RESUMEN

The Osteoarthritis is a chronic disease characterized by a progressive deterioration of the articular cartilage producing a strong inflammatory activity and chronic pain in patients. Horses also show osteoarthritis. Since the activation and progression of the disease are similar to that of human we developed a study model in horses. In this study, we test the effect of Neosaxitoxin, a phycotoxin from Paralytic Shellfish Poison, in the remediation of osteoarthritis equine clinical symptoms such as pain (showed in lameness) and inflammation quantifying the amounts of pro-inflammatory markers like cellular infiltration, TNF-alpha and nitric oxide in the synovial fluid obtained from the horse damaged joint. The outcomes show that Neosaxitoxin blocks pain for long lasting period (average 24.7 days). Furthermore, the amounts of pro-inflammatory markers were reduced and consequently an enhanced horse's well-being was obtained. Neosaxitoxin showed to be a candidate for establishing treatment protocols for OA, being safe and effective as a pain blocker in equine osteoarthritis.

Asunto(s)

Enfermedades de los Caballos , Osteoartritis , Venenos , Animales , Enfermedades de los Caballos/inducido químicamente , Enfermedades de los Caballos/tratamiento farmacológico , Caballos , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/veterinaria , Osteoartritis/inducido químicamente , Osteoartritis/tratamiento farmacológico , Osteoartritis/veterinaria , Dolor/tratamiento farmacológico , Dolor/veterinaria , Saxitoxina/análogos & derivados , Mariscos

RESUMEN

(1) Background: Neosaxitoxin (NeoSTX) has been used as a local anesthetic, but its anti-inflammatory effects have not been well defined. In the present study, we investigate the effects of NeoSTX on lipopolysaccharide (LPS)-activated macrophages. (2) Methods: Raw 264.7 and equine PBMC cells were incubated with or without 100 ng/mL LPS in the presence or absence of NeoSTX (1µM). The expression of inflammatory mediators was assessed: nitric oxide (NO) content using the Griess assay, TNF-α content using the ELISA assay, and mRNA of inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) using a real-time polymerase chain reaction. (3) Results: NeoSTX (1 µM) significantly inhibited the release of NO, TNF-α, and expression of iNOS, IL-1ß, and TNF-α in LPS-activated macrophages of both species studied. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by NeoSTX. Additionally, NeoSTX deactivated polarized macrophages to M1 by LPS without compromising its polarization towards M2. (4) Conclusions: NeoSTX inhibits LPS-induced release of inflammatory mediators from macrophages, and these effects may be mediated by the blockade of voltage-gated sodium channels (VGSC).

Asunto(s)

Mediadores de Inflamación/farmacología , Inflamación/prevención & control , Macrófagos/efectos de los fármacos , Saxitoxina/análogos & derivados , Animales , Humanos , Lipopolisacáridos , Ratones , Células RAW 264.7/efectos de los fármacos , Saxitoxina/farmacología

RESUMEN

Individuals of Mytilus platensis were exposed to Alexandrium catenella to evaluate the accumulation and metabolization of paralytic shellfish toxins (PST) over a period of 25 days. Mussels were collected from the intertidal zone of Cerro Avanzado, Argentine Patagonia. After 16 days, the toxins in the tissues of mussels were detected by the methods of mouse bioassay and high performance liquid chromatography with fluorometric detection (HPLC-FDL). The accumulation kinetics of PST toxins in M. platensis fed with A. catenella fitted to a linear function, in which the accumulation rate was 31.2 µg STX eq kg-1 day-1. After 16 days, the PST toxin level in tissues of mussels reached 1178 µg STX eq kg-1 exceeding the safety limit for human consumption (800 µg STX eq kg-1 tissue), whereas the highest PST toxin level was reached at the end of the experimentation (1613 µg STX eq kg-1) at 25 days. Differences in the toxin profile of the dinoflagellates and the tissues of the mussels confirmed biotransformation of PST in the mussel digestive system. The toxin profile of M. platensis was dominated by the gonyautoxins GTX1 and GTX4, while the toxin profile of A. catenella was dominated by the N-sulfocarbamoyl toxin C2. To our knowledge, this is the first experimentation on a laboratory scale of PST toxins accumulation in M. platensis with a native strain of A. catenella of Argentine Patagonia.

