RESUMEN
Harmful algal blooms are increasing worldwide, including those of Pseudo-nitzschia spp. producing domoic acid off the California coast. This neurotoxin was first shown to cause mortality of marine mammals in 1998. A decade of monitoring California sea lion (Zalophus californianus) health since then has indicated that changes in the symptomatology and epidemiology of domoic acid toxicosis in this species are associated with the increase in toxigenic blooms. Two separate clinical syndromes now exist: acute domoic acid toxicosis as has been previously documented, and a second novel neurological syndrome characterized by epilepsy described here associated with chronic consequences of previous sub-lethal exposure to the toxin. This study indicates that domoic acid causes chronic damage to California sea lions and that these health effects are increasing.
Asunto(s)
Ácido Kaínico/análogos & derivados , Toxinas Marinas/envenenamiento , Neurotoxinas/envenenamiento , Intoxicación/veterinaria , Leones Marinos/fisiología , Convulsiones/veterinaria , Animales , California/epidemiología , Diatomeas , Femenino , Hipocampo/efectos de los fármacos , Ácido Kaínico/análisis , Ácido Kaínico/envenenamiento , Masculino , Giro Parahipocampal/efectos de los fármacos , Intoxicación/epidemiología , Convulsiones/inducido químicamente , Convulsiones/epidemiología , Factores de TiempoRESUMEN
Domoic acid is a rigid analog of the neurotransmitter glutamate and a potent agonist of kainate subtype glutamate receptors. Persistent activation of these receptor subtypes results in rapid excitotoxicity, calcium dependent cell death and neuronal lesions in areas of the brain where kainate pathways are concentrated. To better understand responses to domoic acid induced excitotoxicity, microarrays were used to profile gene expression in mouse brain following domoic acid exposure. Adult female mice were subjected intraperitoneally to domoic acid at the lethal dose 50, killed and dissected at 30, 60 and 240 min post-injection. Total brain RNA from treated mice was compared with time-matched controls on Agilent 22K feature microarrays. Real-time PCR was performed on selected genes. For the 30, 60 and 240 min time points, 3.96%, 3.94% and 4.36% of the genes interrogated were differentially expressed (P-value < or = 0.01), respectively. Rigorous filtering of the data resulted in a set of 56 genes used for trending analysis and K-medians and agglomerative clustering. The earliest genes induced consisted primarily of early response gene families (Jun, Fos, Ier, Egr, growth arrest and DNA damage 45) and the inflammatory response element cyclooxygenase 2. Some later responding genes involved glucocorticoid responses (Gilz, Sgk), cold inducible proteins (Cirbp, Rbm3), Map kinases (Map3k6) and NF-kappaB inhibition. Real-time PCR in male mice from an additional study confirmed the expression of several of these genes across gender. The transcriptional profile induced by domoic acid shared similarity with expression profiles of brain ischemia and other excitotoxins, suggesting a common transcriptional response.
Asunto(s)
Expresión Génica/efectos de los fármacos , Ácido Kaínico/análogos & derivados , Toxinas Marinas/farmacología , Reacción de Fase Aguda , Animales , Femenino , Ácido Kaínico/farmacología , Masculino , Ratones , Análisis por Micromatrices/métodos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
Algal toxins produced by marine and freshwater microalgae present a significant analytical challenge because of their complex structures and frequent occurrence as mixtures of structural congeners, which differ in toxic potencies and are present at varying proportions in contaminated samples. Rapid, sensitive in vitro detection methods specific for each class of algal toxins have been developed over the past decade, including immunoassays, enzyme inhibition assays, receptor assays, and cell assays. This review discusses the conceptual approaches to assay development and provides a detailed assessment of the use of in vitro detection methods for marine and freshwater algal toxins.
