A fast and sensitive technique to study the kinetics and the concentration dependencies of DNA fragmentation during drug-induced apoptosis

Apoptotic cell death with its characteristic coordinated cellular breakdown can be triggered by cytotoxic drugs. One prominent feature that differentiates apoptotic from necrotic cell death is the caspase-mediated activation of an endonuclease that internucleosomally cleaves DNA resulting in the so-called apoptotic DNA ladder. Here we report a new rapid, sensitive and inexpensive column separation technique to study drug-induced DNA fragmentation from 10(6) or less cells. This technique, which is based on a modified plasmid spin column kit, avoids the use of hazardous chemicals. With this procedure and subsequent densitometric analysis it was possible to study the concentration dependencies and the kinetics of drug-induced DNA fragmentation. The applicability of this technique is shown for okadaic acid- and cantharidic-acid-treated pituitary GH(3) cells as well as highly okadaic-acid-resistant sublines. These studies allowed us to compare as well as to differentiate the effects and potencies of these structurally different but functionally quite similar inhibitors of ser/thr phosphatases 1 and 2A.     

Doxorubicin-resistant LoVo adenocarcinoma cells display resistance to apoptosis induction by some but not all inhibitors of ser/thr phosphatases 1 and 2A.

LoVo adenocarcinoma cells are fairly sensitive to cytostatic drugs, e.g. doxorubicin, but can develop drug resistance by expression of a P-glycoprotein-mediated MDR1 phenotype. LoVo cells respond with apoptosis to nanomolar concentrations of okadaic acid and micromolar concentrations of cantharidic acid. Interestingly, LoVoDx cells which had become about 10-fold less sensitive to doxorubicin by incubation in increasing concentrations of this cytostatic drug were also less sensitive to the toxicity of okadaic acid. Resistance to both agents was lost or significantly reduced by incubation in drug-free medium for about 4 months. On the other hand, LoVoDx cells did not lose responsiveness to the structurally different phosphatase inhibitor cantharidic acid but were about twofold more sensitive to the cytotoxic effect of this agent. Thus, MDR expression protects LoVo cells from the toxicity of phosphatase inhibitors that presumably are substrates of the P-glycoprotein, e.g. okadaic acid and its derivatives but not cantharidic acid, despite the fact that both agents are potent inducers of apoptotic cell death via ser/thr phosphatase inhibition.     

Okadaic acid induces apoptosis through the PKR, NF-κB and caspase pathway in human osteoblastic osteosarcoma MG63 cells

Okadaic acid (OA) is the major component of diarrheic shellfish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. However, the underlying regulatory mechanisms involved in OA-induced cell death are not well understood. In the present study, we examined the effects of OA on apoptosis of MG63 cells by characterizing apoptotic morphological changes of the cells and DNA fragmentation. The roles of double-stranded RNA-dependent protein kinase (PKR), nuclear factor-κB (NF-κB) and caspase in OA-mediated apoptosis in MG63 cells were also examined. Results showed that OA induced cytotoxicity and apoptosis in MG63 cells at IC₅₀ of 75 nM. A functional PKR pathway is required to induce apoptosis in response to OA treatment. Blockade of NF-κB by ammonium pyrrolidinedithiocarbamate (PDTC) resulted in down-regulation of apoptosis. The caspase-3 and caspase-8 inhibitors blocked apoptosis in MG63 cells. In conclusion, our results imply that OA can induce MG63 cell apoptosis through the PKR, NF-κB and caspase pathway.     

The phosphatase inhibitor okadaic acid blocks KCl-depolarization-induced rise of cytosolic calcium of rat insulinoma cells (RINm5F)