Asunto(s)

Dinoflagelados/fisiología , Toxinas Marinas , Mytilus/fisiología , Animales , Argentina , Bioensayo , Saxitoxina/análogos & derivados , Alimentos Marinos

RESUMEN

A high sympathetic tone is observed in the development and maintenance of the polycystic ovary (PCO) phenotype in rats. Neosaxitoxin (NeoSTX) specifically blocks neuronal voltage-dependent Na+ channels, and we studied the capacity of NeoSTX administered into the ovary to block sympathetic nerves and PCO phenotype that is induced by estradiol valerate (EV). The toxin was administered with a minipump inserted into the bursal cavity using two protocols: (1) the same day as EV administration and (2) 30 days after EV to block the final step of cyst development and maintenance of the condition. We studied the estrous cycling activity, follicular morphology, steroid plasma levels, and norepinephrine concentration. NeoSTX administered together with EV decreased NA intraovarian levels that were induced by EV, increased the number of corpora lutea, decreased the number of follicular cyst found after EV administration, and decreased the previously increased testosterone plasma levels induced by the PCO phenotype. Estrous cycling activity also recovered. NeoSTX applied after 30 days of EV administration showed near recovery of ovary function, suggesting that there is a specific window in which follicular development could be protected from cystic development. In addition, plasma testosterone levels decreased while those of progesterone increased. Our data strongly suggest that chronic inhibition of sympathetic nerves by a locally applied long-lasting toxin is a new tool to manage the polycystic phenotype in the rat and could be applied to other mammals depending on sympathetic nerve activity.

Asunto(s)

Ovario/inervación , Síndrome del Ovario Poliquístico/prevención & control , Saxitoxina/análogos & derivados , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Dinoflagelados/química , Estradiol/sangre , Ciclo Estral , Estro/metabolismo , Femenino , Humanos , Norepinefrina/sangre , Ovario/efectos de los fármacos , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/fisiopatología , Progesterona/sangre , Ratas , Ratas Sprague-Dawley , Saxitoxina/administración & dosificación , Sistema Nervioso Simpático/fisiopatología

RESUMEN

BACKGROUND: Neosaxitoxin (NeoSTX) and related paralytics shellfish toxins has been successfully used as local anesthetic and muscle relaxants to treat a variety of ailments. The primary mechanism of action of these toxins occurs by blocking voltage-gated sodium channels with compounds such as TTX, lidocaine, or derivatives. However, most of these non-classical sodium channel blockers act with a reduced time effect as well as ensuing neurotoxicity. NEW METHOD: In this report, we show that the use of local NeoSTX injections inactivates the hippocampal neuronal activity reversibly with a by long-term dynamics, without neuronal damage. RESULTS: A single 10 ng/µl injection of NeoSTX in the dorsal CA1 region abolished for up to 48 h memory capacities and neuronal activity measured by the neuronal marker c-fos. After 72 h of toxin injection, the animals fully recover their memory capacities and hippocampal neuronal activity. The histological inspection of NeoSTX injected brain regions revealed no damage to the tissue or reactive gliosis, similar to vehicle injection. Acute electrophysiological recording in vivo shows, also, minimal spreading of the NeoSTX in the cerebral tissue. COMPARISON WITH EXISTING METHODS: Intracerebral NeoSTX injection showed longer effects than other voltage sodium channel blocker, with minimal spreading and no neuronal damage. CONCLUSION: NeoSTX is a new useful tool that reversibly inactivates different brains region for a long time, with minimal diffusion and without neuronal damage. Moreover, NeoSTX can be used as a valuable sodium channel blocker for many studies in vivo and with potential therapeutic uses.

