Bioassay methods for detection of N-palmitoylbrevetoxin-B2 (BTX-B4)

Bottein, Marie‐Yasmine Dechraoui; Fuquay, Jennifer Maucher; Munday, Rex; Selwood, Andrew I.; Ginkel, Roel van; Miles, Christopher O.; Loader, Jared I.; Wilkins, Alistair L.; Ramsdell, John S.

doi:10.1016/j.toxicon.2009.09.022

Cited by 24 publications

(29 citation statements)

References 20 publications

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“…The choices of concentrations were based on previous literature: 500 μM of ouabain was the typical concentration used, and 25 μM of veratridine was the approximate middle of the range (5–50 μM) used [19,20,21,22,23,24,25,26,27,28,29,30]. Cell lines were chosen in the attempt to find a human alternative to the rodent neuroblastoma (Neuro-2A) cell line and to survey other neural tissue-derived cells to determine if they have greater sensitivity to PbTxs without the use of ouabain and veratridine.…”

Section: Resultsmentioning

confidence: 99%

“…Manger and colleagues expanded this test to PbTxs with the observation that PbTx treatment sped up cell death, likely due to synergistic activity between veratridine and PbTxs, both of which bind to and activate VSSCs [21,22]. The development of this assay has allowed for the study of the cytotoxicity of various PbTx analogs and extracts, as well as extracts from other Karenia species [23,24,25,26,27]. Some investigators have found that the Neuro-2A assay is not as sensitive for detecting PbTxs in samples as other assays, such as LC-MS or receptor binding assays [24,28].…”

Section: Introductionmentioning

confidence: 99%

“…The ouabain concentration was typically 500 μM, but the veratridine concentration ranged from 5 to 50 μM. The time from treatment to measurement varied from 12 to 24 h, and cell seeded density ranged 6000–100,000 [19,20,21,22,23,24,25,26,27,28,29,30]. The most commonly used method for determining cell death was the MTT colorimetric assay for determining cell viability, in which metabolically active cells reduce the MTT tetrazolium compound to form a blue color.…”

Section: Introductionmentioning

confidence: 99%

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A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

McCall¹,

Elliott²,

Bourdelais³

2014

Marine Drugs

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Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC50 values for brevetoxin and non-linear triphasic dose response curves. Ouabain/veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC50 values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

McCall¹,

Elliott²,

Bourdelais³

2014

Marine Drugs

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“…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 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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“…Therefore, many researchers have continued to explore sensitive and convenient methods for detecting the toxic substance. Currently, the available detection methods include solid-phase extraction (SPE) for qualitative analysis [12], high-performance liquid chromatography (HPLC) [13], liquid chromatography-mass spectrometry (LC-MS) [14], radioimmunoassay [4], electrochemiluminescence-based immunoassay [15], thin-layer chromatography [16], and immunological methods, such as enzyme-linked immunosorbent assay (ELISA), which utilizes antibodies to quantify the BTX-2 levels [17]. The disadvantages of these assays are the expensive equipment, which require professional staff, the high cost of antibodies with limited stability, and the special storage conditions required for the immunological assay.…”

Section: Introductionmentioning

confidence: 99%

Preparation of a Specific ssDNA Aptamer for Brevetoxin-2 Using SELEX

Tian

Lin

et al. 2016

Journal of Analytical Methods in Chemistry

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The existing assays for detecting brevetoxin (BTX) depend on expensive equipment with a professional operator or on an antibody with limited stability, which requires complex processes, a high cost, and a considerable amount of time. The development of an alternative detection probe is another promising research direction. This paper reports the use of aptamers binding to BTX-2 in an analytical assay using the systematic evolution of ligands by exponential enrichment (SELEX). After 12 rounds of selection, the secondary structures of 25 sequences were predicted. Compared to other aptamers, Bap5 has relatively high affinity with the lowest dissociation constant of 4.83 μM, and IC50 is 73.81 ng mL−1. A good linear regression formula of y = 30.688x − 7.329 with a coefficient correlation of R 2 = 0.9798 was obtained using a biotin-avidin ELISA. Moreover, there is no cross-reaction with the detected marine toxins, except for BTX-2. Thus, Bap5 has potential to detect BTX-2 in shellfish in the future as a substitute for the recognition probe.

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Bioassay methods for detection of N-palmitoylbrevetoxin-B2 (BTX-B4)

Cited by 24 publications

References 20 publications

A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

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

Preparation of a Specific ssDNA Aptamer for Brevetoxin-2 Using SELEX

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