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Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80-400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 10 5 Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays., (© 2021. The Author(s).)

Aim: The present study aims to investigate the effect of flavonoids from stem and leaf of Scutellaria Baicalensis Georgi (SSF) on multi-sites phosphorylation of tau protein in the cerebral cortex and hippocampus of rats induced by okadaic acid (OA) and the regulative mechanism of the protein kinases., Methods: The model of AD-like memory impairment and neuronal injuries was established in male SD rats who were microinjected with OA (200 ng/kg) to establish a memory impairment model and screened for successful model rats by Morris water maze on day 21 after surgery. The successful model rats were continuously administered with intragastric infusion (ig) SSF 25, 50 and 100 mg/kg or Ginkgo biloba leaves flavonoids (GLF) 200 mg/kg for 36 d. The relative protein expressed levels of phosphorylated tau protein at sites of Ser199, Ser202, Ser214, Ser404 and Thr231, protein kinases (CDK5, PKA, pTyr216-GSK3β and pSer9-GSK3β) were detected by Western blotting., Results: The relative protein expressed levels of p-tau(Ser199), p-tau(Ser202), p-tau(Ser214), p-- tau(Ser404), p-tau(Thr231) and pTyr216-GSK3β were significantly increased in both cerebral cortex and hippocampus regions of the model rats subjected to intracerebroventricular injection of OA (P<0.01), while the protein expressed levels of CDK5, PKA and pSer9-GSK3β (P<0.01) were reduced. SSF can dramatically reverse these increments in phosphorylated tau protein levels (P<0.01) and differently regulate the protein expressed levels of CDK5, PKA and GSK3β (P<0.01) in rats' cerebral cortex and hippocampus induced by OA. GLF also exhibit a similar effect to SSF., Conclusion: The results demonstrated that SSF could inhibit the hyperphosphorylation of tau in rats' cerebral cortex and hippocampus induced by microinjection of OA, which may be related to the activities of protein kinase CDK5, PKA and GSK3β., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The present study was designed to investigate the effects of okadaic acid intracerebroventricular (ICV) injection on memory function and expression level of α7 subunit of nicotinic acetylcholine receptor (nAChR) and NR2B subunit of NMDA glutamate receptors in the hippocampus, as well as effect of the antidementic drug memantine on okadaic acid induced changes at systemic and molecular levels in rats. Okadaic acid was dissolved in artificial cerebrospinal fluid (aCSF) and injected ICV 200 ng/10 μl. Vehicle control received 10 μl of aCSF ICV bilaterally. Control and okadaic acid injected rats were divided into two subgroups: treated i.p. with saline or memantine (5 mg/kg daily for 13 days starting from the day of okadaic acid injection). Rats were trained in the dual-solution plus-maze task that can be solved by using place or response strategies. The Western immunoblotting was used to determine relative amount of hippocampal receptors protein levels. Obtained data revealed that okadaic acid ICV injected rats were severely impaired at acquiring the place version of the maze accompanied by significant decline in hippocampal α7 subunit of nACh receptors protein levels. Memantine treatment can prevent okadaic acid induced impairment of hippocampal-dependent spatial memory and accompanied by modulation of the expression level of α7 subunit of nACh and NR2B subunit of NMDA receptors in the hippocampus. Thus, our results support the presumption that α7 nACh receptors may play an important role in the cognitive enhancer effects of memantine and emphasize the role of cholinergic-glutamatergic interactions in memory.

The biotoxin okadaic acid (OA) is a lipophilic secondary metabolite of marine microalgae. Therefore, OA accumulates in the fatty tissue of various shellfish and may thus enter the food chain. The ingestion of OA via contaminated marine species can lead to the diarrhetic shellfish poisoning syndrome characterized by the occurrence of a series of acute gastrointestinal symptoms in humans. In addition, genotoxicity and tumor-promoting properties of OA might constitute a long-term threat to human health. In order to deepen our understanding of the molecular effects of OA, we compared long-term (14 d) and short-term (24 h and 48 h) apoptotic effects of the compound on human HepaRG hepatocarcinoma cells. Cells were treated either with single doses for 24 and 48 h, respectively, or seven times over a period of 14 d, so that the cumulated quantities of OA in the long-term approach were equal to the single doses upon short-term treatment. Both short-term treatment scenarios led to the induction of apoptosis. Specific caspase activation assays and transcriptional analysis of mRNAs encoding proteins involved in the regulation of apoptosis suggest that OA-induced apoptosis occurs presumably by activation of the intrinsic apoptotic pathway. In contrast, effects were much less pronounced in case of long-term treatment. This is possibly linked to cellular protective mechanisms against low amounts of toxins, e.g. transporter-mediated efflux. In conclusion, our results show a clear concentration- and time-dependency of OA-mediated apoptotic effects in HepaRG cells and contribute to the elucidation of molecular effects of OA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The lipophilic phycotoxin okadaic acid (OA) occurs in the fatty tissue and hepatopancreas of filter-feeding shellfish. The compound provokes the diarrhetic shellfish poisoning (DSP) syndrome after intake of seafood contaminated with high levels of the DSP toxin. In animal experiments, long-term exposure to OA is associated with an elevated risk for tumor formation in different organs including the liver. Although OA is a known inhibitor of the serine/threonine protein phosphatase 2A, the mechanisms behind OA-induced carcinogenesis are not fully understood. Here, we investigated the influence of OA on the β-catenin-dependent Wnt-signaling pathway, addressing a major oncogenic pathway relevant for tumor development. We analyzed OA-mediated effects on β-catenin and its biological function, cellular localization, post-translational modifications, and target gene expression in human HepaRG hepatocarcinoma cells treated with non-cytotoxic concentrations up to 50 nM. We detected concentration- and time-dependent effects of OA on the phosphorylation state, cellular redistribution as well as on the amount of transcriptionally active β-catenin. These findings were confirmed by quantitative live-cell imaging of U2OS cells stably expressing a green fluorescent chromobody which specifically recognize hypophosphorylated β-catenin. Finally, we demonstrated that nuclear translocation of β-catenin mediated by non-cytotoxic OA concentrations results in an upregulation of Wnt-target genes. In conclusion, our results show a significant induction of the canonical Wnt/β-catenin-signaling pathway by OA in human liver cells. Our data contribute to a better understanding of the molecular mechanisms underlying OA-induced carcinogenesis.

