Tetrazolium-Based Cell Bioassay for Neurotoxins Active on Voltage-Sensitive Sodium Channels: Semiautomated Assay for Saxitoxins, Brevetoxins, and Ciguatoxins

Manger, R; Leja, L.S.; Lee, S.Y.; Hungerford, James M.; Wekell, Marleen M.

doi:10.1006/abio.1993.1476

Cited by 250 publications

(173 citation statements)

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“…A protocol involving neuroblastoma cells aims to quantify the mitochondrial reduction of Mtt tetrazolium dye as a measure of the viability of veratridine pretreated cells. the lOD for this assay is reported at 250 µg BTX-1/kg shellfish (Manger et al, 1993(Manger et al, , 1994Plakas and Dickey, 2010). Cytotoxicity may occur at higher concentrations than the typical neurotoxic effects.…”

Section: Neurotoxic Shellfish Poisoning (Nsp)mentioning

confidence: 99%

“…Indeed, for most if not all marine biotoxins, there is a dearth of human-relevant methods aiming to address all relevant physiological endpoints in the intoxication cases. For example, although the human neuroblastoma assay (see below) is an excellent biological and human relevant method for assessing the sodium channel-specific interaction of ciguatoxins, brevetoxins, tetrodotoxins or saxitoxins (Manger et al, 1993(Manger et al, , 1994(Manger et al, , 1995, subchronic, chronic, or recurring exposure toxicity cannot be tested. In addition, and despite the fact that strenuous efforts are continuously being put forth, there is a lack of reference materials for toxin analysis.…”

Section: Hazard Monitoring Risk Assessment and Risk Managementmentioning

confidence: 99%

“…the cytotoxicity of NSP toxins is generally recognized, and several cytotoxicity assays have been developed (Bottein et al, 2010;Dechraoui et al, 2007;Dickey et al, 1999;Fairey et al, 1997;louzao et al, 2004;Manger et al, 1995Manger et al, , 1993Manger et al, , 1994trainer et al, 1995). the cytotoxicity of Btx is assumed to result from the interaction with the voltage-gated sodium channels.…”

Section: Neurotoxic Shellfish Poisoning (Nsp)mentioning

confidence: 99%

“…A protocol involving neuroblastoma cells aims to quantify the mitochondrial reduction of tetrazolium dye as a measure of the viability of veratridine pretreated cells (Gallacher and Birkbeck, 1992;Kogure et al, 1988;Manger et al, 1993). this commonly used assay quantifies cells with normal morphology following treatment with PSP toxins with a lOD of 20 µg PSPequiv./kg shellfish (Jellett et al, 1992).…”

Section: Functional Biochemical and Biomolecular Assaysmentioning

confidence: 99%

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A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems

Daneshian

2013

ALTEX

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Summary Aquatic food accounts for over 40% of global animal food products, and the potential contamination with toxins of algal origin -marine biotoxins -poses a health threat for consumers. The gold standards to assess toxins in aquatic food have traditionally been in vivo methodssingle marine biotoxins or combinations of marine biotoxins and intoxication symptomatology as well as monitoring the "success" of controls implemented during fishery and aquaculture production, processing, and retailing. For this reason, a workshop was organized in ermatingen, Switzerland by the Center for Alternatives to Animal testing -europe (CAAteurope) within the framework of the transatlantic think tank for toxicology (t 4 ).In contrast to other food products where hygiene and potential contamination with microbes or mold toxins is the prime issue, the emphasis in aquatic products (shellfish and finfish) is, beyond viral and bacterial contaminations, primarily placed on potential contamination with marine biotoxins owing to the numerous and recurring human intoxications.The gold standards to assess toxins in aquatic food have traditionally been in vivo methods, i.e., the mouse and the rat bioassays. Besides the ethical issues of in vivo bioassays there are specific difficulties, e.g., different exposure route (i.p. versus oral), low inter-species comparability, high intra-species variability, and questionable extrapolation of quantitative risk to humans, thus highlighting the need for the development of more reliable detection and quantification methods. Based on this reasoning, the european Food Safety Authority (eFSA) advocates the use of an analytical method, i.e., LC-MS (Liquid Chromatography coupled Mass Spectrometry), as a substitute for in vivo bioassays for almost all classes of marine toxins. However, although lC-MS is a very sensitive method that has the advantages of being able to detect multiple toxins in a single analysis and being good for confirming the identity of toxins, the quality of quantitative analysis is dependent on the availability of calibration standards. While many new toxin structural analogues have been detected and identified using LC-MS, this technique -as with most other methods -is not good at detecting new toxin types. When new toxin analogues are detected but accurate standards are not yet available, only an approximate quantitation is possible using estimated response factors.For the purpose of risk assessment of food, however, it is imperative to focus on the possible adverse effects, independent of whether they stem from one toxin or a combination of toxins. As toxicity is defined by functional and structural changes of biological systems, the development of a human-relevant in vitro system for appropriate risk assessment of marine biotoxins in seafood stands to reason. Moreover, poor correlation of human intoxications, of acute symptomatology and long-term adverse effects, and of predictive in vitro, in vivo, and analytical detection methodologies of marine biotoxins stems not least from a...