It has been shown that okadaic acid (OA) diminishes insulin secretion of rat pancreatic islets in response to glucose, glyceraldehyde and KCl. Glucose, glyceraldehyde and KCl cause release of insulin by depolarization and subsequent opening of L-type calcium channels. Calcium entry into cells is thought to be related to protein phosphorylation. To evaluate whether or not OA mediated inhibition of insulin secretion in response to depolarization might be due to an interference with calcium uptake, we studied its effect on KCl (30 mM)-induced increases of cytosolic calcium and discharge of insulin in the insulin secreting clonal tumor cell line RINm5F. OA inhibited KCl-stimulated insulin release in concentrations 1 M. In intact RINm5F cells similar concentrations of OA decreased the activity of protein phosphatases PP-1/PP-2A and inhibited the depolarization-induced rise of cytosolic calcium ([Ca²⁺]_i). The latter action could also be achieved with the protein phosphatase inhibitor calyculin A, whereas the OA analogue 1-nor-okadaone, which is without effect on phosphatases, did not affect [Ca²⁺]_i or insulin release. It is concluded that depression of depolarization-induced insulin secretion by OA is due to inhibition of calcium entry along voltage dependent calcium channels. The data also suggest that in RINm5F cells protein phosphatases PP-1/PP-2A are related to the function of voltage-dependent calcium channels.     

Hamster pancreatic beta cell lines with altered sensitivity towards apoptotic signalling by phosphatase inhibitors

Specific inhibitors of serine/threonine phosphatases like okadaic acid can induce apoptotic cell death in the pancreatic beta cell line HIT. Cultivation in stepwise increased concentrations of okadaic acid enabled the isolation of HIT100R cells which proliferate at 100 nM okadaic acid (8 - 10 times the initially lethal concentration). These two cell lines were used to characterize the events triggered by okadaic acid that led to apoptosis. Biochemical markers, e.g. cytochrome c release from mitochondria and increase of caspase-3-like activity, revealed that induction of apoptosis by 100 nM okadaic acid in parental HIT cells started with the release of cytochrome c. In HIT100R cells 500 nM okadaic acid were necessary to induce alterations comparable to those observed with 100 nM okadaic acid in non-resistant HIT cells. In contrast to okadaic acid, the potency of the structurally different phosphatase inhibitor cantharidic acid to induce cytochrome c release, increase of caspase-3-like activity and DNA fragmentation was comparable in HIT and HIT100R cells. Thus, no cross-resistance between these phosphatase inhibitors seemed to exist. Phosphatase activity in extracts from HIT and HIT100R cells did not differ in its total amount or in its sensitivity for okadaic acid. Since higher concentrations of okadaic acid were needed to induce apoptosis in HIT100R cells, a compromised intracellular accumulation of the toxin appeared likely. Functional and structural analysis revealed that this was achieved by the development of the multidrug resistance phenotype in HIT100R cells. The underlying mechanism appeared to be the enhanced expression of the pgp1 but not the pgp2 gene.     

Control of the mutagenicity of aromatic amines by protein kinases and phosphatases I. The protein phosphatase inhibitors okadaic acid and ortho -vanadate drastically reduce the mutagenicity of aromatic amines

The role of protein kinase C and protein phosphatases was examined in the control of mutagenic metabolites of aromatic amines. Various metabolic activating systems derived from rat liver were treated with: 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C modulator; okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatases (PP1 and PP2A); and ortho-vanadate (OV), an inhibitor of tyrosine phosphatases. TPA used over a wide concentration range (10⁻⁹–10⁻⁶ M) did not affect the bacterial mutagenicity of the aromatic amines and of the aromatic amide investigated, 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene (2AAF). At the molecular level, TPA did not affect the function of cytochrome P450s 1A1 or 1A2, which are known key factors for the activation and inactivation of aromatic amines/amides. By contrast the OA and OV treatment of rat hepatocytes, rat liver homogenate, fraction S9 and the nuclear fraction drastically reduced (by >80%) the mutagenicity of the aromatic amines/amide investigated. This is by far the most pronounced change in genotoxicity observed to date via modulation of phosphorylation. Whilst the mutagenicity of the primary toxication product 2-N-OH-acetylaminofluorene (2-N-OH-AAF) in the presence of exogenous activating systems (hepatocytes, S9-fraction, nuclear fraction) was also reduced by OV, OA had no influence. Thus the tyrosine protein phosphatase inhibitor and the serine/threonine protein phosphatase inhibitor influence the genotoxicity of aromatic amines/amides on different levels. Moreover, this shows that the drastic reduction in mutagenicity by OA was due to its influence on a step prior to the presence of the primary toxication product 2-N-OH-AAF. This reduction could be due to changes in the activity of cytochrome P4501A1 and/or 1A2. However, no incorporation of ³²P-labelled phosphate from intracellularly prelabelled [³²P]-ATP into cytochromes P450 1A1 or 1A2 nor any change in their catalytic activities was observed in the presence of OA. Furthermore, a phosphorylation dependent change in the function of P-glycoprotein (known for its role in the transport of diverse xenobiotic substances and their metabolites) was shown not to contribute to the observed decrease in mutagenicity. Our results reveal an important role for protein phosphatase 1 and/or 2A and tyrosine phosphatase(s) in the control of the genotoxicity of aromatic amines and amides. However, the present study does not distinguish between effects mediated by individual proteins affected by these protein phosphatases. Received: 30 April 1997 / Accepted: 20 May 1997     