Background: Early stages of Alzheimer's disease (AD) are characterized by high phosphorylation of microtubule-associated protein tau, which may result from the downregulation of protein phosphatases., New Method: In order to model phosphatase downregulation and analyze its effect on tau aggregation in vitro, we treated neuroblastoma SH-SY5Y cells with okadaic acid (OA), a protein phosphatase inhibitor, and examined high molecular weight phospho-tau species., Results and Comparison With Existing Methods: OA treatment led to the appearance of heat-stable protein species with apparent molecular weight around 100 kDa, which were immunoreactive to anti-tau antibodies against phosphorylated Ser202 and Ser396. As these high molecular weight tau-immunoreactive proteins (HMW-TIPs) corresponded to the predicted size of two tau monomers, we considered the possibility that they represent phosphorylation-induced tau oligomers. We attempted to dissociate HMW-TIPs by urea and guanidine, as well as by alkaline phosphatase treatment, but HMW-TIPs were stable under all conditions tested. These characteristics resemble properties of certain sodium dodecyl sulfate (SDS)-resistant tau oligomers from AD brains. The absence of HMW-TIPs detection by anti-total tau antibodies Tau46, CP27 and Tau13 may be a consequence of epitope masking and protein truncation. Alternatively, HMW-TIPs may represent previously unreported phosphoproteins cross-reacting with tau., Conclusions: Taken together, our data provide a novel characterization of an OA-based cell culture model in which OA induces the appearance of HMW-TIPs. These findings have implications for further studies of tau under the conditions of protein phosphatase downregulation, aiming to explain mechanisms involved in early events leading to AD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Protein phosphorylation states have a pivotal role in regulation of synaptic plasticity and long-term modulation of synaptic transmission. Serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) have a critical effect on various regulatory mechanisms involved in synaptic plasticity, learning and memory. Okadaic acid (OKA), a potent inhibitor of PP1 and PP2A, reportedly leads to cognitive decline and Alzheimer's disease (AD)-like pathology. The aim of this study was to examine the effect of OKA on electrophysiological characteristics of hippocampal dentate gyrus (DG) neurons in vivo. Male Wistar rats were divided into two control and OKA groups. OKA was injected intracerebroventricularly (i.c.v.) into lateral ventricles and after two weeks the long-term potentiation (LTP) and paired-pulse responses recorded from hippocampal perforant path-DG synapses in order to assess short-term and long-term synaptic plasticity. Results of this study revealed that OKA-induced inhibition of PP1 and PP2A activity drastically attenuates the field excitatory postsynaptic potential (fEPSP) slope and population spike (PS) amplitude following paired pulse and high frequency stimulation (HFS) of hippocampal DG neurons indicating pre- and post-synaptic involvement in electrical activity of these neurons. Administration of OKA impaired the short-term and long-term spatial memories conducted by Y-maze and passive avoidance tests, respectively. OKA-induced attenuation in electrophysiological activity and consequent memory deficits also provide a beneficial tool for studying neurodegenerative disorders such as AD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Patients with inherited dilated cardiomyopathy (DCM) often suffer from severe heart failure based on impaired cardiac contractility leading to increased morbidity and mortality. Integrin-linked kinase (ILK) as a part of the cardiac mechanical stretch sensor was found to be an essential genetic regulator of cardiac contractility. Integrin-linked kinase localizes to z-disks and costameres in vertebrate hearts and regulates the activity of the signaling molecule protein kinase B (PKB/Akt) by controlling its phosphorylation. Despite identification of several potential drug targets in the ILK signaling pathway, pharmacological treatment strategies to restore contractile function in ILK-dependent cardiomyopathies have not been established yet. In recent years, the zebrafish has emerged as a valuable experimental system to model human cardiomyopathies as well as a powerful tool for the straightforward high-throughput in vivo small compound screening of therapeutically active substances. Using the ILK deficient zebrafish heart failure mutant main squeeze ( msq ), which shows reduced PKB phosphorylation and thereby impaired cardiac contractile force, we identified here, in an automated small compound screen, the protein phosphatase inhibitors calyculin A and okadaic acid significantly restoring myocardial contractile function by reconstituting PKB phosphorylation in msq ILK-deficient zebrafish embryos.

Objective: In the present study an attempt was made to estimate coefficients of dose response curves for PCC aberrations induced by CalyculinA and Okadaic acid, using 60 Co gamma radiation and 8 MeV pulsed electron beam for biodosimetry application., Materials and Methods: The modified method outlined by Puig et al. 2013 was used to conduct Calyculin A and Okadaic acid induced PCC assay in human blood lymphocytes.Chemical treatment was given for the last 1 h of a 48 h culture. The study was carried out in the dose range 2.5 to 20 Gy using 60 Co gamma rays and 8 MeV pulsed electron beam., Results and Conclusions: Results show a linear dose dependent increase with a slope of 0.047 ± 0.001 from Calycalin A PCC and 0.048 ± 0.002 form Okadaic acid PCC. The slope of the fragments curve was 0.327 ± 0.006 from Calyculin A and 0.328 ± 0.006 from Okadaic acid PCC. Further, dose calibration studies were carried out for 8 MeV electron using Calyculin A PCC assay and the obtained slope from ring yield was 0.054 ± 0.002 and 0.427 ± 0.009 from fragment yield.

Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are histological markers of Alzheimer's disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. Okadaic acid (OKA), a strong inhibitor of protein phosphatase 2A, has been used to induce dementia similar to AD in rats. We herein investigated the effect of intracerebroventricular (ICV) infusion of OKA (100 and 200ng) on hippocampal tau phosphorylation at Ser396, which is considered an important fibrillogenic tau protein site, and on glucose uptake, which is reduced early in AD. ICV infusion of OKA (at 200ng) induced a spatial cognitive deficit, hippocampal astrogliosis (based on GFAP increment) and increase in tau phosphorylation at site 396 in this model. Moreover, we observed a decreased glucose uptake in the hippocampal slices of OKA-treated rats. In vitro exposure of hippocampal slices to OKA altered tau phosphorylation at site 396, without any associated change in glucose uptake activity. Taken together, these findings further our understanding of OKA neurotoxicity, in vivo and vitro, particularly with regard to the role of tau phosphorylation, and reinforce the importance of the OKA dementia model for studying the neurochemical alterations that may occur in AD, such as NFTs and glucose hypometabolism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The N-methyl-D-aspartate (NMDA) receptor is a subtype of ionotropic glutamate receptor that is involved in synaptic mechanisms of learning and memory, and mediates excitotoxic neuronal injury. In this study, we tested the hypothesis that NMDA receptor subunit gene expression is altered in cortex and hippocampus of OKA induced memory impairment. Therefore in the present study, we checked the effect of OKA (ICV) on NMDA receptor regulation and synapse function. The memory function anomalies and synaptosomal calcium ion (Ca(2+)) level were increased in OKA treated rats brain; which was further protected by MK801 (0.05mg/kg. i.p) treatment daily for 13days. To elucidate the involvement of NMDA receptor, we estimated NR1, NR2A and NR2B (subunits) expression in rat brain. Results showed that expression of NR1 and NR2B were significantly increased, but expression of NR2A had no significant change in OKA treated rat brain. We also observed decrease in synapsin-1 mRNA and protein expression which indicates synapse dysfunction. In addition, we detected an increase in MDA and nitrite levels and a decrease in GSH level in synapse preparation which indicates synapse altered redox stress. Moreover, neuronal loss was also confirmed by nissl staining in periventricular cortex and hippocampus. Altered level of oxidative stress markers along with neuronal loss confirmed neurotoxicity. Further, MK801 treatment restored the level of NR1, NR2B and synapsin-1 expression, and protected from neuronal loss and synapse redox stress. In conclusion, Okadaic acid (OKA) induced expression of NR1 and NR2B deteriorates synapse function in rat brain which was confirmed by the neuroprotective effect of MK801., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Green tea polyphenols (GTPs) are now being considered possible protective agents in neurodegenerative diseases such as Alzheimer's disease (AD). Previous studies suggested that GTPs could inhibit amyloid fibril formation and protect neurons from toxicity induced by β-amyloid. However, whether GTPs can ameliorate learning and memory impairments and also reduce tau hyperphosphorylation induced by okadaic acid (OA) in rats remains unclear. The aim of this study was to determine if GTPs have neuroprotection against OA-induced neurotoxicity., Methods: In this work, rats were pretreated with GTPs by intragastric administration for 4 wk. Then OA was microinjected into the right dorsal hippocampus. Morris water maze tests were used to test the ethologic changes in all groups, and tau protein hyperphosphorylation was detected both in vivo and in vitro., Results: The ethologic test indicated that the staying time and swimming distance in the target quadrant were significantly decreased after OA treatment, whereas rats pretreated with GTPs stayed longer in the target quadrant. Methyl thiazolyl tetrazolium assay and lactate dehydrogenase leakage showed that GTPs greatly ameliorated primary hippocampal neurons damage induced by OA. Furthermore, reduced hyperphosphorylated tau protein was detected with GTPs pretreatment., Conclusion: Taken together, our results suggest that GTPs have neuroprotection against OA-induced neurotoxicity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The polyethers yessotoxin (YTX) and okadaic acid (OA) are two marine algal toxins frequently associated as edible shellfish contaminants. Seafood contamination by these compounds, also at low concentrations and for a long period of time, can increase the possibility of their simultaneous and repeated ingestion, with possible synergistic toxic effects. Thus, in vivo toxicity by repeated oral exposure to a combination of fixed doses of YTX and OA (1 mg YTX/kg and 0.185 mg OA/kg, daily for 7 days) was investigated in mice, in comparison to that of each toxin alone. No mortality, signs of toxicity, diarrhea or hematological changes was induced by the toxins co-administration or by the single toxins. Light microscopy revealed changes at gastric level (multifocal subacute inflammation, erosions and epithelial hyperplasia) in 2/5 mice co-administered with the toxins. In animals dosed only with OA, epithelial hyperplasia of forestomach and slight focal subacute inflammation of its submucosa were noted. No changes were induced by the treatment with YTX. Ultrastructural analysis of the heart revealed some cardiomyocytes with "loose packing" of myofibrils and aggregated rounded mitochondria in mice co-administered with the toxins or with YTX; OA-treated mice showed only occasional mitochondrial assemblage and dilated sarcomeres. Thus, the combined oral doses of YTX (1 mg/kg/day) and OA (0.185 mg/kg/day) did not exert cumulative or additive toxic effects in mice, in comparison to the single toxins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Toxins from the okadaic acid (OA) and azaspiracid (AZA) group cause considerable negative health effects in consumers when present in shellfish above certain levels. The main symptoms, dominated by diarrhoea, are caused by damage to the gastrointestinal (GI) tract. Even though OA and AZAs exert toxicity via different mechanisms, it is important to find out whether they may enhance the health effects if present together since they act on the same organs and are regulated individually. In this study, the main issue was the possibility of enhanced lethality in mice upon combined oral exposure to OA and AZA1. In addition, pathological effects in several organs and effects on absorption from the GI tract were studied. Although the number of mice was small due to low availability of AZA1, the results indicate no additive or synergistic effect on lethality when AZA1 and OA were given together. Similar lack of increased toxicity was observed concerning pathological effects that were restricted to the GI-tract. OA and AZA1 were absorbed from the GI-tract to a very low degree, and when given together, uptake was reduced. Taken together, these results indicate that the present practice of regulating toxins from the OA and AZA group individually does not present an unwanted increased risk for consumers of shellfish., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The mouse bioassay is widely used to detect diarrhetic shellfish poisoning (DSP) toxins. To the best of our knowledge, however, there have been no reports specifically on strain differences in susceptibility to DSP toxins. In this study, we investigated the susceptibility of different mice strains to okadaic acid (OA), one of the representative DSP toxins. A lethal dose of OA was injected intraperitoneally (i.p.) into mice. The mice were observed until 24 h after injection. Five inbred strains (A/J, BALB/c, C3H/He, C57BL/6, and DBA/2) and two non-inbred strains (ddY, and ICR) of mice were compared. All the mice were male, weighed 16-20 g, and were 4-5 weeks old. The lethality was 90-100% in the A/J, BALB/c, ddY, and ICR strains, 70-80% in the C3H/He and C57BL/6 strains, and 40% in DBA/2 strain. Survival analysis showed that the BALB/c, C57BL/6, ddY, and ICR strains died earlier and the A/J, C3H/He and DBA/2 strains survived longer. These results indicate that significant differences may exist in the susceptibility of mice strains to OA.

