RESUMO
Due to the rapid elimination of bacteria through normal behaviour of filter feeding and excretion, the decontamination of hazardous contaminating bacteria from shellfish is performed by depuration. This process, under conditions that maximize shellfish filtering activity, is a useful method to eliminate microorganisms from bivalves. The microbiota composition in bivalves reflects that of the environment of harvesting waters, so quite different bacteriomes would be expected in shellfish collected in different locations. Bacterial accumulation within molluscan shellfish occurs primarily in the hepatopancreas. In order to assess the effect of the depuration process on these different bacteriomes, in this work we used 16S RNA pyrosequencing and metagenome prediction to assess the impact of 15 h of depuration on the whole hepatopancreas bacteriome of mussels collected in three different locations.
Assuntos
Bactérias/isolamento & purificação , Bivalves/microbiologia , Frutos do Mar/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Biodiversidade , Genoma Bacteriano , Hepatopâncreas/microbiologia , MetagenomaRESUMO
Understanding the mechanism of action of the yessotoxin (YTX) is crucial since this drug has potential pharmacological effects in allergic processes, tumor proliferation and neurodegenerative diseases. It has been described that YTX activates apoptosis after 24h of treatment, while after 48 h of incubation with the toxin a decrease in cell viability corresponding to cellular differentiation or non-apoptotic cell death was observed. In this paper, these processes were extensively studied by using the erythroleukemia K-562 cell line. On one hand, events of K-562 cell differentiation into erythrocytes after YTX treatment were studied using hemin as positive control of cell differentiation. Cell differentiation was studied through the cyclic nucleotide response element binding (phospho-CREB) and the transferrin receptor (TfR) expression. On the other hand, using rapamycin as positive control, autophagic hallmarks, as non-apoptotic cell death, were studied after toxin exposure. In this case, the mechanistic target of rapamycin (mTOR) and light chain 3B (LC3B) levels were measured to check autophagy activation. The results showed that cell differentiation was not occurring after 48 h of toxin incubation while at this time the autophagy was triggered. Furthermore after 24h of toxin treatment none of these processes were activated. In addition, the role of the type 4A phosphodiesterase (PDE4A), the intracellular target of YTX, was checked. PDE4A-silencing experiments showed different regulation steps of PDE4A in the autophagic processes triggered either by traditional compounds or YTX. In summary, after 48 h YTX treatment PDE4A-dependent autophagy, as non-apoptotic programmed cell death, is activated.
Assuntos
Autofagia/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Oxocinas/toxicidade , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose/efeitos dos fármacos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Inativação Gênica , Humanos , Células K562 , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Venenos de Moluscos , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Yessotoxin at nanomolar concentrations can induce programmed cell death in different model systems. Paraptosis-like cell death induced by YTX in BC3H1 cells, which are insensitive to several caspase inhibitors,has also been reported. This makes yessotoxin of interest in the search of molecules that target cancer cells vulnerabilities when resistance to apoptosis is observed. To better understand the effect of this molecule at the molecular level on tumor cells, we conducted a transcriptomic analysis using 3 human glioma cell lines with different sensitivities to yessotoxin. We show that the toxin induces a deregulation of the lipid metabolism in glioma cells as a consequence of induction of endoplasmic reticulum stress. The endoplasmic reticulum stress in turn arrests the cell cycle and inhibits the protein synthesis. In the three cell lines used we show that YTX induces autophagy, which is involved in cell death. The sensibility of the cell lines used towards autophagic cell death was related to their doubling time, being the cell line with the lowest proliferation rate the most resistant.The involvement of mTOR and BNIP3 in the autophagy induction was also determined.
