A Fluorescent Microplate Assay for Diarrheic Shellfish Toxins

Vieytes, Mercedes R.; Fontal, O.I.; Leira, F.; Sousa, Juan Manuel Vieites Baptista de; Botana, Luís M.

doi:10.1006/abio.1997.2127

Cited by 94 publications

(49 citation statements)

References 18 publications

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“…However, the methanol content in the wells was still under 1%, which is below the level at which the enzyme is affected (Vieytes et al 1997). Four wells for each sample were used, 2 replicates and 2 negative controls where no enzyme was added.…”

Section: Methodsmentioning

confidence: 99%

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Oceanographic settings explain fluctuations in Dinophysis spp. and concentrations of diarrhetic shellfish toxin in the plankton community within a mussel farm area on the Swedish west coast

Godhe¹,

Svensson²,

Rehnstam-Holm³

2002

Mar. Ecol. Prog. Ser.

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The influence of hydrographic, biological and meteorological variables on the abundance of Dinophysis spp. and the concentration of diarrhetic shellfish toxin (DST) in the plankton population were investigated in a mussel (Mytilus edulis) farm area on the Swedish west coast. This location provided an opportunity to simultaneously compare Dinophysis spp. cell numbers, concentration of DST in natural phytoplankton assemblages and toxicity of mussel tissues. Sampling was performed every other day from October 10 to November 5, 2000, and on each occasion, 5 randomly selected sites were sampled. During this period, 3 distinct water masses passed through the vicinity of the mussel farm. The second water mass, characterized by low salinity and nitrogen concentration, was probably advected into the area from surface waters in the nearby Skagerrak. This low salinity water also contained a high abundance of Dinophysis spp., and high concentrations of DST were recorded in the phytoplankton population. Multivariate analysis (projection to latent structures by means of partial least squares, PLS) determined that the principal variables influencing the concentration of DST in the plankton assemblage were the causative species (D. acuminata, D. acuta and D. norvegica) and salinity. The abundance of the 3 Dinophysis spp. was inversely correlated to salinity. A rapid increase in the toxicity of mussels in response to the high levels of DST was observed. The concentration of DST had doubled within 2 d of the appearance of Dinophysis spp. After 8 d, the water mass containing Dinophysis spp. was replaced and cell numbers again returned to low levels. The concentration of DST in the phytoplankton samples remained high for another 2 d after the number of Dinophysis spp. had declined and the toxicity of mussels continued to be high for the remainder of the study. Causes of the rapid intoxication versus slow detoxification of mussels are discussed. These results suggest that present monitoring programs are insufficient to provide early warning of toxic blooms to aquaculturists on the Swedish west coast.

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Section: Methodsmentioning

confidence: 99%

“…The procedure for the sample clean-up followed the protocol for HPLC (high performance liquid chromatography) analysis described by Lee et al (1987). The resulting chloroform extracts were used for detection of DST by the PPIA according to Vieytes et al (1997).…”

Section: Methodsmentioning

confidence: 99%

Oceanographic settings explain fluctuations in Dinophysis spp. and concentrations of diarrhetic shellfish toxin in the plankton community within a mussel farm area on the Swedish west coast

Godhe¹,

Svensson²,

Rehnstam-Holm³

2002

Mar. Ecol. Prog. Ser.

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“…PP2A inhibition assay PP2A inhibition was assayed by using the Toxiline-DSP Test for detection of diarrheic shellfish toxins (DSP), based in the method described by Vieytes et al (1997).…”

Section: Metabolic Rate Assaymentioning

confidence: 99%

“…Fluorescence was measured by using a microplate fluorescence reader FL600 (Bio-Tek/Vermont, USA) after 24 h of incubation with the toxins. Results are presented as percentage of fluorescence versus control; mean values Ϯ SEM, with n Ն 3.PP2A inhibition assay PP2A inhibition was assayed by using the Toxiline-DSP Test for detection of diarrheic shellfish toxins (DSP), based in the method described by Vieytes et al (1997). …”

