okadaic-acid and thiazolyl-blue

To determine the relative toxicity and effects on the cell cycle of okadaic acid and dinophysistoxin-2 in primary hepatocyte cultures.. Cytotoxicity was determined by the MTT method, caspase-3 activity and lactate dehydrogenase release to the medium. The cell cycle analysis was performed by imaging flow cytometry and the effect of the toxins on cell proliferation was studied by quantitative PCR and confocal microscopy.. We show that dinophysistoxin-2 is less toxic than okadaic acid for primary hepatocytes with a similar difference in potency as that observed in vivo in mice after intraperitoneal injection. Both toxins induced apoptosis with caspase-3 increase. They also inhibited the hepatocytes cell cycle in G1 affecting diploid cells and diploid bi-nucleated cells. In proliferating hepatocytes exposed to the toxins, a decrease of p53 gene expression as well as a lower protein level was detected. Studies of the tubulin cytoskeleton in toxin treated cells, showed nuclear localization of this molecule and a granulated tubulin pattern in the cytoplasm.. The results presented in this work show that the difference in toxicity between dinophysistoxin-2 and okadaic acid in cultured primary hepatocytes is the same as that observed in vivo after intraperitoneal injection. Okadaic acid and dinophysistoxin-2 arrest the cell cycle of hepatocytes at G1 even in diploid bi-nucleated cells. p53 and tubulin could be involved in the cell cycle inhibitory effect.

Topics: Analysis of Variance; Animals; Blotting, Western; Caspase 3; Cell Cycle; Cell Proliferation; Flow Cytometry; Hepatocytes; L-Lactate Dehydrogenase; Microscopy, Confocal; Okadaic Acid; Pyrans; Rats; Real-Time Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles

Serine/threonine protein phosphatases are important mediators of general cellular function as well as neurodegenerative processes. We have previously shown inhibition of protein phosphatases to be as neurotoxic as glutamate-induced neuronal death but resistant to neuroprotection by estrogens. In this study, the mechanism by which phosphatase inhibition via okadaic acid (OA) induced neurotoxicity is explored. Neurons were exposed to OA or glutamate in the presence or absence of various protein kinases inhibitors, and/or one of four estrogens. Both OA and glutamate induced cell death via increased reactive oxygen species, protein carbonylation, lipid peroxidation, caspase-3 activity, and mitochondrial dysfunction. All estrogens attenuated glutamate-mediated responses, but not OA-induced responses. In addition, inhibition of protein kinase C and mitogen-activated protein kinase pathway was neuroprotective against glutamate but not OA toxicity. Interestingly, inhibition of mitogen-activated protein kinase pathway with PD98096 or U0126 caused a decrease in reactive oxygen species production suggesting that activation of ERK1/2 could further exacerbate the oxidative stress caused by glutamate-induced toxicity; however, these inhibitors had no effect on OA-induced toxicity. Collectively, these results indicate that both glutamate and OA neurotoxicities are mediated by persistent activation of ERK1/2 and/or protein kinase C and a resulting oxidative stress, and that protein phosphatase activity is an important and necessary aspect of estrogen-mediated neuroprotection.

Topics: Animals; Caspase 3; Caspase 7; Cell Death; Cells, Cultured; Cerebral Cortex; Cytosol; Enzyme Inhibitors; Estrogens; Female; Glutamic Acid; Lipid Peroxidation; Mitogen-Activated Protein Kinases; Neurons; Neuroprotective Agents; Okadaic Acid; Pregnancy; Protein Carbonylation; Rats; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles

Because of the critical role of the nuclear transcription factor NF-kappaB in inflammation, viral replication, carcinogenesis, antiapoptosis, invasion, and metastasis, specific inhibitors of this nuclear factor are being sought and tested as treatments. NF-kappaB activation is known to require p65 phosphorylation at serine residues 276, 529, and 536 before it undergoes nuclear translocation. Small protein domains, termed protein transduction domains (PTDs), which are able to penetrate cell membranes can be used to transport other proteins across the cell membrane. We have identified two peptides from the p65 subunit of NF-kappaB (P1 and P6 were from amino acid residues 271-282 and 525-537, respectively) that, when linked with a PTD derived from the third helix sequence of antennapedia, inhibited tumor necrosis factor (TNF)-induced NF-kappaB activation in vivo. Linkage to the PTD was not, however, required to suppress the binding of the p50-p65-heterodimer to the DNA in vitro. PTD-p65-P1 had no effect on TNF-induced AP-1 activation. PTD-p65-P1 suppressed NF-kappaB activation induced by lipopolysaccharide, interleukin-1, okadaic acid, phorbol 12-myristate 13-acetate, H(2)O(2), and cigarette smoke condensate as well as that induced by TNF. PTD-p65-P1 had no effect on TNF-induced inhibitory subunit of NF-kappaB(IkappaBalpha) phosphorylation, IkappaBalpha degradation, or IkappaBalpha kinase activation, but it blocked TNF-induced p65 phosphorylation and nuclear translocation. NF-kappaB-regulated reporter gene expression induced by TNF, TNF receptor 1, TNF receptor-associated death domain, TNF receptor-associated factor-2, NF-kappaB-inducing kinase, IkappaBalpha kinase, and p65 was also suppressed by these peptides. Suppression of NF-kappaB by PTD-p65-P1 enhanced the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results demonstrate the identification of a p65 peptide that can selectively inhibit NF-kappaB activation induced by various inflammatory stimuli, down-regulate NF-kappaB-mediated gene expression, and up-regulate apoptosis.

Topics: Active Transport, Cell Nucleus; Alkaline Phosphatase; Amino Acid Sequence; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell-Free System; Coloring Agents; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Genes, Reporter; Humans; Hydrogen Peroxide; I-kappa B Kinase; I-kappa B Proteins; In Situ Nick-End Labeling; Interleukin-1; Lipopolysaccharides; Microscopy, Phase-Contrast; Molecular Sequence Data; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; Peptide Fragments; Peptides; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles; Time Factors; Transcription Factor RelA; Transfection; Tumor Necrosis Factor-alpha; Up-Regulation

The specific inhibitory activity exerted by okadaic acid on protein phosphatase 2A was used to assess the presence of okadaic acid in mussels, using a commercially available protein phosphatase 2A preparation. Under the conditions used, okadaic acid inhibits the enzymatic activity dose-dependently, with an IC50 = 0.26 ng/ml (0.32 nM). The assay is accurate and reproducible. Okadaic acid was detected in concentrations as low as 0.063 ng/ml in aqueous solutions and 2 ng/g in mussel digestive glands. Thirty naturally contaminated mussel samples were submitted to the protein phosphatase 2A inhibition assay as well as to an ELISA assay and to a MTT cytotoxicity assay, with similar results. The proposed assay is sensitive, rapid and does not require expensive equipment. These characteristics make it a good candidate for employment in the routine assessment of okadaic acid shellfish contamination.

Topics: Animals; Bivalvia; Carcinogens; Coloring Agents; Digestive System; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Food Contamination; Lethal Dose 50; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Reproducibility of Results; Shellfish; Tetrazolium Salts; Thiazoles; Water Pollutants, Chemical

The suitability of a cytotoxicity assay based on the MTT colorimetric method has been evaluated for the detection of okadaic acid in mussels. On KB cells, okadaic acid exhibited a dose-dependent cytotoxic effect, the IC50 being inversely related to the exposure time (IC50 = 6.3 ng/ml, 4.0 ng/ml and 1.1 ng/ml after 24, 48 and 72 hr of contact, respectively). Using a contact time of 24 hr, the MTT cytotoxicity assay is suitable for revealing okadaic acid concentrations in mussel samples as low as 50 ng/g of digestive glands, with a sensitivity higher than that of the commercially available kits for enzyme-linked immunosorbent assay (ELISA). In the okadaic acid concentration range from 50 to 1500 ng/g of digestive glands the MTT cytotoxicity assay showed satisfactory accuracy and reproducibility. A high degree of correlation was found between the okadaic acid content of 16 naturally contaminated samples measured by the MTT cytotoxicity assay and by an ELISA.

Topics: Animals; Bivalvia; Colorimetry; Coloring Agents; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; Injections, Intraperitoneal; KB Cells; Lethal Dose 50; Mice; Okadaic Acid; Oxidation-Reduction; Reproducibility of Results; Shellfish Poisoning; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured