okadaic-acid and Necrosis

Diarrhetic Shellfish Poisoning (DSP) is a specific type of food poisoning, characterized by severe gastrointestinal illness due to the ingestion of filter feeding bivalves contaminated with a specific suite of toxins. It is known that the problem is worldwide and three chemically different groups of toxins have been historically associated with DSP syndrome: okadaic acid (OA) and dinophysistoxins (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). PTXs and YTXs have been considered as DSP toxins because they can be detected with the bioassays used for the toxins of the okadaic acid group, but diarrhegenic effects have only been proven for OA and DTXs. Whereas, some PTXs causes liver necrosis and YTXs damages cardiac muscle after intraperitoneal injection into mice. On the other hand, azaspiracids (AZAs) have never been included in the DSP group, but they cause diarrhoea in humans. This review summarizes the origin, characterization, structure, activity, mechanism of action, clinical symptoms, method for analysis, potential risk, regulation and perspectives of DSP and associated toxins produced by marine dinoflagellates.

Topics: Animals; Dinoflagellida; Humans; Liver; Macrolides; Mice; Molecular Structure; Mollusk Venoms; Myocardium; Necrosis; Okadaic Acid; Oxocins; Pyrans; Rats; Shellfish; Shellfish Poisoning; Toxicity Tests

Okadaic acid (OA) and dinophysistoxins (DTXs) are the main toxins responsible for diarrhetic shellfish poisoning (DSP) intoxications during harmful algal blooms (HABs). Although the genotoxic and cytotoxic responses to OA have been evaluated in vitro, the in vivo effects of these toxins have not yet been fully explored. The present work fills this gap by evaluating the in vivo effects of the exposure to the DSP-toxin-producing dinoflagellate Prorocentrum lima during the simulation of an early HAB episode in the mussel Mytilus galloprovincialis. The obtained results revealed that in vivo exposure to this toxic microalgae induced early genotoxicity in hemocytes, as a consequence of oxidative DNA damage. In addition, the DNA damage observed in gill cells seems to be mainly influenced by exposure time and P. lima concentration, similarly to the case of the oxidative damage found in hemocytes exposed in vitro to OA. In both cell types, the absence of DNA damage at low toxin concentrations is consistent with the notion suggesting that this level of toxicity does not disturb the antioxidant balance. Lastly, in vivo exposure to growing P. lima cell densities increased apoptosis but not necrosis, probably due to the presence of a high number of protein apoptosis inhibitors in molluscs. Overall, this work sheds light into the in vivo genotoxic and cytotoxic effects of P. lima. In doing so, it also demonstrates for the first time the potential of the modified (OGG1) comet assay for assessing oxidative DNA damage caused by marine toxins in marine invertebrates.

Topics: Animals; Apoptosis; Cell Survival; Comet Assay; Dinoflagellida; DNA Damage; Dose-Response Relationship, Drug; Flow Cytometry; Gills; Hemocytes; Marine Toxins; Mytilus; Necrosis; Okadaic Acid

Okadaic acid (OA) is the predominant biotoxin responsible for diarrhetic shellfish poisoning (DSP) syndrome in humans. While its harmful effects have been extensively studied in mammalian cell lines, the impact on marine organisms routinely exposed to OA is still not fully known. Few investigations available on bivalve molluscs suggest less genotoxic and cytotoxic effects of OA at high concentrations during long exposure times. In contrast, no apparent information is available on how sublethal concentrations of OA affect these organisms over short exposure times. In order to fill this gap, this study addressed for the first time in vitro analysis of early genotoxic and cytotoxic effects attributed to OA in two cell types of the mussel Mytilus galloprovincialis. Accordingly, hemocytes and gill cells were exposed to low OA concentrations (10, 50, 100, 200, or 500 nM) for short periods of time (1 or 2 h). The resulting DNA damage, as apoptosis and necrosis, was subsequently quantified using the comet assay and flow cytometry, respectively. Data demonstrated that (1) mussel hemocytes seem to display a resistance mechanism against early genotoxic and cytotoxic OA-induced effects, (2) mussel gill cells display higher sensitivity to early OA-mediated genotoxicity than hemocytes, and (3) mussel gill cells constitute more suitable systems to evaluate the genotoxic effect of low OA concentrations in short exposure studies. Taken together, this investigation provides evidence supporting the more reliable suitability of mussel gill cells compared to hemocytes to evaluate the genotoxic effect of low short-duration exposure to OA.