Assuntos
Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Oxocinas/toxicidade , Proteínas Proto-Oncogênicas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colesterol/metabolismo , Análise por Conglomerados , Regulação para Baixo/efeitos dos fármacos , Glioma/metabolismo , Glioma/patologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Venenos de Moluscos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Marine sponges have evolved the capacity to produce a series of very efficient chemicals to combat viruses, bacteria, and eukaryotic organisms. It has been demonstrated that several of these compounds have anti-neoplastic activity. The highly toxic sponge Crambe crambe has been the source of several molecules named crambescidins. Of these, crambescidin-816 has been shown to be cytotoxic for colon carcinoma cells. To further investigate the potential anti-carcinogenic effect of crambescidin-816, we analysed its effect on the transcription of HepG2 cells by microarray analysis followed by experiments guided by the results obtained. EXPERIMENTAL APPROACH: After cytotoxicity determination, a transcriptomic analysis was performed to test the effect of crambescidin-816 on the liver-derived tumour cell HepG2. Based on the results obtained, we analysed the effect of crambescidin-816 on cell-cell adhesion, cell-matrix adhesion, and cell migration by Western blot, confocal microscopy, flow cytometry and transmission electron microscopy. Cytotoxicity and cell migration were also studied in a variety of other cell lines derived from human tumours. KEY RESULTS: Crambescidin-816 had a cytotoxic effect on all the cell lines studied. It inhibited cell-cell adhesion, interfered with the formation of tight junctions, and cell-matrix adhesion, negatively affecting focal adhesions. It also altered the cytoskeleton dynamics. As a consequence of all these effects on cells crambescidin-816 inhibited cell migration. CONCLUSIONS AND IMPLICATIONS: The results indicate that crambescidin-816 is active against tumour cells and implicate a new mechanism for the anti-tumour effect of this compound.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Compostos de Espiro/farmacologia , Comunicação Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/patologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Invasividade Neoplásica , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/patologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Resveratrol is a phytoalexin that has been shown to inhibit cell proliferation of several cancer cell lines. In some cases this inhibition was specific for the transformed cells when compared with normal cells of the same tissue. To test whether this was the case in rat hepatocytes, we exposed primary rat hepatocytes in culture and transformed rat hepatic cells to this compound and studied its effect on cell proliferation, measuring deoxy-bromouridine incorporation and total DNA. We also studied the effect of resveratrol on the cell cycle of normal and transformed rat hepatocytes. We observed that resveratrol inhibited proliferation in a dose-dependent manner in both cases, with no differential action in the transformed cells compared to the normal ones. This compound arrested the cell cycle in G0/G1 in primary hepatocytes, while it arrested the cell cycle in G2/M in transformed cells. Transformed hepatocytes showed accumulation of cells in the S phase of the cell cycle.
Assuntos
Antioxidantes/farmacologia , Proliferação de Células/efeitos dos fármacos , DNA/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Células Cultivadas , DNA/biossíntese , Fase G2/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Ratos , ResveratrolRESUMO
Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit α of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit α of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250 µg per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94 µmol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2 months at -20 °C.
Assuntos
Ensaios Enzimáticos/métodos , Mariposas/genética , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Larva/genética , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/isolamento & purificaçãoRESUMO
Palytoxin (PLT) is a highly toxic nonpeptidic marine natural product, with a complex chemical structure. Its mechanism of action targets Na,K-ATPase. Fluorescence polarization (FP) is a spectroscopic technique that can be used to determine molecular interactions. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, FP was used to develop a detection method based on the interaction between the Na,K-ATPase and the PLT. The Na,K-ATPase was labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of protein-dye conjugate was measured when the amount of PLT in the medium was modified. The assay protocol was first developed using ouabain as a binding molecule. The final result was a straight line that correlates FP units and PLT concentration. Within this line the PLT equivalents in a natural sample can be quantified. A selective cleaning procedure to mussel samples and dinoflagellates cultures was also developed to avoid the matrix effect. The LOQ (limit of quantification) of the method is 10nM and the LOD (limit of detection) is 2 nM. This new PLT detection method is easier, faster, and more reliable than the other methods described to date.