Aims: The aim of the present study is to investigate the status of proinflammatory cytokine in the brain of intracerebroventricular (i.c.v.) okadaic acid (OKA) induced memory impaired rat., Main Methods: OKA (200 ng) intracerebroventricular (i.c.v.) was administered in rats. Memory was assessed by Morris water maze test. Biochemical marker of neuroinflammation (TNF-α, IL-β), total nitrite, mRNA (RT PCR) and protein expression (WB) of iNOS and nNOS were estimated in rat brain areas., Key Findings: OKA caused memory-impairment in rats with increased expression of proinflammatory cytokine TNF-α and IL-1β and total nitrite in brain regions hippocampus and cortex. The expression of mRNA and protein of iNOS was increased while; the expressions were decreased in case of nNOS. Pretreatment with antidementic drugs donepezil (5 mg/kg, p.o.) and memantine (10 mg/kg, p.o) for 13 days protected i.c.v. OKA induced memory impairment and changes in level of TNF-α, IL-β, total nitrite and expressions of iNOS and nNOS in OKA treated rat., Significance: This study suggests that neuroinflammation may play a vital role in OKA induced memory impairment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Several types of animal models have been developed to investigate Alzheimer's disease (AD). Okadaic acid (OA), a potent inhibitor of phosphatases 1 and 2A, induces characteristics that resemble AD-like pathology. Memory impairment induced by intra-hippocampal injection of OA has been reported, accompanied by remarkable neuropathological changes including hippocampal neurodegeneration, a paired helical filament-like phosphorylation of tau protein, and formation of β-amyloid containing plaque-like structures. Rats were submitted to bilateral intrahippocampal okadaic acid-injection (100 ng) and, 12 days after the surgery, behavioral and biochemical tests were performed. Using this model, we evaluated spatial cognitive deficit and neuroglial alterations, particularly astroglial protein markers such as glial fibrillary acidic protein (GFAP) and S100B, metabolism of glutamate, oxidative parameters and alterations in MAPKs. Our results indicate significant hippocampal changes, including increased GFAP, protein oxidation, and phosphorylation of p38(MAPK); and decreases in glutathione content, transporter EAAT2/GLT-1, and glutamine synthetase activity as well as a decrease in cerebrospinal fluid S100B. No alterations were observed in glutamate uptake activity and S100B content. In conclusion, the OA-induced model of dementia caused spatial cognitive deficit and oxidative stress in this model and, for the first time to our knowledge, specific astroglial alterations. Findings contribute to understanding diseases accompanied by cognitive deficits and the neural damage induced by AO administration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mitochondrial abnormalities have been identified in a large proportion of neurodegenerative diseases. Recently we have reported that intracerebroventricular (ICV) administration of okadaic acid (OKA) causes memory impairment in rat. However involvement of mitochondrial function in OKA induced memory impairment and neuronal damage has not been determined. OKA (200 ng) was administered by ICV route. After 13th day of OKA administration memory function was evaluated by Morris Water Maze test. Following completion of behavioral studies on 16th day, mitochondrial membrane potential, Ca(2+) and reactive oxygen species were evaluated in mitochondrial preparation of cortex, hippocampus, striatum and cerebellum of rat brain. While ATP, mitochondrial activity, lipid peroxidation and nitrite were investigated in synaptosomal preparation of rat brain areas. The activities and mRNA expression of apoptotic factors, caspase-3 and caspase-9, were studied in rat brain regions. The neuronal damage was also confirmed by histopathological study. OKA treated rats showed memory impairment including increased Ca(2+) and reactive oxygen species and decreased mitochondrial membrane potential, ATP and mitochondrial activity in mitochondrial preparation. There was a significant increase in lipid peroxidation and nitrite in synaptosomal preparations. Preventive treatment daily for 13 days with antidementic drugs, donepezil (5 mg/kg, p.o) and memantine (10 mg/kg, p.o), significantly attenuated OKA induced mitochondrial dysfunction, apoptotic cell death, memory impairment and histological changes. Mitochondrial dysfunction appeared as a key factor in OKA induced memory impairment and apoptotic cell death. This study indicates that clinically used antidementic drugs are effective against OKA induced adverse changes at behavioral, cellular, and histological levels and mitochondrial dysfunction., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The marine biotoxin okadaic acid (OA), produced by dinoflagellates, can accumulate in various bivalve molluscs. In humans, oral consumption of shellfish contaminated with OA induces acute toxic effects like diarrhea, nausea, vomiting and abdominal pain. However, tumorigenic and embryotoxic effects of OA have been also described. Current toxicokinetic studies with mice were performed with high cytotoxic oral doses leading presumably to a paracellular passage of OA through the gastrointestinal barrier. There are no studies available analyzing the absorption at low concentrations, which represent a realistic dietary exposure, making a reliable risk assessment difficult. Therefore, we performed a low-dose study using the human intestinal Caco-2 cell model to simulate the intestinal barrier. Low level exposure of 20-200 nM OA to the cell monolayer allows an only limited passage from the "luminal" to the "blood side". Furthermore, we could detect a significant efflux of OA, which led to the suggestion that active transport mechanisms are involved in the elimination process of OA. In conclusion, our results indicate that besides the well known defense mechanisms of humans against this marine biotoxin--vomiting and diarrhea--further detoxification mechanisms are available to limit the absorption of toxic OA., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes cognitive and behavioral deterioration in the elderly. Neurofibrillary tangles (NFTs) are one of the pathological hallmarks of AD that has been shown to correlate positively with the severity of dementia in the neocortex of AD patients. In an attempt to characterize an in vivo AD tauopathy model, okadaic acid (OA), a protein phosphatase inhibitor, was microinfused into the right lateral dorsal hippocampus area of ovariectomized adult rat. Cognitive deficiency was seen in OA-treated rats without a change in motor function. Both silver staining and immunohistochemistry staining revealed that OA treatment induces NFTs-like conformational changes in both the cortex and hippocampus. Phosphorylated tau as well as cyclin-dependent kinase 5 (cdk5) and its coactivator, p25, were significantly increased in these regions of the brain. Oxidative stress was also increased with OA treatment as measured by protein carbonylation and lipid peroxidation. These data suggest that the unilateral microinfusion of OA into the dorsal hippocampus causes cognitive deficiency, NFTs-like pathological changes, and oxidative stress as seen in AD pathology via tau hyperphosphorylation caused by inhibition of protein phosphatases., (Copyright © 2010. Published by Elsevier B.V.)