Asunto(s)

Región CA1 Hipocampal/efectos de los fármacos , Neuronas/efectos de los fármacos , Saxitoxina/análogos & derivados , Bloqueadores de los Canales de Sodio/administración & dosificación , Memoria Espacial/efectos de los fármacos , Amnesia/inducido químicamente , Animales , Región CA1 Hipocampal/fisiología , Masculino , Neuronas/fisiología , Ratas Sprague-Dawley , Reconocimiento en Psicología/efectos de los fármacos , Saxitoxina/administración & dosificación

RESUMEN

Local anesthesia is an effective method to control pain. Neosaxitoxin is a phycotoxin whose molecular mechanism includes a reversible inhibition of voltage-gated sodium channels at the axonal level, impeding nerve impulse propagation. The present study was designed to evaluate the clinical efficacy of Neosaxitoxin as a local long-acting pain blocker in horse bucked shins, and it was found to effectively control pain. While Neosaxitoxin and Gonyautoxin, another Paralytic Shellfish Poison (PSP) toxin, have been successfully used in humans as long-lasting pain blockers, this finding marks the first time a PSP has been shown to have an established effect in veterinary medicine.

Asunto(s)

Anestésicos Locales/uso terapéutico , Enfermedades de los Caballos/tratamiento farmacológico , Dolor/veterinaria , Periostitis/veterinaria , Saxitoxina/análogos & derivados , Anestésicos Locales/administración & dosificación , Animales , Caballos , Infusiones Subcutáneas/veterinaria , Cojera Animal/tratamiento farmacológico , Dolor/tratamiento farmacológico , Periostitis/tratamiento farmacológico , Saxitoxina/administración & dosificación , Saxitoxina/uso terapéutico

RESUMEN

Improvements in pain management techniques in the last decade have had a major impact on the practice of total knee arthroplasty (TKA). Gonyautoxin are phycotoxins, whose molecular mechanism of action is a reversible block of the voltage-gated sodium channels at the axonal level, impeding nerve impulse propagation. This study was designed to evaluate the clinical efficacy of Gonyautoxin infiltration, as a long acting pain blocker in TKA. Fifteen patients received a total dose of 40 µg of Gonyautoxin during the TKA operation. Postoperatively, all patients were given a standard painkiller protocol: 100 mg of intravenous ketoprofen and 1000 mg of oral acetaminophen every 8 hours for 3 days. The Visual Analog Scale (VAS) pain score and range of motion were recorded 12, 36, and 60 hours post-surgery. All patients reported pain of 2 or less on the VAS 12 and 36 hours post-surgery. Moreover, all scored were less than 4 at 60 hours post-surgery. All patients achieved full knee extension at all times. No side effects or adverse reactions to Gonyautoxin were detected in the follow-up period. The median hospital stay was 3 days. For the first time, this study has shown the effect of blocking the neuronal transmission of pain by locally infiltrating Gonyautoxin during TKA. All patients successfully responded to the pain control. The Gonyautoxin infiltration was safe and effective, and patients experienced pain relief without the use of opioids.

Asunto(s)

Artroplastia de Reemplazo de Rodilla/efectos adversos , Manejo del Dolor/métodos , Saxitoxina/análogos & derivados , Anciano , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dolor/etiología , Dimensión del Dolor , Rango del Movimiento Articular , Saxitoxina/uso terapéutico

RESUMEN

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na⁺ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

Asunto(s)

Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , Saxitoxina/análogos & derivados , Saxitoxina/metabolismo , Dinoflagelados/metabolismo , Simulación del Acoplamiento Molecular , Canal de Sodio Activado por Voltaje NAV1.4/química , Saxitoxina/química

RESUMEN

A growing body of evidence suggests that learned fear may be related to the function of the interoceptive insular cortex. Using an auditory fear conditioning paradigm in rats, we show that the inactivation of the posterior insular cortex (pIC), the target of the interoceptive thalamus, prior to training produced a marked reduction in fear expression tested 24h later. Accordingly, post-training anisomycin infused immediately, but not 6h after, also reduced fear expression tested the following day, supporting a role for the pIC in consolidation of fear memory. The long-term (ca. a week) and reversible inactivation of the pIC with the sodium channel blocker neosaxitoxin, immediately after fear memory reactivation induced a progressive decrease in the behavioral expression of conditioned fear. In turn, we observed that fear memory reactivation is accompanied by an enhanced expression of Fos and Zif268, early genes involved in neural activity and plasticity. Taken together these data indicate that the pIC is involved in the regulation of fear memories.