Assuntos
Acrilamidas/análise , Polarização de Fluorescência/métodos , Animais , Bivalves/química , Venenos de Cnidários , Dinoflagellida/química , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Temperatura , Extratos de Tecidos/químicaRESUMO
AIMS: To determine the relative toxicity and effects on the cell cycle of okadaic acid and dinophysistoxin-2 in primary hepatocyte cultures. MAIN METHODS: Cytotoxicity was determined by the MTT method, caspase-3 activity and lactate dehydrogenase release to the medium. The cell cycle analysis was performed by imaging flow cytometry and the effect of the toxins on cell proliferation was studied by quantitative PCR and confocal microscopy. KEY FINDINGS: We show that dinophysistoxin-2 is less toxic than okadaic acid for primary hepatocytes with a similar difference in potency as that observed in vivo in mice after intraperitoneal injection. Both toxins induced apoptosis with caspase-3 increase. They also inhibited the hepatocytes cell cycle in G1 affecting diploid cells and diploid bi-nucleated cells. In proliferating hepatocytes exposed to the toxins, a decrease of p53 gene expression as well as a lower protein level was detected. Studies of the tubulin cytoskeleton in toxin treated cells, showed nuclear localization of this molecule and a granulated tubulin pattern in the cytoplasm. SIGNIFICANCE: The results presented in this work show that the difference in toxicity between dinophysistoxin-2 and okadaic acid in cultured primary hepatocytes is the same as that observed in vivo after intraperitoneal injection. Okadaic acid and dinophysistoxin-2 arrest the cell cycle of hepatocytes at G1 even in diploid bi-nucleated cells. p53 and tubulin could be involved in the cell cycle inhibitory effect.
Assuntos
Ciclo Celular/efeitos dos fármacos , Hepatócitos/metabolismo , Ácido Okadáico/toxicidade , Piranos/toxicidade , Análise de Variância , Animais , Western Blotting , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Hepatócitos/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Microscopia Confocal , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Sais de Tetrazólio , TiazóisRESUMO
Okadaic acid is one of the toxins responsible for the human intoxication known as diarrhetic shellfish poisoning, which appears after the consumption of contaminated shellfish. The main diarrhetic shellfish poisoning toxins are okadaic acid, dinophysistoxin-1, -2, and -3. In vivo, after intraperitoneal injection, dinophysistoxin-2 is approximately 40% less toxic than okadaic acid in mice. The cytotoxic and genotoxic effect of okadaic acid varies very significantly in different cell lines, so similar responses could be expected for dinophysistoxin-2. In order to determine whether this was the case, we studied the effect of okadaic acid and dinophysistoxin-2 in two hepatic cell lines (HepG2 and Clone 9). The cytotoxicity of these toxins, as well as their effects on the cell cycle and its regulation on both cell lines, were determined. Okadaic acid and dinophysistoxin-2 resulted to be equipotent in clone 9 cultures, while okadaic acid was more potent than dinophysistoxin-2 in HepG2 cell cultures. Both toxins had opposite effects on the cell cycle; they arrested the cell cycle of clone 9 cells in G2/M inducing aberrant mitosis while arresting the cell cycle of HepG2 in G0/G1. When the effect of the toxins on p53 subcellular distribution was studied, p53 was detected in the nuclei of both cell types. The effect of the toxins on the gene expression of cyclins and cyclin-dependent kinases was different for both cell lines. The toxins induced an increase in gene expression of cyclins A, B, and D in clone 9 cells while they induced a decrease in cyclins A and B in HepG2 cells. They also induced a decrease in cyclin-dependent kinase 1 in HepG2 cells.
Assuntos
Ciclo Celular/efeitos dos fármacos , Fígado/efeitos dos fármacos , Ácido Okadáico/toxicidade , Piranos/toxicidade , Animais , Proteína Quinase CDC2/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciclinas/efeitos dos fármacos , Ciclinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Injeções Intraperitoneais , Fígado/citologia , Fígado/patologia , Mitose/efeitos dos fármacos , Ratos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.