One of the pathological hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs), which are composed of abnormally hyperphosphorylated tau, but the mechanism of tau hyperphosphorylation in AD is still unclear. To investigate the effects of estrogens on tau phosphorylation, SH-SY5Y cells were treated with okadaic acid (OA), a serine/threonine phosphatase inhibitor, to induce tau phosphorylation and the effects of estrogen were observed by co-treatment with 17beta-estradiol (E2). We found that OA induced in vitro tau hyperphosphorylation, which was prevented by E2 in a dose-dependent manner. This effect of E2 was partially blocked by an estrogen receptor (ER) antagonist, ICI 182,780. In addition to tau hyperphosphorylation, inhibition of serine/threonine phosphorylation induced upregulation of cdk5 levels, which was attenuated by E2 in a manner that was counteracted by ICI 182,780. Our results show that cdk5 is involved in OA-induced tau hyperphosphorylation, and estrogens ameliorate the tau hyperphosphorylation, which may be mediated in part by ER., (Published by Elsevier B.V.)

Background and Purpose: Buprenorphine displays attributes of opioids, but also some features distinct from them. We examined spinal and supraspinal signal transduction of buprenorphine-induced anti-nociception in mice compared with morphine and fentanyl., Experimental Approach: The opioid receptor antagonist naloxone, Pertussis toxin (PTX), G(z) protein antisense and nociceptin/orphanin-FQ receptor agonist nociceptin, and antagonist, JTC-801, were injected supraspinally (intracerebroventricular) and spinally (intrathecal). Also the cell-permeable Ser/Thr protein phosphatase inhibitor okadaic acid was given supraspinally., Key Results: Spinal naloxone (20 microg) or PTX (1 microg) attenuated morphine, fentanyl and buprenorphine (s.c.) anti-nociception. Supraspinal naloxone or PTX attenuated morphine and fentanyl, but not buprenorphine anti-nociception. Spinal G(z) protein antisense did not alter buprenorphine, morphine or fentanyl anti-nociception and supraspinal G(z)-antisense did not alter morphine or fentanyl anti-nociception. However, supraspinal G(z)-antisense (not random sense) reduced buprenorphine anti-nociception. Peripheral JTC-801 (1 mgxkg(-1), i.p.) enhanced the ascending (3 mgxkg(-1)) and descending (30 mgxkg(-1)) portions of buprenorphine's dose-response curve, but only spinal, not supraspinal, nociceptin (10 nmolxL(-1)) enhanced buprenorphine anti-nociception. Intracereboventricular okadaic acid (0.001-10 pg) produced a biphasic low-dose attenuation, high-dose enhancement of buprenorphine(3 or 30 mgxkg(-1), s.c.) anti-nociception, but did not affect morphine or fentanyl anti-nociception., Conclusions and Implications: Buprenorphine has an opioid component to its supraspinal mechanism of analgesic action. Our present results reveal an additional supraspinal component insensitive to naloxone, PTX and nociceptin/orphanin-FQ, but involving G(z) protein and Ser/Thr protein phosphatase. These data might help explain the unique preclinical and clinical profiles of buprenorphine.

Algal bloom with consequent production of marine toxins contaminating bivalves is increasing in costal regions worldwide because of sea water quality worsening. Contamination of seafood by diarrheic shellfish poisoning toxins (DSP) together with metals is frequently reported, a phenomenon not fully explained yet. In this context, metal ions were assayed in clams collected from the banned area of Boughrara, Tunisia, contaminated by Gymnodinium and other algae such as Dinophysis sp, accumulated by these bivalves. The presence of toxic metals ions such as Chromium (Cr) and Cadmium (Cd) in meat, shells, and water released by the clams prompted us to experiment in Caco-2 intestinal cell line toxic effects of these heavy metals ions in combination with okadaic acid, one DSP present in clams to assess the potential global toxicity. Cr and Cd produce additive effects in (i) reactive oxygen species production, (ii) cytotoxicity as assessed by the mitochondrial activity testing method (MTT test), and (iii) DNA lesions evaluated by agarose gel electrophoresis and acridine orange staining. Exaggerated DNA fragmentation is observed, suggesting an overloading of repair capacity of Caco-2 cells. The apoptosis suggested by a DNA fragment sizing (180-200 bp) in agarose gel and mechanisms underlying these additive effects in Caco-2 cells still need to be more comprehensively explained.

Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8-31.3 ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8 ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis.

Nuclear receptor corepressor (N-CoR) is a critical regulator of neural stem cell differentiation. Nuclear localization of N-CoR is a feature of undifferentiated neural stem cells and cytoplasmic translocation of N-CoR leads to astrocytic differentiation. Comparative proteomic analysis of microdissected glioblastoma multiforme (GBM) specimens and matched normal glial tissue reveals increased expression of N-CoR in GBM. In GBM primary cell cultures, tumor cells with nuclear localization of N-CoR demonstrate an undifferentiated phenotype, but are subject to astroglial differentiation upon exposure to agents promoting phosphorylation of N-CoR and its subsequent translocation to the cytoplasm. Treatment of glioma cell lines with a combination of retinoic acid and low-dose okadaic acid decreases the corepressor effect of N-CoR and has a striking synergistic effect on growth inhibition. The identification of N-CoR in GBM provides insights into the tumorigenesis process and supports the development of differentiation-based therapeutic strategies.

When substituting the mouse bioassay for lipophilic marine algal toxins in shellfish with analytical methods, science based factors of relative toxicity for all analogues that contribute to health risk to consumers are necessary. The aim of this paper is to establish the relative intraperitoneal toxicity of dinophysistoxin-2 (DTX-2) compared with okadaic acid (OA). The study was performed as an open, randomised parallel group trial with a four level response surface design within each of the two parallels. In accordance with the response surface design model, the LD50 for DTX-2 and OA was 338 and 206 microg/kg, respectively. By use of common regression analysis, the LD50 of DTX-2 and OA were estimated to 352 microg/kg and 204 microg/kg, respectively. The deviations between the LD50 estimates by the two methods was 4% for DTX-2 and less than 1% for OA. Taken together, these results indicate that the relative toxicity of DTX-2 is about 0.6, compared to OA. Results from the PP2A assay correspond very well with the results obtained by the mouse bioassay. The IC50 concentrations for DTX-2 and OA were 5.94 and 2.81 ng/mL, respectively. This indicates that OA is about twice as toxic as DTX-2. Since inhibition of PP2A is acknowledged as the main mechanism of toxicity of the OA group toxins, this supports the establishment of a relative toxicity factor of DTX-2 of 0.6 compared with OA.