Asunto(s)
Eucariontes/química , Toxinas Biológicas/análisis , Animales , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunoensayo , Técnicas para Inmunoenzimas , Receptores de Droga/efectos de los fármacos , Toxinas Biológicas/toxicidadRESUMEN
This research describes the diel phasing of the cell cycle in the dinoflagellate, Amphidinium operculatum Claparéde and Lachmann, and investigates the mechanisms that serve to link the cell cycle to the diel cycle. Unlike many dinoflagellates, A. operculatum has a naturally high division rate of approximately 1 division day(-1), which yields a nearly synchronous population, making it useful for population studies. When grown on a 16:8 h light/dark cycle, S-phase begins 10 h and mitosis 14-16 h after the onset of light, as determined by flow cytometry. Alterations in the timing of the dark/light and light/dark transitions showed that the cell cycle is entrained by the dark/light transition, with the light/dark cue being uninvolved. Cells in logarithmic phase growth also undergo diel changes in cell size (9-14 &mgr;m), reaching a maximum size late in the light phase, concurrent with mitosis. Stationary phase cells or cells blocked in G1 of the cell cycle with a cell cycle inhibitor, olomoucine, showed no size changes or reduced size changes over the diel cycle, suggesting a coupling of cell size to the cell division cycle. In Euglena, cAMP-dependent signaling appears to mediate diel phasing of the cell cycle. Therefore, the role of cAMP in cell cycle control in A. operculatum was investigated. Measurement of intracellular cAMP by radioimmunoassay (RIA) revealed that cAMP concentrations varied on a diel basis, but increases observed appeared to correlate with cell size increases, and did not correlate with light cues at the dark/light or light/dark transition. However, when cells were treated with the cAMP phosphodiesterase inhibitor, IBMX, cell cycle progression was inhibited at both the G1/S and the G2/M phase transitions. This supports a role for cAMP-dependent signaling in the dinoflagellate cell cycle and is in agreement with the documented role of cAMP in the cell cycle control of higher eukaryotes.
RESUMEN
Certain marine algae produce potent toxins that impact human health through the consumption of contaminated shellfish and finfish and through water or aerosol exposure. Over the past three decades, the frequency and global distribution of toxic algal incidents appear to have increased, and human intoxications from novel algal sources have occurred. This increase is of particular concern, since it parallels recent evidence of large-scale ecologic disturbances that coincide with trends in global warming. The extent to which human activities have contributed to their increase therefore comes into question. This review summarizes the origins and health effects of marine algal toxins, as well as changes in their current global distribution, and examines possible causes for the recent increase in their occurrence.
Asunto(s)
Eucariontes/metabolismo , Toxinas Marinas/efectos adversos , Animales , Eucariontes/química , Peces , Enfermedades Transmitidas por los Alimentos/epidemiología , Enfermedades Transmitidas por los Alimentos/etiología , Humanos , Toxinas Marinas/análisis , MariscosRESUMEN
Over 400 California sea lions (Zalophus californianus) died and many others displayed signs of neurological dysfunction along the central California coast during May and June 1998. A bloom of Pseudo-nitzschia australis (diatom) was observed in the Monterey Bay region during the same period. This bloom was associated with production of domoic acid (DA), a neurotoxin that was also detected in planktivorous fish, including the northern anchovy (Engraulis mordax), and in sea lion body fluids. These and other concurrent observations demonstrate the trophic transfer of DA resulting in marine mammal mortality. In contrast to fish, blue mussels (Mytilus edulus) collected during the DA outbreak contained no DA or only trace amounts. Such findings reveal that monitoring of mussel toxicity alone does not necessarily provide adequate warning of DA entering the food web at levels sufficient to harm marine wildlife and perhaps humans.
Asunto(s)
Diatomeas , Eutrofización , Leones Marinos , Animales , Bivalvos/microbiología , Encefalopatías/inducido químicamente , Encefalopatías/veterinaria , California , Cromatografía Liquida , Peces/microbiología , Cadena Alimentaria , Humanos , Ácido Kaínico/análogos & derivados , Ácido Kaínico/análisis , Ácido Kaínico/envenenamiento , Toxinas Marinas/análisis , Toxinas Marinas/envenenamiento , Espectrometría de Masas , Mortalidad , Neurotoxinas/análisis , Neurotoxinas/envenenamiento , Intoxicación/veterinaria , Leones Marinos/microbiologíaRESUMEN
More than 20 countries have either established or proposed regulatory limits for one or more of the paralytic shellfish poisoning (PSP) toxins as they occur in seafood products. PSP toxin levels are generally estimated using the standard AOAC mouse bioassay, yet because of various limitations of this method [e.g. high variability (+/-20%), low sensitivity, limited sample throughput and use of live animals], there remains a need for alternative testing protocols. A sensitive and selective, high capacity assay was developed for the PSP toxins which exploits the highly specific interaction of these toxins with their biological receptor (i.e. voltage-dependent sodium channel) and is thus based on functional activity. This receptor binding assay provides a radioactive endpoint, and is performed in a microtiter filter plate format with results determined by standard liquid scintillation counting within 24 hr. The Ki for the assay is 3.66 +/- 0.86 nM saxitoxin, with a limit of detection of c. 5 ng saxitoxin/ml in a sample extract. Good quantitative agreement of the assay with both mouse bioassay and high-performance liquid chromatographic analysis of crude extracts of contaminated shellfish, as well as PSP toxin-producing algae, was observed. Our findings indicate that the receptor binding assay has a strong predictive value for toxicity determined by mouse bioassay, and that this approach warrants consideration as a rapid, reliable and cost-effective alternative to live animal testing for detection and estimation of PSP-related toxicity in seafood and toxic algae.
Asunto(s)
Ensayo de Unión Radioligante/métodos , Saxitoxina/análisis , Mariscos , Animales , Química Encefálica , Cromatografía Líquida de Alta Presión , Eucariontes/química , Masculino , Ratones , Moluscos/química , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Saxitoxina/metabolismo , Canales de Sodio/metabolismo , Sinaptosomas/química , Sinaptosomas/metabolismoRESUMEN
Following four outbreaks of paralytic shellfish poisoning on Kodiak Island, Alaska, during 1994, medical records of ill persons were reviewed and interviews were conducted. Urine and serum specimens were analyzed at three independent laboratories using four different saxitoxin binding assays. High-performance liquid chromatography was used to determine the presence of specific toxin congeners. Among 11 ill persons, three required mechanical ventilation and one died. Mean peak systolic and diastolic blood pressure measurements were 172 (range 128-247) and 102 (range 78-165) mmHg, respectively, and blood pressure measurements corresponded with ingested toxin dose. All four different laboratory methodologies detected toxin in serum at 2.8-47 nM during acute illness and toxin in urine at 65-372 nM after acute symptom resolution. The composition of specific paralytic shellfish poisons differed between mussels and human biological specimens, suggesting that human metabolism of toxins had occurred. The results of this study indicate that saxitoxin analogues may cause severe hypertension. In addition, we demonstrate that saxitoxins can be detected in human biological specimens, that nanomolar serum toxin levels may cause serious illness and that human metabolism of toxin may occur. Clearance of paralytic shellfish poisons from serum was evident within 24 hr and urine was identified as a major route of toxin excretion in humans.
Asunto(s)
Bivalvos , Brotes de Enfermedades , Hipertensión/inducido químicamente , Parálisis/inducido químicamente , Intoxicación/epidemiología , Saxitoxina/envenenamiento , Adolescente , Adulto , Alaska/epidemiología , Animales , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Intoxicación/etiología , Saxitoxina/análisis , Saxitoxina/metabolismo , Bloqueadores de los Canales de Sodio , Canales de Sodio/efectos de los fármacosRESUMEN
Calcium regulates progression through several checkpoints in the cell cycle, including the G1/S-phase transition, G2/M-phase transition, and exit from mitosis. In the GH4C1 rat pituitary cell line, calcium mobilizing polypeptides and calcium channel activation inhibit cell proliferation. This report examines the effects of maitotoxin (MTX), an activator of type L voltage-dependent calcium channels (L-VDCC), on calcium influx and cell cycle progression in GH4C1 cells. MTX causes both a block from G1 to S-phase and a concentration-dependent accumulation of cells in G2+M. MTX does not increase the mitotic index; thus, sustained calcium channel activation by MTX results in an accumulation of cells in G2. In order to temporally localize the MTX-induced G2 block relative to cell cycle regulatory events at the G2/M transition, we assessed the relative activity of two cell cycle regulatory protein kinases, CDC2 and CDK2, in MTX-treated cells. CDC2-specific histone kinase activity in MTX-treated cells is lower than either in cells blocked in mitosis with the microtubule destabilizing agent demecolcine or in randomly cycling cells. In contrast, the activity of CDK2 is highest in MTX-treated cells, consistent with a G2 block prior to CDC2 activation. Together, these results implicate with a G2 block prior to CDC2 activation. Together, these results implicate calcium as an intracellular signal required for progression through G2 phase of the cell cycle prior to CDC2 kinase activation.