Asunto(s)

Conducta Animal/fisiología , Corteza Cerebral/fisiología , Condicionamiento Psicológico/fisiología , Miedo/fisiología , Interocepción/fisiología , Memoria/fisiología , Animales , Anisomicina/farmacología , Conducta Animal/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Condicionamiento Psicológico/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Inhibidores Enzimáticos/farmacología , Miedo/efectos de los fármacos , Genes fos/fisiología , Interocepción/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Saxitoxina/análogos & derivados , Saxitoxina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Tálamo

RESUMEN

Among toxin-producing dinoflagellates of the genus Alexandrium, Alexandrium ostenfeldii is the only species able to produce paralytic shellfish poisoning (PSP) toxins, spirolides (SPXs) and gymnodimines (GYMs). In this study we characterized and compared three A. ostenfeldii strains isolated from the Baltic, Mediterranean, and southern Chile Seas with respect to their toxin profiles, morphology, and phylogeny. Toxin analyses by HPLC-FD and LC-HRMS revealed differences in the toxin profiles of the three strains. The PSP toxin profiles of the southern Chile and Baltic strains were largely the same and included gonyautoxin (GTX)-3, GTX-2, and saxitoxin (STX), although the total PSP toxin content of the Chilean strain (105.83 ± 72.15 pg cell(-1)) was much higher than that of the Baltic strain (4.04 ± 1.93 pg cell(-1)). However, the Baltic strain was the only strain that expressed detectable amounts of analogues of GYM-A and GYM-B/-C (48.27 ± 26.12 pg GYM-A equivalents cell(-1)). The only toxin expressed by the Mediterranean strain was 13-desmethyl SPX-C (13dMeC; 2.85 ± 4.76 pg cell(-1)). Phylogenetic analysis based on the LSU rRNA showed that the studied strains belonged to distinct molecular clades. The toxin profiles determined in this study provide further evidence of the taxonomic complexity of this species.

Asunto(s)

Dinoflagelados/aislamiento & purificación , Compuestos Heterocíclicos con 3 Anillos/aislamiento & purificación , Hidrocarburos Cíclicos/aislamiento & purificación , Iminas/aislamiento & purificación , Compuestos de Espiro/aislamiento & purificación , Chile , Cromatografía Líquida de Alta Presión , Dinoflagelados/clasificación , Compuestos Heterocíclicos con 3 Anillos/toxicidad , Hidrocarburos Cíclicos/toxicidad , Iminas/toxicidad , Toxinas Marinas/análisis , Toxinas Marinas/toxicidad , Océanos y Mares , Filogenia , Filogeografía , Saxitoxina/análogos & derivados , Saxitoxina/aislamiento & purificación , Saxitoxina/toxicidad , Intoxicación por Mariscos/etiología , Intoxicación por Mariscos/patología , Compuestos de Espiro/toxicidad

RESUMEN

The paralytic shellfish toxin (PST) profiles of Gymnodinium catenatum Graham have been reported for several strains from the Pacific coast of Mexico cultured under different laboratory conditions, as well as from natural populations. Up to 15 saxitoxin analogues occurred and the quantity of each toxin depended on the growth phase and culture conditions. Previous analysis of toxin profiles of G. catenatum isolated from Mexico have been based on post-column oxidation liquid chromatography with fluorescence detection (LC-FLD), a method prone to artefacts and non-specificity, leading to misinterpretation of toxin composition. We describe, for the first time, the complete toxin profile for several G. catenatum strains from diverse locations of the Pacific coast of Mexico. The new results confirmed previous reports on the dominance of the less potent sulfocarbamoyl toxins (C1/2); significant differences, however, in the composition (e.g., absence of saxitoxin, gonyautoxin 2/3 and neosaxitoxin) were revealed in our confirmatory analysis. The LC-MS/MS analyses also indicated at least seven putative benzoyl toxin analogues and provided support for their existence. This new toxin profile shows a high similarity (> 80%) to the profiles reported from several regions around the world, suggesting low genetic variability among global populations.

