultivated under the same conditions. Morphological features of each clone, including cell size and valve and marginal pore numbers, were examined by scanning electron microscopy. The toxic potential of each clone was determined by protein phosphatase type 2A (PP2A) inhibition test and fibroblast cell line FR3T3 bioassay. Scanning electron microscopy showed that variation in morphological features of clones within and between sites was minimal and not significant. However, equivalent okadaic acid content, determined by PP2A assay, was different within and between clones isolated from the four islands. Cytotoxicity bioassay with the FR3T3 cell line confirmed the variation on global toxic potential within and between the eight P. lima clones. This test also suggested the presence of other toxic compounds without PP2A inhibiting activity in crude extracts of some clones.

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Okadaic acid was isolated from a strain of Prorocentrum arenarium Faust (Prorocentrales, Dinophyceae) collected from Europa Island (40 degrees 22'E, 22 degrees 20'S, SW Indian Ocean). The presence of okadaic acid in the algal extract was suspected after cytotoxicity and phosphatase 2A inhibition testing. It was confirmed by ADAM derivatization, immunoaffinity extraction and liquid chromatography with fluorimetric detection analysis as well as by liquid microchromatography with mass spectrometric detection. Results indicate that the P. arenarium strain was toxinogenic and could be potentially involved in the toxin production associated with the human diseases, diarrhetic shellfish poisoning and possibly ciguatera fish poisoning in the SW Indian Ocean area.

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Under certain environmental conditions, marine and freshwater phytoplankton may produce phycotoxins inhibitors of serine/thréonine protein phosphatases 1, 2A and 3. In the marine environment, dinoflagellates produce fatty polyethers: okadaic acid and its derivatives, the dinophysistoxins, which accumulate in shellfish and can cause diarrhetic shellfish poisoning (DSP) when ingested. In freshwater, the toxins are microcystins and nodularin, 7 or 5 amino acid cyclic peptides and are hepatotoxic. These toxins have caused massive poisoning of wild animals or domestic livestock and now are a health threat for humans through use of drinking and recreation water. Moreover, all these toxins are potent tumor promoters but belong to a new class, different from the TPA class, because they do not act on Protein Kinase C. Although the mutagenicity Ames test responds negatively, several results show their genotoxic potential, and therefore they are a health hazard through chronic exposition to low doses. Finally, okadaic acid, through its easy penetration in all cellular types can be used as a tool to study mechanisms involved in protein phosphorylation/dephosphorylation processes.

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Okadaic acid (OA), a toxin involved in diarrhetic shellfish poisoning (DSP), has been shown to be a potent tumor promoter in mouse skin and glandular stomach. However, more recent studies tended to show that OA can also act as a genotoxic. In this study, using the 32P-postlabelling method, DNA adduct formation was obtained in two cell lines (BHK21 C13 fibroblasts and HESV keratinocytes) after treatment by OA for 24 h with a dose range between 0.01 and 5 nM. Nineteen adducts were observed with BHK21 C13 cells and 15 with HESV ones. Low doses did not show adduct formation. Intermediate doses have given the most important number of adducts and with higher doses, the number of adducts decreased dose dependently. Ten adducts were similar in the two strains while 9 were specific of BHK21 C13 cell line and 5 of HESV one. The highest total DNA adduct level from origin parts was estimated at 95.6 adducts/10(9) nucleotides for BHK21 C13 fibroblasts (1 nM OA treatment) and 31.1 adducts/10(9) nucleotides for HESV keratinocytes (0.5 nM OA treatment). In this case, the major adduct (number 3) represented 20% for the fibroblastic cell line and 30% for the keratinocytic strain. The genotoxic effect of OA showed in this study should lead to a more careful survey of DSP outbreaks.

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The specific cytotoxic effects of phycotoxins on BHK 21 C13 fibroblasts in culture were studied with maitotoxin (MTX), okadaic acid (OA) and crude extracts from Gambierdiscus toxicus Adachi and Fukuyo and Prorocentrum lima Ehrenberg. These dinoflagellates produce MTX and OA respectively. MTX and G. toxicus crude extracts caused large blebs within minutes after exposure, in a dose-dependent manner. F-actin fibres decreased in number, then disappeared. An increase in fluorescence was observed at the periphery of the nucleus. For one strain of G. toxicus F-actin granules were also observed. Cells exposed to OA or P. lima crude extracts became polygonal in shape and then round. At low doses this effect occurred only after a 24-hr exposure. F-actin fibres crossing the cytoplasm were reduced in size and number then disappeared, while peripheral F-actin fibres became shorter giving the cell first a polygonal and then a round shape. According to these specific responses we have defined an MTX-like effect which is distinct from an OA-like effect. In both cases the amount of F-actin diminished at doses that did not affect cell viability.

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Maitotoxin (MTX) induces an increase of [Ca2+]i and of phosphoinositide breakdown in various cell types. The [Ca2+]i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the "signal" induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca2+]i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10-20 min (G1) or more (G2). No [Ca2+]i change could be detected during mitosis. The [Ca2+]i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.