Asunto(s)
Proteína Quinasa CDC2/antagonistas & inhibidores , Agonistas de los Canales de Calcio/farmacología , Ciclo Celular/efectos de los fármacos , Toxinas Marinas/farmacología , Oxocinas , Animales , Proteína Quinasa CDC2/metabolismo , Calcio/fisiología , Canales de Calcio/metabolismo , División Celular/fisiología , Línea Celular/citología , Línea Celular/enzimología , ADN/biosíntesis , Conductividad Eléctrica , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Mitosis/efectos de los fármacos , Hipófisis/citología , Protamina Quinasa/antagonistas & inhibidores , Protamina Quinasa/metabolismo , Ratas , Fase S/efectos de los fármacos , Transducción de Señal/fisiología , Timidina/metabolismo , Tritio/metabolismoRESUMEN
The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.
Asunto(s)
Ciguatoxinas/aislamiento & purificación , Ácido Kaínico/análogos & derivados , Toxinas Marinas/aislamiento & purificación , Fármacos Neuromusculares Despolarizantes/aislamiento & purificación , Oxocinas , Animales , Unión Competitiva , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Ciguatoxinas/metabolismo , Ciguatoxinas/toxicidad , Ácido Kaínico/aislamiento & purificación , Ácido Kaínico/metabolismo , Ácido Kaínico/toxicidad , Toxinas Marinas/metabolismo , Toxinas Marinas/toxicidad , Fármacos Neuromusculares Despolarizantes/metabolismo , Fármacos Neuromusculares Despolarizantes/toxicidad , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Rana pipiens , Ratas , Ratas Sprague-DawleyRESUMEN
Maitotoxin (MTX) is a water-soluble polyether, isolated from the marine dinoflagellate Gambierdiscus toxicus, that stimulates hormone release and Ca2+ influx. We have investigated the action by which MTX induces Ca2+ influx and stimulates prolactin (PRL) release from GH4C1 rat pituitary cells. PRL release elicited by MTX is abolished in a concentration-dependent manner by nimodipine, a dihydropyridine (DHP) antagonist of type L voltage-dependent calcium channels (L-VDCC), indicating that MTX-enhanced PRL release occurs via activation of type L-VDCC. As an initial approach to determine whether MTX interacts directly with VDCC, we examined whether MTX affects the binding of [3H]PN 200-110, a DHP class antagonist, in intact GH4C1 cells. MTX increased the Bmax of [3H]PN 200-110 binding to intact GH4C1 cells from 4.6 +/- 0.03 to 12.5 +/- 2.2 fmol/10(6) cells, without changing the Kd. This indicates that MTX does not bind to the DHP site, but rather suggests that MTX may have an allosteric interaction with the DHP binding site. The effect of MTX on DHP binding was largely (65%) calcium-dependent. We next examined whether MTX alters the membrane potential of GH4C1 cells using the potential sensitive fluorescent dye bisoxonol. Addition of 100 ng/ml MTX to GH4C1 cells caused a membrane depolarization within 2.5 min which reached a plateau at 5 min. The MTX-induced depolarization was not prevented by substitution of impermeant choline ions for Na+. It was similarly unaffected by K+ channel blockers or by depleting the K+ chemical concentration gradient with gramicidin, a monovalent cation pore-forming agent. By contrast, low extracellular Ca2+ totally abolished the depolarization response, and nimodipine at 100 nM substantially reduced the MTX-induced membrane depolarization. These results indicate that the predominant effect of MTX on depolarization is Ca2+ influx through L-VDCC. Taken together, our results indicate that MTX-enhanced PRL release occurs exclusively via activation of type L-VDCC in GH4C1 cells. We suggest that MTX induces an initial slow calcium conductance, possibly via an allosteric interaction with a component of the VDCC complex, which, in turn, initiates a positive feedback mechanism involving calcium-dependent membrane depolarization and voltage-dependent activation of calcium channels.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Membrana Celular/fisiología , Toxinas Marinas/farmacología , Oxocinas , Animales , Canales de Calcio/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Colina/farmacología , Relación Dosis-Respuesta a Droga , Isradipino/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Nimodipina/farmacología , Neoplasias Hipofisarias , Potasio/farmacología , Ratas , Sodio/farmacología , Células Tumorales CultivadasRESUMEN
Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.