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Section: Neurotoxic Shellfish Poisoning (Nsp)mentioning

confidence: 99%

Section: Hazard Monitoring Risk Assessment and Risk Managementmentioning

confidence: 99%

Section: Neurotoxic Shellfish Poisoning (Nsp)mentioning

confidence: 99%

Section: Functional Biochemical and Biomolecular Assaysmentioning

confidence: 99%

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A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems

Daneshian

2013

ALTEX

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“…Cells were maintained at 37ºC in an incubator (Thermo Scientifics), with injection of 5% CO 2 . In order to determine the cytotoxicity of A. chilensis extracts on the tumor cell line of mouse neuroblastoma N2a, the inhibition of cell growth was evaluated at 24 h post-exposure, by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Invitrogen) colorimetric assay, according to the method described by Manger et al (1993) for determination of cell viability. A density of 15x10 4 cells ml -1 in 200 μL of culture medium supplemented with 5% FBS was grown in 96 wells-plates (Nunc) at 37ºC and 5% CO 2 for 24 h. Later, 30 μl of different concentrations of A. chilensis extract (from 3.9 μg ml -1 to 1500 μg ml -1 ) were added.…”

Section: Cytotoxicity Test On Mammal Cellsmentioning

confidence: 99%

Characterization of bioactive molecules isolated from sea cucumber Athyonidium chilensis

Sottorff¹,

Aballay²,

Hernández³

et al. 2013

Rev. biol. mar. oceanogr.

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4Agri-Food Laboratories-Concepción, SGS Chile Ltda. Américo Vespucio 820, Parque Industrial Las Arucas, Talcahuano, ChileResumen.-En la última década se ha incrementado el interés por la búsqueda de moléculas con potencial biomédico y amigable para el ambiente. Desde esta perspectiva, la búsqueda de principios bioactivos de origen marino ha permitido valorar la diversidad biológica presente en los ecosistemas acuáticos. En el Phylum Equinodermata, se destaca la familia Holothuridae, por su capacidad de sintetizar moléculas como saponinas y otros metabolitos secundarios de alto interés farmacológico debido a su capacidad hemolítica, antitumoral, anti-inflamatoria, antimicrobiana, citostática y antineoplásica. El interés del presente trabajo se centró en la caracterización de un extracto purificado obtenido desde Athyonidium chilensis (Holothuria) mediante técnicas cromatográficas y de espectrometría de masas, con la posterior evaluación de su potencial bioactivo sobre modelos in vitro. Como resultado de estos análisis, se identificaron 2 saponinas. La primera con un peso molecular de 1522 Da y altamente conjugada con monosacáridos. La segunda con un peso molecular de 764 Da fue identificada como holoturinósido D. Respecto a los resultados de la actividad biológica del extracto purificado, éste mostró actividad antibacteriana, antifúngica y citotóxica frente a una línea celular de neuroblastoma. Los resultados de este estudio se convierten en la primera caracterización de moléculas con actividad biológica desde Athyonidium chilensis. Palabras clave: Holothuria, holoturinósido D, saponinaAbstract.-In the last decade, the interest for searching molecules with biomedical potential as well as innocuous for the environment has been increased. Under this perspective, the search for bioactive principles of marine origin has allowed to value the biological diversity present in aquatic systems. In phylum Echinodermata, the family Holothuridae is distinguished by its capacity of synthesizing molecules such as saponins and other secondary metabolites of high pharmacological interest because of their interesting haemolytic, antitumor, anti-inflammatory, antimicrobial, cytostatic and antineoplastic capacity. The aim of the present study was focused on the characterization of a purified extract obtained from Athyonidium chilensis (Holothuria) by chromatographic and mass spectrometric techniques and the later assessment of its bioactive potential on in vitro models. As a result of these analyses 2 saponins were identified. The first with a molecular weight of 1522 Da and highly conjugated with monosaccharides. The second with a molecular weight of 764 Da was identified as holothurinoside D. The biological activity of the purified extract showed antibacterial, antifungal and cytotoxic activity on a neuroblastoma cell line. Outcomes of this study correspond to the first characterization of molecules with biological activity from Athyonidium chilensis.

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Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin

1999

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Tetrazolium-Based Cell Bioassay for Neurotoxins Active on Voltage-Sensitive Sodium Channels: Semiautomated Assay for Saxitoxins, Brevetoxins, and Ciguatoxins

Cited by 250 publications

References 0 publications

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems

Characterization of bioactive molecules isolated from sea cucumber Athyonidium chilensis

Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin

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