Asunto(s)

Dinoflagelados/química , Saxitoxina/análogos & derivados , Cromatografía Líquida de Alta Presión , México , Saxitoxina/análisis , Espectrometría de Masas en Tándem

RESUMEN

INTRODUCTION AND HYPOTHESIS: Neosaxitoxin is a phycotoxin whose molecular mechanism of action shows a reversible inhibition of voltage-gated sodium channels at the axonal level, impeding nerve impulse propagation. This study was designed to evaluate the clinical efficacy of neosaxitoxin as a long-acting pain blocker in the treatment of bladder pain syndrome (BPS). METHODS: Five patients with a diagnosis of BPS received a total dose of 80 µg of neosaxitoxin in an isoosmotic solution of 0.9 % NaCl, pH 6.5. Infiltration was performed via cystoscopy under spinal anesthesia. Questionnaires were administered immediately before and 7, 30 and 90 days after the procedure to measure the patients' reported pain severity and quality of life. RESULTS: This study, for the first time, showed the effect of blocking the neuronal transmission of pain by local infiltration of neosaxitoxin into the bladder submucosa. All five patients successfully responded to the treatment. Furthermore, the analgesic effect lasted for the entire 90 days of follow-up without the need for a second infiltration, and no adverse reactions to neosaxitoxin were detected. CONCLUSIONS: Neosaxitoxin infiltration was shown to be a safe and effective intervention to control pain related to BPS. It was well tolerated by patients, who experienced extended pain relief and associated beneficial effects over a follow-up of 90 days. These results confirm the effectiveness of neosaxitoxin as a long-acting local pain blocker.

Asunto(s)

Bloqueantes Neuromusculares/uso terapéutico , Dolor Intratable/tratamiento farmacológico , Saxitoxina/análogos & derivados , Vejiga Urinaria/inervación , Adulto , Cistoscopía , Femenino , Humanos , Persona de Mediana Edad , Bloqueantes Neuromusculares/administración & dosificación , Saxitoxina/administración & dosificación , Saxitoxina/uso terapéutico , Síndrome

RESUMEN

Neosaxitoxin (NeoSTX) is a specific reversible blocker of voltage gated sodium channels on excitable cells. In the last decade, it has been tested in a number of interesting clinical trials, however there is still little information available on mammalian toxicity. Rats were treated for 12 weeks with doses of 1, 3 or 6 µg/kg of subcutaneous NeoSTX. At weeks 12 and 17, animals were sacrificed and blood samples collected for hematological and biochemical analysis. Organs were harvested for weight determination and histopathological assessments. The lowest acute toxicity via the intraperitoneal (ip) route was (30.35 µg/kg) and there was no significant difference between intramuscular and subcutaneous routes (11.4 and 12.41 µg/kg). The NeoSTX adiministration did not produce lethality at week 12 and after five weeks of suspension. NeoSTX 6 µg/kg ip produced reductions (p < 0.05) in body weight and food intake, and increased blood level of total and direct bilirubin, GGT and SGOT at week 12; all of these were reversed in the recovery period. NeoSTX 1 and 3 µg/kg ip did not show significant changes with the control group. Histopathological presentations were normal in all groups. This study revealed that NeoSTX is safe in vivo, giving a reliable security margin for its use like a local anesthetic.