The challenging possibility of selectively inducing mitotic death in tumor cells by combining genotoxic agents with the inhibition of G2 checkpoints of the cell cycle is the subject of intensive investigation. We show that very low concentrations (3.5 and 5 nM) of okadaic acid induce mitotic death in two glioblastoma cell lines, in the absence of genotoxic agents. At the concentrations used, the main target of okadaic acid action is protein phosphatase 2A (PP2A), an enzyme deeply involved in the negative control of cell-cycle progression. The peculiar susceptibility of glioblastoma cells to induction of mitotic death by very low concentrations of okadaic acid must be related to an impairment of PP2A activity and to a specific deficiency in some cell-cycle checkpoints. In addition to its ability to induce abnormal mitoses in actively proliferating glioblastoma cells, okadaic acid possesses the ability to force semi-confluent glioblastoma cells to the M phase of the cell cycle, where they show the same abnormalities observed in actively proliferating glioblastoma cells. In semi-confluent cells the induction of mitotic death involves the activity of both the extracellular signal regulated kinases (ERKs) and the M-phase promoting factor: okadaic acid overstimulates ERK activity, and PD98059 (inhibitor of ERK activation) as well as roscovitine (S)-isomer (specific inhibitor of M-phase promoting factor activity) counteract the induction of mitotic death. Our results show that, without the use of genotoxic agents, it is possible to induce mitotic death in glioblastoma cells by activating the same uncontrolled pathways responsible for the uncontrolled proliferation.

In experimental alcoholic liver disease, protein degradation by the ATP-ubiquitin-proteasome pathway is inhibited. Failure of the proteasome to eliminate cytoplasmic proteins leads to the accumulation of oxidized and otherwise modified proteins. One possible explanation for the inhibition of the proteasome is hyperphosphorylation of proteasome subunits. To examine this possibility, the 26S proteasomes from the liver of rats fed ethanol and a pair-fed control were studied by isolating the proteasomes in a purified fraction. The effect of ethanol on the phosphorylation of proteasomal subunits was compared with the hyperphosphorylation of the proteasomes caused by okadaic acid given to rats in vivo. Ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome. The 2D electrophoresis and Western blot analysis of the purified 20S and 26S proteasomes from the ethanol-fed rats indicated that hyperphosphorylation of proteasomal subunits had occured. The proteasomal alpha type subunits C9/alpha3 and C8/alpha7 were hyperphosphorylated compared to the controls. Chymotrypsin-like activity was also inhibited by okadaic acid treatment similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel revealed many hyperphosphorylated bands in the proteasomes from the okadaic acid treated and the ethanol fed rat livers compared with the controls. In conclusion hyperphosphorylation of the proteasome subunits occurs in the ethanol treated proteasomal subunits which could be one mechanism of the inhibition of the 26S proteasome caused by ethanol feeding.

A short-term toxicity study after 7 days oral daily administration of yessotoxin (YTX; 2 mg/kg/day), homoYTX (1 mg/kg/day), 45-hydroxy-homoYTX (1 mg/kg/day) and of the main diarrhoetic shellfish toxin okadaic acid (OA; 1 mg/kg/day) was carried out in mice. Symptoms, lethality, food consumption, body and organ weights, gross pathology and histopathology of the main organs and tissues, leukocytes formula as well as plasmatic levels of transaminases, lactate dehydrogenase and creatinine phosphokinase were evaluated. Heart tissue was studied also hystochemically for the presence of apoptotic nuclei and by transmission electron microscopy. No mortality, signs of toxicity or cumulative effects were induced by the repeated oral exposure to YTXs. Only ultrastructural changes in the cardiac muscle cells near the capillaries, such as package of rounded mitochondria and alteration of the cells boundary were observed, without any increase of lactate dehydrogenase, an index of cardiac damage. OA induced diarrhoea, body weight loss, reduced food consumption, and the death of 2/5 mice after 5 days. Necroscopy and/or light microscopy analysis revealed toxic effects mainly at forestomach (ulceration and hyperplasia), liver and, indirectly to body weight loss of mice, atrophic signs in the lymphoid organs and exocrine pancreas. Electron microscopy of heart tissue showed alterations of mitochondria and fibers in myocardiocytes, although no apoptotic change was recorded.

We have previously shown that the intrahippocampal microinjection of okadaic acid (OKA), a potent inhibitor of serine/threonine protein phosphatases, induces epileptic seizures, neuronal death, and the hyperphosphorylation of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor. We administered OKA by reverse microdialysis in the hippocampus of awake and halothane-anesthetized rats, with simultaneous collection of microdialysis fractions and recording of the EEG activity, and subsequent histological analysis. OKA produced intense behavioral and persistent EEG seizure activity in the awake rats but not in the anesthetized animals, and did not significantly alter the extracellular concentration of glutamate and aspartate detected in the microdialysis fractions. One day after the experiment a remarkable neurodegeneration of CA1 hippocampal region was observed in both the awake and the anesthetized rats. We conclude that the OKA-induced epilepsy cannot be ascribed to increased extracellular glutamate, but to an increased sensitivity of NMDA receptor. We propose that halothane protected against the epilepsy because it blocks NMDA receptor overactivation, and that the neurodegeneration of CA1 region is independent of this overactivation and due probably to alterations of cytoskeletal proteins consequent to the OKA-induced hyperphosphorylation.

The acute toxicity of yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxy-homoyessotoxin (45-OH-homoYTX) has been studied in comparison to that of okadaic acid (OA), the main diarrhogenic toxin, both after intraperitoneal (i.p.) and oral administration. After i.p. administration, homoYTX and YTX showed similar lethality (LD(50)=444 microg/kg and 512 microg/kg), higher than that of OA (LD(50)=225 microg/kg), while 750 microg/kg of 45-OH-homoYTX did not cause death. OA induced the already known toxic signs: before death, mice were motionless and cyanotic; small intestine and liver damage were shown at post-mortem. Mice treated with YTX and homoYTX were restless and jumped before death; necroscopy did not show major changes. After oral treatment, 2 mg/kg of OA induced diarrhoea and body weight loss, causing 4/5 deaths; necroscopy and/or histology revealed degenerative lesions to small intestine, forestomach and liver (confirmed by increased plasma transaminase), but no myocardium alterations. On the contrary, the oral treatment with YTX (1 and 2 mg/kg) and its derivatives (1 mg/kg) did not cause any death or signs of toxicity, except some ultrastructural myocardiocyte alterations, adjacent to capillaries, such as cytoplasmic protrusions (YTX, 1 and 2 mg/kg), fibrillar alteration (YTX, 1 mg/kg) or mitochondria assemblage (45-OH-homoYTX). Altogether, our data show that YTX and its derivatives are less toxic than OA after acute oral and i.p. treatments, at doses which may represent up to 100 times of the possible human daily intake.