Assuntos
Cálcio/metabolismo , Ciguatoxinas/toxicidade , Citosol/efeitos dos fármacos , Dinoflagellida/química , Toxinas Marinhas/toxicidade , Actinas/metabolismo , Actinas/ultraestrutura , Linhagem Celular Tumoral , Citosol/metabolismo , Citosol/ultraestrutura , HumanosRESUMO
Paralytic shellfish poisoning (PSP) toxins are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. Shellfish contaminated with these toxins create significant public health threat and economic losses to the shellfish industry. For this reason, several methods of high performance liquid chromatography (HPLC) with fluorescence detection have been developed in order to gain better knowledge of toxins profiles in shellfish and dinoflagellates samples. These methods have been subjected to continuous modifications to improve and shorten the run time of analysis in the routine monitoring control. In this paper, different samples are analyzed by pre- and post- column HPLC methods to compare toxin profiles. All PSP toxins were individually identified and quantified within the post-column oxidation method. However, although the pre-column oxidation method is significantly more sensitive and detects lower toxin levels, it provides a total amount of toxins that co-elute together, as GTX2 and 3, GTX1 and 4 and dcGTX2 and dcGTX3. The results obtained by both HPLC methods showed similar toxin concentration (expressed in mug/mL) in mussel samples, however when dinoflagellates samples were analyzed the toxin profile and concentration were different. In summary, the post-column oxidation method is accurate to determine the amount of each individual PSP toxin and to know the real toxic profile of non-transformed samples. In addition, this method is easy and faster to screen a large number of samples. The pre-column HPLC method is useful when mussel samples are analyzed even though the time required to prepare the samples is longer.
Assuntos
Toxinas Marinhas/análise , Intoxicação por Frutos do Mar , Cromatografia Líquida de Alta Pressão , Oxirredução , Padrões de Referência , Espectrometria de FluorescênciaRESUMO
Gambierol is a marine polyether ladder toxin derived from the dinoflagellate Gambierdiscus toxicus. To date, gambierol has been reported to act either as a partial agonist or as an antagonist of sodium channels or as a blocker of voltage-dependent potassium channels. In this work, we examined the cellular effect of gambierol on cytosolic calcium concentration, membrane potential and sodium and potassium membrane currents in primary cultures of cerebellar granule cells. We found that at concentrations ranging from 0.1 to 30 microM, gambierol-evoked [Ca(2+)]c oscillations that were dependent on the presence of extracellular calcium, irreversible and highly synchronous. Gambierol-evoked [Ca(2+)]c oscillations were completely eliminated by the NMDA receptor antagonist APV and by riluzole and delayed by CNQX. In addition, the K(+) channel blocker 4-aminopyridine (4-AP)-evoked cytosolic calcium oscillations in this neuronal system that were blocked by APV and delayed in the presence of CNQX. Electrophysiological recordings indicated that gambierol caused membrane potential oscillations, decreased inward sodium current amplitude and decreased also outward IA and IK current amplitude. The results presented here point to a common mechanism of action for gambierol and 4-AP and indicate that gambierol-induced oscillations in cerebellar neurons are most likely secondary to a blocking action of the toxin on voltage-dependent potassium channels and hyperpolarization of sodium current activation.
Assuntos
Cálcio/metabolismo , Cerebelo/efeitos dos fármacos , Ciguatoxinas/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Grânulos Citoplasmáticos/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Canais de Sódio/metabolismoRESUMO
BACKGROUND AND PURPOSE: Pectenotoxins are macrocyclic lactones found in dinoflagellates of the genus Dinophysis, which induce severe liver damage in mice after i.p. injection. Here, we have looked for the mechanism(s) underlying this hepatotoxicity. EXPERIMENTAL APPROACH: Effects of pectenotoxin (PTX)-1, PTX-2, PTX-2 seco acid (PTX-2SA) and PTX-11 were measured in a hepatocyte cell line with cancer cell characteristics (Clone 9) and in primary cultures of rat hepatocytes. Cell morphology was assessed by confocal microscopy; F- and G-actin were selectively stained and cell viability measured by Alamar Blue fluorescence. KEY RESULTS: Clone 9 cells and primary hepatocytes showed a marked depolymerization of F-actin with PTX-1, PTX-2 and PTX-11 (1-1000 nM) associated with an increase in G-actin level. However, morphology was only clearly altered in Clone 9 cells. PTX-2SA had no effect on the actin cytoskeleton. Despite the potent F-actin depolymerizing effect, PTX-1, PTX-2 or PTX-11 did not decrease the viability of Clone 9 cells after 24-h treatment. Only prolonged incubation (> 48 h) with PTXs induced a fall in viability, and under these conditions, morphology of both Clone 9 and primary hepatocytes was drastically changed. CONCLUSIONS AND IMPLICATIONS: Although the actin cytoskeleton was clearly altered by PTX-1, PTX-2 and PTX-11 in the hepatocyte cell line and primary hepatocytes, morphological assessments indicated a higher sensitivity of the cancer-like cell line to these toxins. However, viability of both cell types was not altered.