Asunto(s)
Éteres Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/fisiología , Hipófisis/citología , Hipófisis/fisiología , Huso Acromático/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Cromosomas/efectos de los fármacos , Cromosomas/ultraestructura , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Immunoblotting , Microscopía Electrónica , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Índice Mitótico/efectos de los fármacos , Índice Mitótico/fisiología , Ácido Ocadaico , Fosforilación/efectos de los fármacos , Hipófisis/efectos de los fármacos , Ratas , Proteína de Retinoblastoma/metabolismo , Proteína de Retinoblastoma/fisiología , Huso Acromático/efectos de los fármacos , Huso Acromático/ultraestructura , Timidina/metabolismo , TritioRESUMEN
This laboratory has previously demonstrated that PRL-secreting cells are virtually nonexistent on day 3 but appear in appreciable numbers on day 4 of neonatal life. The purpose of the present study was to determine whether this explosive appearance of PRL cells is due to maternal influences specific to the first 4 days of lactation. Litters of 1-day-old rat pups were placed with foster mothers that had been lactating for either 1 or 4 days. Four days later (at 5 days of age), the anterior pituitaries from these pups were removed, dispersed into individual cells with trypsin, and subjected to reverse hemolytic plaque assays for PRL and GH release. We found that the proportion of PRL-releasing cells in pituitaries from pups placed with day 1 mothers (4.2 +/- 0.6%; mean +/- SE; n = 4 experiments) was similar to that in 5-day-old pups that had not been fostered (3.4 +/- 0.6%; n = 3 experiments). In contrast, placing 1-day-old pups with day 4 mothers significantly reduced (P less than 0.01) the fraction that released PRL to 0.6 +/- 0.2% of all pituitary cells (n = 4 experiments). This effect appeared to be specific to PRL cells, since the percentage of all pituitary cells that secreted GH was not different between litters fostered onto day 1 or day 4 dams (40.0 +/- 3.8% and 41.1 +/- 3.9%, respectively; n = 3 experiments). Furthermore, the suppression of PRL cell expression did not result from a gross nutritional deficit, since the rates of body wt gain during the foster period were not different between the two groups (12.4 +/- 0.5 and 12.9 +/- 0.8 g/litter.day, respectively; n = 4 experiments). In a second design, newborn pups were placed with females that had been lactating for 0, 2, 4, or 7 days and analyzed for PRL- and GH-releasing cells at 5 days of age. In two trials of this experiment, the fractions of pituitary cells that secreted PRL were 5.2, 6.3, 5.9, and 1.2%, respectively for trial 1 and 3.3, 4.1, 0.7, and 0.6%, respectively for trial 2. As in the first experiment, the proportion of GH-releasing cells and the rate of growth were not different among the four groups. Taken together, these results indicate that normal differentiation of PRL cells in neonatal rats is triggered by a maternal signal present during the first few days of lactation, and that the magnitude of this signal declines with the progression of lactation.
Asunto(s)
Animales Recién Nacidos/metabolismo , Lactancia/fisiología , Hipófisis/metabolismo , Prolactina/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Diferenciación Celular , Femenino , Hormona del Crecimiento/metabolismo , Hipófisis/citología , Ratas , Ratas EndogámicasRESUMEN
Two feeding studies with young mice and one in situ intestinal perfusion study with adult rats were conducted to evaluate the influences of intrinsic (oyster) and extrinsic sources of cadmium and zinc on iron metabolism. When oyster was included in the diets, less cadmium accumulated in small intestines whether the cadmium was supplied as cadmium chloride or cadmium intrinsic to oyster. Increasing the zinc concentration of diets containing 2 ppm cadmium reduced cadmium retention in the small intestine regardless of whether the zinc supplied was intrinsic to oyster or as zinc carbonate. Dietary cadmium (20 ppm) reduced iron retention in the small intestine. Increasing dietary intrinsic zinc from 290 to 450 ppm reduced iron retention in small intestine whereas zinc carbonate did not. Inclusion of oyster in low cadmium diets reduced iron retention in the liver. Short-term in situ studies indicated salt sources of cadmium and zinc reduce uptake of iron from the intestine. Iron concentrations in the blood peaked between 20 and 55 min after exposure whether the iron was supplied alone or in combination with cadmium or zinc. The results suggest foods containing high concentrations of cadmium and zinc may reduce the availability of iron.