Asunto(s)

Saxitoxina/análogos & derivados , Bloqueadores de los Canales de Sodio/toxicidad , Animales , Recuento de Células Sanguíneas , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Saxitoxina/administración & dosificación , Saxitoxina/sangre , Saxitoxina/toxicidad , Bloqueadores de los Canales de Sodio/administración & dosificación , Bloqueadores de los Canales de Sodio/sangre , Equilibrio Hidroelectrolítico/efectos de los fármacos

RESUMEN

Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na(+) channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H(+)) or sodium (Na(+)) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na(+) and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na(+) recognition domain was conserved in both SxtF/M, indicating that Na(+) can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na(+) incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na(+) in toxin export, as well as the motifs L(398)XGLQD(403) (SxtM) and L(390)VGLRD(395) (SxtF) in toxin recognition.

Asunto(s)

Proteínas Bacterianas/metabolismo , Cylindrospermopsis/metabolismo , Toxinas Marinas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico Activo , Simulación por Computador , Cylindrospermopsis/química , Cylindrospermopsis/genética , Toxinas Marinas/química , Toxinas Marinas/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Familia de Multigenes , Filogenia , Conformación Proteica , Saxitoxina/análogos & derivados , Saxitoxina/genética , Saxitoxina/metabolismo

RESUMEN

Paralytic shellfish poisoning (PSP) toxins are a group of naturally occurring neurotoxic alkaloids produced among several genera of primarily freshwater cyanobacteria and marine dinoflagellates. Although saxitoxin (STX) and analogs are all potent Na(+) channel blockers in vertebrate cells, the functional role of these compounds for the toxigenic microorganisms is unknown. Based upon the known importance of monovalent cations (such as sodium) in the maintenance of cellular homeostasis and ion channel function, we examined the effect of high extracellular concentrations of these ions on growth, cellular integrity, toxin production and release to the external medium in the filamentous freshwater cyanobacterium, Raphidiopsis brookii D9; a gonyautoxins (GTX2/3) and STX producing toxigenic strain. We observed a toxin export in response to high (17 mM) NaCl and KCl concentrations in the growth medium that was not primarily related to osmotic stress effects, compared to the osmolyte mannitol. Addition of exogenous PSP toxins with the same compositional profile as the one produced by R. brookii D9 was able to partially mitigate this effect of high Na⁺ (17 mM). The PSP toxin biosynthetic gene cluster (sxt) in D9 has two genes (sxtF and sxtM) that encode for a MATE (multidrug and toxic compound extrusion) transporter. This protein family, represented by NorM in the bacterium Vibrio parahaemolyticus, confers resistance to multiple cationic toxic agents through Na⁺/drug antiporters. Conserved domains for Na⁺ and drug recognition have been described in NorM. For the D9 sxt cluster, the Na⁺ recognition domain is conserved in both SxtF and SxtM, but the drug recognition domain differs between them. These results suggest that PSP toxins are exported directly in response to the presence of monovalent cations (Na⁺, K⁺) at least at elevated concentrations. Thus, the presence of both genes in the sxt cluster from strain D9 can be explained as a selective recognition mechanism by the SxtF/M transporters for GTX2/3 and STX. We propose that these toxins in cyanobacteria could act extracellularly as a protective mechanism to ensure homeostasis against extreme salt variation in the environment.

Asunto(s)

Cianobacterias/patogenicidad , Potasio/farmacología , Saxitoxina/análogos & derivados , Saxitoxina/metabolismo , Intoxicación por Mariscos/etiología , Sodio/farmacología , Monensina/farmacología , Saxitoxina/análisis