Cytoskeletal disruption is the key factor that triggers apoptosis in okadaic acid-treated neuroblastoma cells

Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.     

The Discodermia calyx Toxin Calyculin A Enhances Cyclin D1 Phosphorylation and Degradation, and Arrests Cell Cycle Progression in Human Breast Cancer Cells

Cyclin D1 is a key regulator of the cell cycle that is over expressed in more than half of breast cancer patients. The levels of cyclin D1 are controlled primarily through post-translational mechanisms and phosphorylation of cyclin D1 at T286 induces its proteasomal degradation. To date, no studies have explored the involvement of phosphatases in this process. Here we treated human breast cancer cells with the structurally distinct toxins calyculin A, okadaic acid, and cantharidin, which are known to inhibit Ser/Thr phosphatases of the PPP family. At low nanomolar concentrations calyculin A induced T286 phosphorylation and degradation of cyclin D1 via the proteosome in MDA-MB-468 and MDA-MB-231 cells. Cyclin D1 degradation also was dose-dependently induced by okadaic acid and catharidin, implicating a negative regulatory role for type-2A phosphatases. These effects occurred without increasing phosphorylation of p70S6K, cyclin D3, or myosin light chain that were used as endogenous reporters of cellular PP2A and PP1 activity. A reverse phase phosphoprotein array analysis revealed increased phosphorylation of only 6 out of 33 Ser/Thr phosphosites, indicating selective inhibition of phosphatases by calyculin A. Calyculin A treatment induced cell cycle arrest in MDA-MB-468 and MCF-7 breast cancer cells. These findings suggest that a specific pool of type-2A phosphatase is inhibited by calyculin A leading to the degradation of cyclin D1 in human breast cancer cells. The results highlight the utility of toxins as pharmacological probes and points to the T286 cyclin D1 phosphatase inhibited by calyculin A as a possible target for chemotherapy to treat triple negative breast cancer.     

Contrasting effects of calyculin A and okadaic acid on the respiratory burst of human neutrophils

The involvement of serine/threonine protein-phosphatases in the production of superoxide (respiratory burst) by human neutrophils was investigated using calyculin A, a potent inhibitor of both protein phosphatases type 1 and 2A, and okadaic acid, which preferentially inhibits protein phosphatase type 2A. Treatment of neutrophils with calyculin A (25–75 nM) or okadaic acid (1–4 μM) had no stimulatory effect but potently enhanced total superoxide production induced by an optimal fMLP (N-formyl-methionyl-leucoyl-phenylalanine) concentration (0.1 μM). The maximal increase platacuaed with 50–75 nM calyculin A and 2–4 μM okadaic acid, reaching approximately 120 and 200% of control values, respectively. Unlike calyculin A, okadaic acid also primed the initial rate of superoxide production, suggesting that protein phosphatases may down-regulate both initiation and termination of respiratory burst. Optimal stimulation of the respiratory burst by PMA (160 nM) was inhibited by calyculin A and okadaic acid, with an IC₅₀ of 60 nM and 2 μM, respectively, although both drugs caused protein hyperphosphorylation. The inhibition was partially prevented by a nonstimulatory concentration of A23187, indicating a role of calcium in the inhibitory effects of the drugs. Unlike the optimal respiratory burst, suoptimal respiratory burst induced by PMA (1–7 nM) was enhanced by calyculin A and okadaic acid. Unprimed and primed respiratory burst were depressed by a selective antagonist of protein kinase C (GF 109203X), indicating positive regulation of these responses by protein kinase C. Thus, the use of calyculin A and okadaic acid distinguishes two regulatory processes of superoxide production. The respiratory burst induced by low PMA concentrations of fMLP was up-regulated by both calyculin A and okadaic acid, in keeping with a down-regulatory role of protein phosphatases in these responses. By contrast, intense protein kinase C activation by PMA triggered a respiratory burst which was depressed by both drugs, pointing to positive regulation of the respiratory burst by protein phosphatases.     