Assuntos
Citoesqueleto/efeitos dos fármacos , Furanos/toxicidade , Hepatócitos/metabolismo , Piranos/toxicidade , Actinas/metabolismo , Animais , Células Cultivadas , Células Clonais , Corantes Fluorescentes/metabolismo , Macrolídeos , Masculino , Microscopia Confocal , Faloidina/metabolismo , Ratos , Ratos Sprague-Dawley , Xantenos/metabolismoRESUMO
BACKGROUND AND PURPOSE: Palytoxin (PLT) is a potent toxin that binds to the Na,K-ATPase. Palytoxin is highly neurotoxic and increases the cytosolic calcium concentration ([Ca(2+)](c)) while decreasing intracellular pH (pH(i)) in neurons (Vale et al., 2006; Vale-Gonzalez et al., 2007). It is also a tumour promoter that activates several protein kinases. EXPERIMENTAL APPROACH: The role of different protein kinases in the effects of palytoxin on [Ca(2+)](c), pH(i) and cytoxicity was investigated in cultured neurons. KEY RESULTS: Palytoxin-induced calcium load was not affected by inhibition of calcium-dependent protein kinase C (PKC) isoforms but it was partially ameliorated by blockade of calcium-independent PKC isozymes. Inhibition of the extracellular signal-regulated kinase (ERK) 2 eliminated the palytoxin-induced rise in calcium and intracellular acidification, whereas inhibition of MEK greatly attenuated the palytoxin effect on calcium without modifying the PLT-evoked intracellular acidification. Blockade of c-Jun N-terminal protein kinases (JNK) somewhat decreased the palytoxin-effect on calcium, whereas inhibition of the p38 mitogen activated protein kinases (MAPKs) delayed the onset of the palytoxin-evoked rise in calcium and acidification. Furthermore, the cytotoxicity of palytoxin was completely blocked by inhibition of ERK 2 and partially prevented by inhibition of MEK. PLT increased phosphorylated ERK immunoreactivity in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: MAPKs, specifically ERK 2, link palytoxin cytotoxicity with its effects on calcium homeostasis after inhibition of the Na,K-ATPase. Binding of palytoxin to the Na,K-ATPase would alter signal transduction pathways, even in non-dividing cells, and this finding is related to the potent neurotoxicity of this marine toxin.
Assuntos
Acrilamidas/farmacologia , Cálcio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Venenos de Cnidários/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The phycotoxin palytoxin (PTX) binds to Na,K-ATPase, inhibiting its activity and converting the pump into a channel. These mechanisms are poorly understood. We examined the effect of PTX on membrane potential (E(m)), intracellular calcium concentration ([Ca2+]i) and intracellular pH (pH(i)) in primary cultures of cerebellar granule cells (CGC) and compared PTX and ouabain actions in the same cellular parameters. In this system, PTX caused depolarization, intracellular calcium increase and acidification. This is similar to the effect of ouabain. Preincubation of the cells with ouabain, before addition of PTX, altered E(m), [Ca2+]i, and pH(i) in a fashion similar to that of ouabain alone. This suggest a direct interaction of PTX with the Na,K-ATPase. Therefore, we used a resonant mirror biosensor to evaluate the binding of PTX and ouabain to immobilized Na,K-ATPase. Ouabain binding to immobilized Na,K-ATPase was concentration-dependent. No binding of PTX to Na,K-ATPase was observed with up to 10 microM, or with PTX addition in the presence of ATP. The fact that ouabain binds to the pump in an immobilized conformation whereas not binding of PTX was observed indicates that PTX and ouabain do not share the same binding site, and PTX binding may require the tridimensional pump structure.
Assuntos
Acrilamidas/toxicidade , Técnicas Biossensoriais/métodos , Neurônios/efeitos dos fármacos , Ouabaína/toxicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Acrilamidas/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Venenos de Cnidários , Cães , Concentração de Íons de Hidrogênio , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Ouabaína/metabolismo , SuínosRESUMO
Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from mussels that does not interfere with this detection method. YTX concentrations were measured in spiked mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally contaminated mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.