RESUMEN

The aim of this study was to investigate the unusual gas-phase dissociation behavior of two epimer pairs of protonated gonyautoxins (GTX) following electrospray ionization in comparison to their deprotonated counterparts. The chemical structures of the investigated GTX1-4 variants vary in their substitution pattern at N-1 and the stereochemical orientation of the hydroxysulfate group at C-11 (11α for GTX1/2 versus 11ß for GTX3/4). The direct comparison of mass spectra in positive and negative ion modes illustrated two distinct features: first, an intriguing difference between protonated 11α and 11ß species, where 11α conformations exhibited almost complete dissociation of [M + H](+) ions via facile SO(3) elimination, while 11ß species remained mostly intact as [M + H](+); and second, the lack of such differences for the deprotonated counterparts. In this study, we propose an acid-catalyzed elimination mechanism from density functional theory calculations, initiated by a proton transfer of a guanidinium proton to the hydroxysulfate group with simultaneous SO(3) release, which is only possible for the 11α conformation based on intramolecular distances. The same mechanism explains the lack of a comparable SO(3) loss in the negative ion mode. CID experiments supported this proposed mechanism for GTX1 and GTX2. Computational modeling of product ions seen in the CID spectra of GTX3 and GTX4 established that the lowest energy dissociation pathway for the 11ß epimers is elimination of water with the possibility for further SO(3) release from the intermediate product. Experimental data for structurally analogous decarbamoyl gonyautoxins confirmed the evidence for the GTX compounds as well as the proposed elimination mechanisms.

Asunto(s)

Gases/química , Protones , Saxitoxina/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Modelos Moleculares , Saxitoxina/química , Estereoisomerismo

RESUMEN

BACKGROUND AND OBJECTIVES: Wound infiltration with available local anesthetics generally provides analgesia for less than 8 hrs. The site 1 sodium-channel toxin neosaxitoxin (neoSTX) produced analgesia for over 24 hrs in animals and human volunteers. In this randomized, double-blind trial, we examined the postoperative course of patients undergoing laparoscopic cholecystectomy under a standardized general anesthesia with wound infiltration using either neoSTX or bupivacaine. We hypothesized that neoSTX would reduce pain compared with bupivacaine at 12 hrs postoperatively. METHODS: Patients received preincisional infiltration of laparoscope entry sites with 20 mL containing either neoSTX (total dose, 100 µg) or bupivacaine 0.25% (total dose, 50 mg). The primary outcome measure was the visual analog pain score at 12 hrs postoperatively. Secondary outcomes included repeated pain scores at rest and with movement,analgesic use, functional recovery, and adverse effects. Groups were compared using Mann-Whitney U tests for pain scores, Fisher exact test for proportions of patients with severe pain and complete analgesia, and Kaplan-Meier curves for time to full recovery. RESULTS: Among 137 subjects, 69 were randomized to neoSTX and 68 to bupivacaine. Median pain scores at rest and with movement 12 hrs postoperatively were lower in the neoSTX group compared with the bupivacaine group (P<0.01). Additional pain measures and recovery parameters also favored neoSTX. No serious adverse events occurred,and no adverse events were more frequent in the neoSTX group. CONCLUSIONS: NeoSTX shows promise as a long-acting local anesthetic. Future studies will examine dose response, combination formulations, and safety with dose escalation.

Asunto(s)

Analgesia , Anestesia Local , Bupivacaína/administración & dosificación , Colecistectomía Laparoscópica , Dolor Postoperatorio/prevención & control , Saxitoxina/análogos & derivados , Adulto , Analgesia/métodos , Anestesia Local/métodos , Bupivacaína/farmacocinética , Colecistectomía Laparoscópica/efectos adversos , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Dolor Postoperatorio/etiología , Dolor Postoperatorio/metabolismo , Saxitoxina/administración & dosificación , Saxitoxina/farmacocinética

RESUMEN

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

Asunto(s)

Venenos de Cnidarios/toxicidad , Unión Neuromuscular/efectos de los fármacos , Escifozoos/química , Transmisión Sináptica/efectos de los fármacos , Animales , Pollos , Venenos de Cnidarios/aislamiento & purificación , Lidocaína/metabolismo , Masculino , Toxinas Marinas , Ratones , Unión Neuromuscular/metabolismo , Oxocinas/aislamiento & purificación , Oxocinas/toxicidad , Fentolamina/metabolismo , Fosfolipasas A2/aislamiento & purificación , Fosfolipasas A2/toxicidad , Saxitoxina/análogos & derivados , Saxitoxina/aislamiento & purificación , Saxitoxina/toxicidad , Manejo de Especímenes , Tetrodotoxina/aislamiento & purificación , Tetrodotoxina/toxicidad , Conducto Deferente/efectos de los fármacos