Short-term oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice.

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.     

DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line

Okadaic acid (OA) is a shellfish toxin produced by dinoflagellates, in mussels. It is a potent tumour promoter and represents a potential threat to human health even at low concentrations. OA targets mainly the gastrointestinal tract in acute poisoning, causing diarrhoea. Therefore the present investigations were designed to study the ability of okadaic acid to induce cytotoxicity and DNA lesions in a human colonic cell line (Caco-2). Incubation of Caco-2 cells with OA (3.75-60 ng/ml, i.e. 4.6 x 10(-3)-7.5 x 10(-2) microM) causes a significant reduction in cell viability. Moreover, okadaic acid inhibits protein and DNA synthesis with, respectively, IC50 of 16 and 6.5 ng/ml after 24 h incubation. It also provokes cell cycle arrest, characterised by an increase in the number of S phase cells, correlated with a significant decrease in G0/G1 phase cells at high concentration. One of the main results obtained in these investigations is the apoptosis induced by OA in Caco-2 cells of intestinal origin, shown by DNA laddering in agarose gel electrophoresis (250-1000 base pairs). OA also induces clastogenic effects evaluated by DNA fragmentation analysis using the method of Higuchi and Aggarwal (52% for 60 ng/ml) and comet assay (increase of the frequency of comets and their tails length). Therefore, the cell death induced by OA seems clearly to be concentration-dependent after 24 h of incubation. The cytotoxic properties of okadaic acid and its ability to damage DNA result in cell death, mainly by apoptosis. Since consumption of shellfish contaminated with acceptable okadaic acid concentrations exposes colonic cells to harmful concentrations of this toxin, the possibility that OA would display its toxic effects on intestinal cells in vivo should be evaluated in human primary intestinal cells and human intestinal slices for cytotoxic effects, DNA fragmentation and apoptosis.     

山茱萸环烯醚萜苷对冈田酸拟阿尔茨海默病细胞模型的抗凋亡作用研究

目的:研究山茱萸环烯醚萜苷(CIG)对蛋白磷酸酶抑制剂冈田酸(OA)拟阿尔茨海默病(AD)细胞模型中神经细胞凋亡的影响及其作用机制。方法:CIG与人神经母细胞瘤细胞系SK-N-SH细胞预孵育24h,再用OA10nmol与神经细胞共孵育6h,用TUNEL法观察凋亡细胞的变化,用蛋白免疫印记法观察凋亡因子B细胞淋巴瘤(Bcl-2)、Bcl-2相关X蛋白(Bax)和细胞凋亡蛋白酶(Caspase-3)的表达。结果:正常对照细胞铺展良好,未出现凋亡细胞;OA模型组凋亡细胞显著增多,Bax和Cas-pase-3的表达水平显著增高,Bcl-2的表达水平显著下降;CIG(100和200μg.mL-1)给药组凋亡细胞较OA模型组显著减少,Bax和Caspase-3表达水平显著降低,Bcl-2表达显著增高。结论:CIG能通过影响凋亡调节因子而抑制神经细胞凋亡,提示该药可能具有治疗AD的良好应用前景。     

Structurally different members of the okadaic acid class selectively inhibit protein serine/threonine but not tyrosine phosphatase activity.