Assuntos
Bivalves/metabolismo , Éteres Cíclicos/isolamento & purificação , Oxocinas/isolamento & purificação , Animais , Bivalves/química , Éteres Cíclicos/análise , Éteres Cíclicos/química , Polarização de Fluorescência/métodos , Venenos de Moluscos , Oxocinas/análise , Oxocinas/química , Diester Fosfórico Hidrolases/metabolismo , Reprodutibilidade dos TestesRESUMO
Most common phycotoxin poisoning syndromes have important neurological symptoms. However, little is known of the cellular and molecular targets of many of the phycotoxins that produce those human intoxications. We explore the effect of representative toxins on the membrane potential in human neuroblastoma cells by using a fluorimetric assay. Results presented in this study demonstrate that maitotoxin, palytoxins, brevetoxins, and ciguatoxins triggered a dose-dependent membrane depolarization. Mechanisms responsible for the toxins-induced changes in membrane potential are always related to a direct action of the compounds on membrane ion fluxes. This initial screening of the phycotoxins effect is the starting point to lately develop functional methods of detection.
Assuntos
Toxinas Marinhas/farmacologia , Neurônios/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Potenciais da Membrana/efeitos dos fármacos , Neurônios/patologia , Fatores de TempoRESUMO
Biotoxins produced by harmful marine microalgae (phycotoxins) can be accumulated into seafood, representing a great risk for public health. Some of these phycotoxins are responsible for a variety of gastrointestinal disturbances; however, the relationship between their mechanism of action and toxicity in intestinal cells is still unknown. The actin cytoskeleton is an important and highly complicated structure in intestinal cells, and on that basis our aim has been to investigate the effect of representative phycotoxins on the enterocyte cytoskeleton. We have quantified for the first time the loss of enterocyte microfilament network induced by each toxin and recorded fluorescence images using a laser-scanning cytometer and confocal microscopy. Our data show that pectenotoxin-6, maitotoxin, palytoxin and ostreocin-D cause a significant reduction in the actin cytoskeleton. In addition, we found that the potency of maitotoxin, palytoxin and ostreocin-D to damage filamentous actin is related to Ca(2+) influx in enterocytes. Those results identify the cytoskeleton as an early target for the toxic effect of those toxins.
Assuntos
Acrilamidas/toxicidade , Actinas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/toxicidade , Citoesqueleto/efeitos dos fármacos , Furanos/toxicidade , Mucosa Intestinal/citologia , Toxinas Marinhas/toxicidade , Oxocinas/toxicidade , Piranos/toxicidade , Animais , Cálcio/metabolismo , Venenos de Cnidários , Fluoresceínas , Citometria de Varredura a Laser , Macrolídeos , Microscopia Confocal , CoelhosRESUMO
A human mast cell line (HMC-1) has been used to study the effect of cytosolic alkaline pH in exocytosis. Compound 48/80, concanavalin A, and thapsigargin do not induce histamine release in HMC-1 cells. Although thapsigargin does not activate histamine release, it does show a large increase in cytosolic Ca(2+), and no change in cytosolic pH. However, when HMC-1 cells were activated with ionomycin, a significant histamine release takes place, and this effect is higher in the presence of thapsigargin. Both drugs show an additive effect on cytosolic Ca(2+) levels. Ammonium chloride (NH(4)Cl) does activate cytosolic alkalinization and histamine release, with no increase in cytosolic Ca(2+). NH(4)Cl does block the release of internal Ca(2+) by thapsigargin, not by ionomycin, and decreases Ca(2+) influx stimulated by these drugs. Under conditions in which the alkalinization induced by NH(4)Cl is blocked by acidification with sodium propionate, histamine release is inhibited. The release of histamine is also observed when NH(4)Cl is added after propionate addition, regardless of the final pH value attained. Our results show that a shift in pH alkaline values, even with final pH below 7.2 is enough to activate histamine release. A shift to less acidic values is a sufficient signal to activate the cells.
Assuntos
Cloreto de Amônio/farmacologia , Cálcio/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Exocitose/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Linhagem Celular , Histamina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ionomicina/farmacologia , Tapsigargina/farmacologiaRESUMO
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.