RESUMEN

Major toxins from skin extracts of 18 specimens of six Atelopus toad species collected in Panama were analyzed. Chiriquitoxin was identified using (1)H NMR in A. limosus and A. glyphus for the first time. Zetekitoxin in A. zeteki and tetrodotoxin in A. varius, A. chiriquiensis and A. zeteki were identified again. Furthermore, A. certus was suggested to contain a water-soluble toxin other than tetrodotoxin.

Asunto(s)

Bufonidae , Piel/química , Toxinas Biológicas/análisis , Animales , Bioensayo , Cromatografía Liquida , Mezclas Complejas/química , Mezclas Complejas/toxicidad , Dosificación Letal Mediana , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Panamá , Saxitoxina/análogos & derivados , Saxitoxina/análisis , Saxitoxina/química , Saxitoxina/toxicidad , Especificidad de la Especie , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , Tetrodotoxina/análisis , Tetrodotoxina/química , Tetrodotoxina/toxicidad , Toxinas Biológicas/química , Toxinas Biológicas/toxicidad

RESUMEN

Paralytic shellfish toxins (PST) are a collection of over 26 structurally related imidazoline guanidinium derivatives produced by marine dinoflagellates and freshwater cyanobacteria. Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is the major phase II conjugation reaction in mammalian liver. In this study, using human liver microsomes, the in vitro paralytic shellfish toxins oxidation and sequential glucuronidation are achieved. Neosaxitoxin (neoSTX), Gonyautoxin 3/2 epimers (GTX3/GTX2) and Saxitoxin (STX) are used as starting enzymatic substrates. The enzymatic reaction final product metabolites are identified by using HPLC-FLD and HPLC/ESI-IT/MS. Four metabolites from GTX3/GTX2 epimers precursors, three of neoSTX and two of STX are clearly identified after incubating with UDPGA/NADPH and fresh liver microsomes. The glucuronic-Paralytic Shellfish Toxins were completely hydrolysed by treatment with beta-glucuronidase. All toxin analogs were identified comparing their HPLC retention time with those of analytical standard reference samples and further confirmed by HPLC/ESI-IT/MS. Paralytic Shellfish Toxins (PST) were widely metabolized by human microsomes and less than 15% of the original PST, incubated as substrate, stayed behind at the end of the incubation. The apparent V(max) and Km formation values for the respective glucuronides of neoSTX, GTX3/GTX2 epimers and STX were determined. The V(max) formation values for Glucuronic-GTX3 and Glucuronic-GTX2 were lower than Glucuronic-neoSTX and Glucuronic-STX (6.8+/-1.9x10(-3); 8.3+/-2.8x10(-3) and 9.7+/-2.8x10(-3)pmol/min/mg protein respectively). Km of the glucuronidation reaction for GTX3/GTX2 epimers was less than that of glucuronidation of neoSTX and STX (20.2+/-0.12; 27.06+/-0.23 and 32.02+/-0.64microM respectively). In conclusion, these data show for the first time, direct evidence for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. The PST oxidation and glucuronidation pathway showed here, is the hepatic conversion of its properly glucuronic-PST synthesized, and the sequential route of PST detoxication in human.

Asunto(s)

Toxinas Marinas/metabolismo , Intoxicación por Mariscos/metabolismo , Biotransformación , Cromatografía Líquida de Alta Presión , Glucuronidasa/metabolismo , Glucurónidos/biosíntesis , Glucurónidos/química , Glucurónidos/metabolismo , Humanos , Inactivación Metabólica , Cinética , Microsomas Hepáticos/metabolismo , Estructura Molecular , Oxidación-Reducción , Saxitoxina/análogos & derivados , Saxitoxina/química , Saxitoxina/metabolismo , Espectrometría de Masa por Ionización de Electrospray