The relative potencies of four main types of okadaic acid class compounds as inhibitors of the catalytic subunits of protein serine/threonine phosphatases 1 and 2A and the protein tyrosine phosphatase 1 were determined. These four types of compounds are okadaic acid, calyculin A, microcystin-LR, and tautomycin, which are isolated from different natural sources, a black sponge Halichondria okadai, a marine sponge Discodermia calyx, a blue-green alga Microcystis aeruginosa, and Streptomyces spirover ticillatus, respectively. While okadaic acid was a more effective inhibitor of protein phosphatase 2A (IC50, 0.07 nM) than protein phosphatase 1 (IC50, 3.4 nM), other compounds of the okadaic acid class were equally effective against the two protein serine/threonine phosphatases. The order of potency was microcystin greater than calyculin A greater than tautomycin, and the IC50S ranged from 0.1 to 0.7 nM. None of the okadaic acid class compounds inhibited protein tyrosine phosphatase 1 activity at concentrations up to 0.01 mM. These results indicate that the compounds of the okadaic acid class are selective inhibitors of protein serine/threonine but not tyrosine phosphatases.     

Okadaic acid modulates exocytotic and transporter-dependent release of dopamine in bovine retina in vitro

Bovine retinas were isolated for the study of the modulation of exocytotic and transporter-dependent release of dopamine (DA) in vitro. Endogenous DA was measured in the medium using HPLC with electrochemical detection under successive incubations with transfers in fresh medium every 30 min.As expected, potassium caused a calcium-dependent exocytotic liberation of DA. Amphetamine or tyramine induced a calcium-independent release by reversing DA transport across the plasma membrane. Okadaic acid, a specific inhibitor of phosphatases 1 and 2A, induced a slight but significant DA release in the absence of calcium. Furthermore, the toxin increased potassium-, amphetamine-or tyramine-induced DA release independently of extracellular calcium. In addition, okadaic acid completely annulled the ability of a calcium-free extracellular environment to inhibit the potassium-induced DA release. Finally, the toxin prevented the time-dependent decline in the efficacies of amphetamine or tyramine to release DA.In agreement with proposed schemes described for rat striatum, the results of the present study confirmed the existence of distinct release modes of DA in bovine retina. The results obtained with okadaic acid suggest that phosphatase 1 and/or phosphatase 2A constitute part of a direct or indirect mechanism to inhibit both exocytotic and transporter-dependent DA release.     

Micronucleus induction in mussels exposed to okadaic acid.

Some toxins present in the marine environment are capable of inducing mutagenicity and/or carcinogenicity. Among these toxins, okadaic acid (OA) is gaining considerable interest since it induces DNA based modifications at low concentrations and accumulates in filter-feeding marine animals, including those used for human consumption. This study aims to evaluate the genotoxicity of OA in the haemocytes of the mussel Perna perna, using the micronucleus assay. Fifty-four mussels were separated into three groups of 18 animals. One group received 0.3 microg of OA diluted in 10 microl of ethanol and ultrapure water while the other groups were considered as controls and were exposed to a solvent plus seawater mixture. A significantly higher frequency of micronuclei was observed in haemocytes from the OA-exposed group. There were no statistical differences between the two control groups.     

Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells

Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.     

蛋白丝/苏氨酸磷酸酶1和2A抑制药对大鼠三叉神经元电压依赖性钠通道的影响(英文)

目的通过研究蛋白丝/苏氨酸磷酸酶1和2A特异性抑制药冈田酸对大鼠三叉神经元电压依赖性钠电流的影响,探讨磷酸酶在细胞信号转导中的作用。方法在成年大鼠三叉神经元上进行全细胞膜片钳记录。结果1μmol·L-1冈田酸显著抑制总钠电流(INa-T) ,仅轻微抑制毒素不敏感型钠电流(INa-TTX-R) ,其抑制率分别为(20±13) %(n=9,P<0 .05)和(4±3) %(n=6, P<0 .05)。冈田酸对INa-T的激活曲线产生明显的超极化位移,半激活电压从给药前的-(13±8)mV升至给药后的-(16±7)mV(P<0 .05) ,但是对INa-TTX-R的激活曲线没有影响。结论①蛋白丝/苏氨酸磷酸酶参与了大鼠三叉神经元电压依赖性钠通道的调节。②大鼠三叉神经元存在多种电压依赖性钠通道,它们对冈田酸具有不同的敏感性。     

Characterization of distinct apoptotic changes induced by okadaic acid and yessotoxin in the BE(2)-M17 neuroblastoma cell line.