Topics: Animals; Apoptosis; Comet Assay; Cytotoxins; DNA Damage; Dose-Response Relationship, Drug; Environmental Monitoring; Flow Cytometry; Gills; Hemocytes; Marine Toxins; Mytilus; Necrosis; Okadaic Acid; Time Factors

We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions.

Topics: Animals; Apoptosis; Cells, Cultured; Cricetinae; Cricetulus; Diffusion; Female; Flow Cytometry; Hydrogen Peroxide; Magnetic Resonance Spectroscopy; Microscopy, Confocal; Necrosis; Okadaic Acid; Ovary; Water

Oxidative stress can be involved in several cellular responses, such as differentiation, apoptosis and necrosis. Dehydrocrotonin (DCTN, diterpene lactone) from Croton cajucara, Brazilian medicinal plant, slightly induced NBT-reducing activity. In presence of protein phosphatase inhibitors significant differentiation of HL60 cells was observed. Flow cytometry analysis demonstrated that apoptosis was induced when the cells were treated with okadaic acid (OKA) and plus trans-dehydrocrotonin (t-DCTN) this effect was two-fold increased. Unlike, when the cells were treated only with t-DCTN, necrosis was observed. On the other hand, the necrosis induced by t-DCTN could be due to oxidative stress, revealed by increase of GSH content. Therefore, this differentiation pathway involves the modulation of protein phosphatases and this inhibition promotes the t-DCTN action on apoptosis induction.

Topics: Annexin A5; Apoptosis; Brazil; Cell Differentiation; Croton; Diterpenes; Diterpenes, Clerodane; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Flow Cytometry; Glutathione; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Necrosis; Okadaic Acid; Oxidative Stress; Phosphoprotein Phosphatases; Plants, Medicinal; Vanadates

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.

Topics: Animals; Apoptosis; Carcinogens; Cell Division; Cell Size; CHO Cells; Cricetinae; Cricetulus; Dinoflagellida; DNA Damage; Flow Cytometry; Necrosis; Okadaic Acid

8-Cl-cAMP and 8-NH2-cAMP induced MCF-7 cell death. The type(s) of cell death were studied in more detail and compared with the cell death type (apoptosis) induced by okadaic acid, an inhibitor of serine/threonine phosphatases. By morphological criteria dying cells showed loss of cell-cell interactions and microvilli, condensation of nuclear chromatin and segregation of cytoplasmic organelles. By in situ nick end-labelling, using digoxigenin-conjugated dUTP as probe, a large fraction of 8-Cl-cAMP, 8-NH2-cAMP and 8-Cl-adenosine-exposed cells stained positively in the advanced stages of death. In the early phase of chromatin condensation the cells stained negatively. Specific (internucleosomal) DNA fragmentation was not observed. The MCF-7 cell death induced by 8-Cl-cAMP and 8-NH2-cAMP was not mediated by activation of the cAMP kinase since more stable cAMP analogues (8-CPT-cAMP and N6-benzoyl-cAMP) or forskolin failed to induce death. Furthermore, 8-Cl-cAMP action was counteracted by adenosine deaminase and 3-isobutyl-1-methylxanthine, and mimicked by 8-Cl-adenosine, a major metabolite of 8-Cl-cAMP. It is concluded that 8-Cl- and 8-NH2-cAMP can induce morphological and biochemical effects resembling apoptotic cell death in MCF-7 cells through their conversion into potent cytotoxic metabolite(s).

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Adenocarcinoma; Adenosine Deaminase; Amino Acid Sequence; Apoptosis; Biotransformation; Breast Neoplasms; Chromatin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA Damage; Ethers, Cyclic; Female; Humans; Marine Toxins; Microvilli; Molecular Sequence Data; Necrosis; Okadaic Acid; Organelles; Oxazoles; Phosphoprotein Phosphatases