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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.

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Gambierdiscus toxicus is a marine dinoflagellate involved in the food-borne disease ciguatera. Its toxicity is mainly due to maitotoxin, a hydrophilic toxin, the chemical structure of which has recently been described. This toxin increases internal Ca(2+) concentration and triggers phosphoinositide breakdown. Three cytotoxic tests were set for the detection and quantification of toxicity in G. toxicus extracts, further purified by HPLC: the neutral red uptake assay (NRU), observation of morphological alterations on baby hamster kidney fibroblasts (BHK 21/C13) and the measurement of internal Ca(2+) concentration on human lymphocytes (Jurkat D). In order to evaluate cytotoxicity tests, results were compared with the mouse bioassay. A positive correlation was observed between the minimum lethal dose (MLD), estimated at 24 hr by the mouse bioassay, and the IC(50), estimated by the NRU assay, for extracts of increasing degree of purity. By the use of the NRU assay, a precise detection and quantification of toxicity in multiple extracts was possible after 8 hr. Cell blebbing was generally highest for extracts that strongly inhibited NRU. The analysis of the correlation between the two cytological tests facilitates monitoring of the progression of purification. An indication of the presence of maitotoxin activity was supported by the increase of internal Ca(2+) concentration in Jurkat D lymphocytes, which was maximal in cells exposed to the purest extracts.

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Maitotoxin (MTX), a potent marine toxin involved in ciguatera poisoning, inhibited sea urchin egg fertilization in a dose-dependent manner with an IC50 of 7.5 x 10(-3) MU (mouse-unit)/ml. It did not affect male gametes fertilizing capabilities but provoked exocytosis in female gametes. It induced a K+ loss simultaneously with a Na+ entry into unfertilized eggs and increased the Ca2+ influx at higher concentrations. On isolated cortex preparations, high concentrations of MTX reduced the rate of ATP-dependent Ca2+ accumulation into reticulum compartments and caused a leakage of Ca2+ from a preparation pre-loaded with 45Ca2+. Verapamil (10(-4) M) similarly blocked the increase of egg permeability to Ca2+ and the effect on Ca2+ sequestering into intracellular compartment, induced by MTX. Ion transport perturbations which evolved relatively slowly are probably not the direct cause of fertilization inhibition which could be related to a modification of the plasma membrane of the female gametes by this hydrophilic toxin.

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Crassolide, a monocyclic diterpene isolated and purified from the soft coral Lobophytum crassum, inhibited the cell cleavage of sea urchin eggs without affecting fertilization. The effect was observed with concentrations above 2 x 10(-5)m in egg suspensions. Addition of crassolide between 5 and 40 min post-fertilization totally blocked the first cleavage, which in the control occurs 1 hr after fertilization. When added between 50 and 60 min post-fertilization, crassolide produced polynucleated cells in embryos. Crassolide did not affect the egg permeability to Na(+) and Ca(2+), but caused an increase of 0.2 units in the intracellular pH of fertilized eggs coupled with a proton efflux. Crassolide, which does not affect Ca(2+) influx or permeability at the level of storage in reticular vesicles, could be used as a negative control when analysing calcium changes in short-term toxicological studies. The relationship between the pH increase and the cell cleavage needs further investigation.

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Two plant flavanones, (Sigmoidin A and B) having noticeable antibacterial activity, were assayed using a preparation for the study of sea urchin egg cleavage. When added after insemination, both toxins inhibit egg division with a half maximal dose of 7.5 microM for Sigmoidin A and 12 microM for Sigmoidin B. The first Ca2+ signal following fertilization was not modified by the molecules, however, the intracellular storage of calcium in isolated non-mitochondrial compartments was reduced in a dose-dependent manner by Sigmoidin A and Sigmoidin B. Both trigger a complete discharge of the sequestered calcium. In vivo the flavanones dramatically reduced the capacity of storage of non-mitochondrial intracellular calcium compartments necessary to the cyclical elevation of cytosolic free calcium during the cell cycle.

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Acrolein, a potent cilioinhibitor of cigarette smoke was tested on synchronously dividing cultures. Mitosis could be blocked whenever the addition of acrolein was made during the cell cycle but this effect was totally reversible (between 0.06 microgram/ml to 1 microgram/ml). Nucleic acid syntheses were more affected than protein syntheses but the syntheses were only delayed and they resumed simultaneously during the same cycle.

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Autoradiographic and biochemical experiments have revealed the presence, in vegetative cells of Acetabularia, of an apicobasal gradient of penetration and incorporation of labelled DNA precursors into the chloroplasts. Staining of chloroplasts with the DNA-specific fluorochrome DAPI has shown that the number of chloroplasts without DNA increases from the apex towards the base of the cell. All together, our findings support the existence of an apicobasal gradient of chloroplast DNA synthesis and distribution in Acetabularia.

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