Apoptotic changes induced by okadaic acid and yessotoxin in BE(2)-M17 neuroblastoma cells have been evaluated and quantified by combining classical methods and fast and sensitive fluorimetric microplate assays. The phosphatase inhibitor okadaic acid induced rapid time- and dose-dependent apoptotic changes in this cell line, which were evident after 1h at concentrations equal or higher than 500 nM. Decreased mitochondrial membrane potential by okadaic acid (IC(50)=350 nM at 1h) was followed by cell detachment (IC(50)=400 nM at 1h), changes in total nucleic acids content (50% of controls after 1h with 1000 nM okadaic acid), caspase-3 activation (3- to 4-fold increase at 6h) and increased Annexin-V binding (1.5-fold at 6h). Yessotoxin induced similar changes in BE(2)-M17 cells, although significant differences were found in the time-course and degree of apoptotic events induced by this phycotoxin, indicating a lower potency for yessotoxin when compared with okadaic acid. This is the first report on apoptogenic activity of yessotoxin.     

Synergistic effects of some metals contaminating mussels on the cytotoxicity of the marine toxin okadaic acid

Okadaic acid (OA), a marine toxin is cytotoxic and promotes tumours in mouse skin. It is a specific and potent inhibitor of protein synthesis and also inhibits phosphatases A1 and A2 in vitro. In the present study, we investigated the influence of metals found at acceptable levels in mussels as environmental pollutants on the cytotoxicity of OA in Vero cells. Among the metals found in mussels (Mytilus edulis), the most represented, in terms of molar quantities per gram of dried weight are aluminium (230 nmol/g), copper (58 nmol/g), lead (16 nmol/g), mercury (14 nmol/g) and cadmium (7.4 nmol/g). A solution containing these five metals Al³⁺, Cu²⁺, Pb²⁺, Hg²⁺ and Cd²⁺ combined at the concentrations detected in mussels, stimulated protein synthesis (+25%, P < 0.01), whereas different dilutions of this solution in the presence of okadaic acid (15 ng/ml, i.e. 18.7 × 10⁻⁹ M) increased the percentage of protein synthesis inhibition from 35 to 79%. The metals also increased the lactate dehydrogenase (LDH) release into the medium and the lipid peroxidation induced by this algal toxin. In addition, these metals reduced the cell viability for an incubation period of 24 h especially at the two higher concentrations. These results indicate that metals (Al³⁺, Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺) in concentration ranges largely below the acceptable levels, synergistically increase the cytotoxicity of low concentrations of OA in cultured cells. Received: 11 January 1999 / Accepted: 29 April 1999     

Effect of okadaic acid on glucose regulation

Okadaic acid is the main toxin responsible for the natural phenomena known as diarrheic shellfish poisoning (DSP). This toxin is a tumor promoter C38 polyether fatty acid that contains acidic and hydrophobic moieties and is cyclic. Okadaic acid is a potent inhibitor of important classes of protein serine/threonine phosphatases such as protein phosphatase 1 and 2A. The toxin binds in a hydrophobic groove adjacent to the active site of the protein phosphatases and interacts with basic residues within the active site. Therefore okadaic acid causes increases in phosphorylation of proteins that affect a diverse array of cellular processes. For instance, this toxin modulates metabolic parameters in intact cells. In this sense it stimulates lipolysis, and inhibits fatty acid synthesis in adipocytes however increases glucose output and gluconeogenesis in hepatocytes. Additionally, okadaic acid reaches cytotoxic concentrations in the intestinal tissues in accordance with the diarrhea. Recent studies suggested that toxic effects of okadaic acid might be related to modification of nutrients, ionic and water absorption across the small intestine presumably by altering the transporter system. The subject of this review is limited to the effect of okadaic acid on glucose regulation and its cellular as well as clinical implications.