Okadaic acid (OA)-induced germinal vesicle breakdown (GVBD) and localization of protein phosphatase-1 (PP1) in oocyte nuclei are suggestive of PP1's role in regulating oocyte GVBD. To explore this possibility, we microinjected protein phosphatase (PP) inhibitors OA, anti-PP1 antibody (anti-PP1), PP1 inhibitor I2, and anti-PP2A antibody (anti-PP2A) into nuclei of roscovitine (ROSC)-arrested mouse oocytes. Oocytes were also injected with recombinant PP1 in the absence of ROSC. Oocytes were assessed for GVBD and metaphase II (MII) development at 2 and 18 hr post-injection. Data were analyzed using Cochran-Mantel-Haenszel Statistics adjusted for time. Microinjection of OA significantly enhanced GVBD in comparison to controls at 2 and 18 hr (P < 0.01), yet had no effect on MII development. Similarly, microinjection of anti-PP1 resulted in significantly higher levels of GVBD compared to controls at 2 and 18 hr (P < 0.01). Interestingly, anti-PP1 microinjection also tended to enhance MII development at 18 hr in comparison to controls (P < 0.09). Microinjection of I2, anti-PP2A, and PP1 had no effect on GVBD or MII development. If reduction of PP1 activity was important for GVBD, one would anticipate an endogenous means of regulating PP1 activity at this developmental stage. In somatic cells, phosphorylation of PP1 at Thr320 causes PP1 inactivation. Germinal vesicle-intact oocytes did not contain phosphorylated PP1, as determined using a specific Thr320-Phospho-PP1 antibody, Western blot analysis, and confocal immunocytochemistry. At or around the time of GVBD, oocyte PP1 became phosphorylated at Thr320, which remained phosphorylated through MII development. These data indicate that inhibition of intra-nuclear PP1, through specific antibody neutralization, mimics OA-stimulated GVBD, providing the first direct evidence that nuclear PP1 is involved in regulation of oocyte nuclear membrane integrity. In addition, phosphorylation of PP1 occurs at/or around GVBD indicating that inactivation of PP1 is an important intracellular event in regulation of nuclear envelope dissolution at GVBD., (Copyright 2003 Wiley-Liss, Inc.)

This paper describes the distribution and excretion of okadaic acid (OA) administered orally to mice and examined by immunostaining method. Five min after administration, OA was systemically distributed, being detected in the lung, liver, heart, kidney, and small and large intestine. The swollen small intestinal villi contained OA, causing the separation of epithelial cells from villi and erosion, which developed within 1h. Bleeding and edema in the lung were also found, and the distribution of OA coincided with these injuries. Although a considerable amount of OA was accumulated in the liver, no symptoms, such as bleeding, were observed. The detection of OA continued for 2 weeks in the liver and blood vessels. Excretion from kidney, cecum and large intestine began even after 5 min of the administration, and the excretion from intestine continued for 4 weeks.

Disturbance in phosphorylation/dephosphorylation can trigger apoptosis. Little is known as to its effects on mesencephalic dopamine neurons, the major neurons lost in Parkinson's disease. In this study, okadaic acid (OKA), a phosphatase 1 and 2A inhibitor, with greater potency toward 2A, was toxic to mesencephalic dopamine and gamma-aminobutyric acid (GABA) neurons, however, dopamine neurons were 4-fold more sensitive. The EC(50) for dopamine versus GABA toxicity was 1.5 versus 6.5 nM, respectively, and was consistent with an inhibition of phosphatase 2A. Dopamine neurons were also more sensitive to calyculin-A, a phosphatase inhibitor equipotent toward 1 and 2A. OKA-methyl-ester, which lacks phosphatase inhibitory activity, was without effect. DNA laddering typical of apoptosis was observed in cultures at a concentration that was specifically toxic to dopamine neurons (5 nM). In contrast to the sensitivity of mesencephalic neurons to phosphatase inhibition, inhibition of protein kinase activity with staurosporine or K252a showed little toxicity and protected neurons from OKA. Consistent with in vitro findings, infusion of 32 to 320 pmol of OKA into the left striatum of rats caused a dose-dependent loss of striatal dopamine without any loss of GABA 1 week following infusion. Acutely, OKA increased tyrosine hydroxylase activity, a phosphatase 2A substrate, and increased dopamine turnover. The above-mentioned findings demonstrate that dysregulation of phosphatase activity is detrimental to mesencephalic neurons, with dopamine neurons, in vitro and in vivo, being relatively more sensitive to phosphatase 2A inhibition. Disturbances in the phosphorylation control of proteins unique to dopamine neurons may contribute to their enhanced vulnerability to OKA exposure.

The diarrhetic shellfish toxin, okadaic acid, administered to rats by intragastric intubation, caused intestinal damage, diarrhea and death, but had no detectable effect on the liver. In contrast, okadaic acid administered intravenously had little effect on intestinal function, but caused a rapid dissolution of hepatic bile canalicular actin sheaths, congestion of blood in the liver, hypotension and death at high doses. In isolated rat hepatocytes, okadaic acid induced disruption of the canalicular sheaths as well as of the keratin intermediate filament network. Both of these cytoskeletal changes could be prevented by addition of a cytoprotective flavonoid, naringin, to the isolated hepatocytes, whereas intravenously or intragastrically administered naringin failed to protect against the effects of okadaic acid in vivo. Freshly isolated colonocytes already had fragmented keratin and tubulin cytoskeletons, died rapidly and were not further afflicted by okadaic acid. Naringin had no protective effect on isolated colonocytes or on intestinal function in vivo, but the nonspecific protein kinase inhibitor, K-252a, and the protein-tyrosine-phosphatase inhibitor, vanadate, significantly reduced the extent of colonocytic keratin fragmentation, and an inhibitor of apoptotic caspases, zVAD.fmk, was strongly protective. Further studies of hepatic and intestinal cytoprotectants should focus on conditions that limit their effectiveness in vivo.

Our biopharmacological approach suggests that the now well-documented inhibitory effects of genistein on the maturation of mammalian oocytes do not seem to be related to its effect on tyrosine kinases. Indeed, we show that both tyrphostin B46 and Lavendustin A, two selective inhibitors of protein tyrosine kinases, fail to inhibit meiosis reinitiation. According to recent findings, the G2/M arrest induced by genistein could be due to inhibition of the kinase activity of cdc2. We were therefore mainly interested in dissecting the cytological effects of genistein on mouse primary and secondary oocytes. Genistein exerts the same cytological effects as IBMX on primary oocytes: their germinal vesicle is maintained in a central position, the cytoplasmic microtubule network is stabilized, the central GV immobilization is overcome by demecolcine and they complete normal maturation after their transfer to culture medium. The GV-arresting activity of genistein is also bypassed by OA but combination of both drugs results in a dramatic reorganization of the cytoskeleton leading to a huge membrane bulging, which is quite different to apoptotic-related blebbing. MAP Kinase activation is correlated with meiosis reinitiation. When applied after GVBD has taken place, genistein does not inhibit MAPK activation, metaphase spindle formation and metaphase-to-anaphase transition, but prevents the barrel-shaped MI spindle from undergoing its peripheral migration and the oocytes from extruding their first polar body. It may thus be concluded that the checkpoint control for anaphase onset is unaffected by the drug. On the contrary, our results suggest that spindle anaphase A to spindle anaphase B transition, spindle degradation, mid-body formation and cytokinesis are triggered by a genistein-sensitive mechanism that might be a mid-anaphase checkpoint. Finally, we confirm that genistein induces transition to interphase in metaphase II oocytes but never induces cortical granule exocytosis, the cytoplasmic hallmark of activation.

We have studied the effect of two protein phosphatase inhibitors on thyrotropin (TSH)-stimulated triiodothyronine (T3) production by sheep thyroid cells grown in primary culture. Incubation of sheep thyrocytes with okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type 1 (PP1) and type 2A (PP2A) protein phosphatases, resulted in an increase of TSH-stimulated T3 production. This effect was detected using concentrations as low as 0.1 pM with OA and 1 fM with CL-A. An inhibitory effect on T3 production, due to cellular death, was observed with 6 nM OA and 1 nM CL-A. In the absence of TSH, OA or CL-A had no effect on T3 production by thyrocytes. Forskoline (10 microM), an activator of adenylate cyclase, increased the basal and TSH-stimulated T3 release by sheep thyroid cells; this effect was increased by OA in cells grown in the basal state but not in the presence of TSH. These results suggest that the marine toxins OA and CL-A, two potent inhibitors of PP-1 and PP-2A, have significant stimulatory effects on T3 secretion promoted by TSH and FK. These observations indicate that these proteins could be important mediators of thyroid hormone production.

Ceramide is a lipid second messenger which is generated in response to stimulation of a number of surface receptors, treatment with chemotherapeutic agents, or ionising radiation. Depending on the target cell, ceramide induces diverse biological responses including apoptosis, cell-cycle arrest, differentiation, and also proliferation. We studied the effect of ceramide on the degradation of IkappaB, the cytoplasmic inhibitor of the transcription factor NF-kappaB. We show that ceramide treatment results in reduced levels of phosphorylated IkappaBalpha and degradation of both IkappaBalpha and IkappaBbeta. Ceramide synergised with okadaic acid (OA), a compound which interferes with the protein phosphatase 2A-controlled component of the NF-kappaB activation pathway, enhancing OA-induced IkappaB degradation. Ceramide also synergised with phorbol 12-myristate 13-acetate, which mimics protein kinase C activation. Finally, we show that the synergistic effect of ceramide with OA or phorbol ester can be observed in primary lymph node T-cells as well as in transformed T-cells., (Copyright 1999 Academic Press.)

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.

It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.

Effects of okadaic acid (OA) on mucosal damage were examined in rat colon. OA was sprinkled on rat colon mucosa under observation with an electronic-endoscopic system, and OA was also applied to the in vivo microscopic field. The OA-induced changes in transepithelial conductance (Gt) were measured by the Ussing voltage clamp technique. By endoscopic observation, the luminal sprinkling of OA (60 nmol/kg) evoked transient microthrombi in the submucosal venule, which was followed by mucosal edema. Histological study after endoscopic observation showed submucosal fluid retention, suggesting an increase of vascular permeability. The microthrombi were also detected by in vivo microscopy. By electrophysiological study after endoscopic observation with and without OA addition, the basal Gt values were 54+/-6.2 and 36.2+/-4.2 mS/cm2, respectively (P < 0.01). Furthermore in control rats, the serosal addition of OA evoked an increase in Gt in a concentration-dependent manner without increasing lactate dehydrogenase release. 2,4,6-Triaminopyrimidinium inhibited OA-induced Gt change by 60%. These results indicate that OA evokes an increase in paracellular permeability of epithelium. We conclude that the developed microthrombi are the first key event of OA-induced mucosal damage, followed by an increase in permeability in the submucosal venule and in the paracellular pathway of the epithelium.

Mussel samples from four locations along the Norweigian coast were extracted by methods for diarrheic shellfish toxins (DST) and tested by chemical and biological methods, including histopathology. All samples had previously been found to be highly toxic in mice, with symptoms indicating the presence of non-diarrheagenic toxins in the mouse bioassay. Chemical analysis revealed that the DST okadaic acid (OA) and dinophysistoxin-1 (DTX1) were present each one in one sample, but only a minor part of the total toxicity could be attributed to these toxions. In the other two samples, OA and DTX1 were absent. Incubation of the mussel extracts from all four samples with freshly prepared hepatocytes indicated the presence of unknown toxin(s) which may not be classified within the DST complex. Purified mussel samples were given to baby mice both via intraperitoneal (i.p.) injections and by oral intubation. Oral toxicity was about 25-50 times lower than toxicity obtained by i.p. injections, a result in accordance with acute toxic properties of many toxins. Risk assessment of the unknown toxin(s) requires chemical identification, but the preliminary results obtained indicate a large margin of safety, based on the large amounts of mussel extracts necessary to yield toxic effects in the intestine and liver in experimental animals upon oral exposure versus human intake.

Neonatal rat cortical astroglial cells in primary culture synthesize and secrete interleukin-1 beta (IL-1) and nerve growth factor (NGF). Treatment of astrocytes with okadaic acid (OA), an inhibitor of phosphoprotein phosphatases, dramatically increased both IL-1 and NGF mRNA content (about 50-fold) with maximal induction seen at 20-30 nM OA. The induction of IL-1 mRNA preceded that of NGF mRNA and was maximal after 9 h of treatment. OA increased IL-1 mRNA half-life by about 10-fold similar to the reported stabilization of the NGF mRNA. Addition of an IL-1 receptor antagonist dose-dependently inhibited the secretion of NGF stimulated by OA and IL-1. The results indicate that OA profoundly stimulates IL-1 expression in glial cells by enhancing IL-1 mRNA stability and that glial cell-derived IL-1 acts in a paracrine/autocrine manner to stimulate NGF production.