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confidence: 99%

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

Espiña

Louzao

Cagide

et al. 2010

British J Pharmacology

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The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytesb ph_512 337.. 344 Begoña Espiña Japan Food Research Laboratories, Tama, Tokyo 206-0025, JapanBackground and purpose: Okadaic acid (OA) and microcystins (MCs) are structurally different toxins with the same mechanism of action, inhibition of serine/threonine protein phosphatases (PPs). Methyl okadaate (MeOk), a methyl ester derivative of OA, was considered almost inactive due to its weak inhibition of PP1 and PP2A. Here, we have investigated the activity and potency of MeOk in hepatic cells in comparison with that of OA and MCs. Experimental approach: We tested the effects of MeOK, OA and microcystin-leucine and arginine (MC-LR) on the metabolic rate, the actin cytoskeleton and glucose uptake in a rat hepatocyte cell line (Clone 9) and in primary cultured rat hepatocytes. PP2A was assayed to compare OA and MeOk activity. Key results: MeOk disrupted the actin cytoskeleton and depressed the metabolic rate of both types of rat hepatocytes, being six-fold less potent than OA in Clone 9 cells but nearly six-fold more potent in primary cultured hepatocytes. However, unlike OA, MeOk did not change glucose uptake in these cells, suggesting a weak inhibition of PP2A, as confirmed in direct assays of PP2A activity. Conclusions and implications:Although MeOk was originally described as a weakly bioactive molecule, it clearly depressed the metabolic rate and disrupted the cytoskeleton in primary and immortalized rat hepatocytes. Furthermore, MeOk affected primary hepatocytes at much lower concentrations than those affecting immortalized cells. These effects were unrelated to PP2A inhibition. Our results suggest the risk to public health from MeOk in foodstuffs should be re-evaluated.British Journal of Pharmacology (2010) 159, 337-344; doi:10.1111/j.1476-5381.2009 published online 15 December 2009 Keywords: actin cytoskeleton; glucose uptake kinetics; metabolic rate; methyl okadaate; microcystin; okadaic acid; phycotoxins; protein phosphatase and rat hepatocytes Abbreviations: DSP, diarrheic shellfish poisoning; MC-LR, microcystin-leucine and arginine; MeOk, methyl okadaate; OA, okadaic acid; PP1, protein phosphatase-1; PP2A, protein phosphatase-2A IntroductionMany natural toxins such as okadaic acid (OA) potently inhibit the catalytic activity of serine/threonine protein phosphatases (PPs), of which PP1 and PP2A are the most abundant in cells. Both PPs have a wide spectrum of activity being able to dephosphorylate a large number of substrates in vitro (Fernandez et al., 2002). The list of PPs inhibitors includes a structurally diverse group of compounds: polyether fatty acid substances similar to okadaic acid (OA), cyclic peptides like microcystins (MCs) and nodularins, terpenoids such as cantharidin and thyrsiferyl 23-acetate and polyketides, such as tautomycin and calyculin A (Fernandez et al., 2002). OA is the best representative of the diarrheic shellfish poisoning ...

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“…This method is under increasing criticism not only from an ethical point of view but also because of its limited sensitivity and inability to distinguish between toxins of different groups (Quilliam and Wright, 1995;Hess et al, 2006). A number of alternative methods have been proposed ranging from cytotoxicity assays (Aune et al, 1991;Amzil et al, 1992.;Tubaro et al, 1996a), protein phosphatase assays (Luu et al, 1993;Honkanen et al, 1996;Simon and Vernoux, 1994;Tubaro et al, 1996b;Isobe et al, 1995;Vieytes et al, 1997), liquid chromatography (LC) coupled with fluorescence detection and LC coupled with mass spectrometry, capillary electrophoresis and numerous immunosorbent methods (James et al, 2000). Some of these methods lack the required sensitivity and others require long sample preparation.…”

Section: Introductionmentioning

confidence: 99%

Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors

Stewart

Elliott

Walker³

et al. 2009

Toxicon

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a b s t r a c tOkadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.

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A Fluorescent Microplate Assay for Diarrheic Shellfish Toxins

Cited by 94 publications

References 18 publications

Oceanographic settings explain fluctuations in Dinophysis spp. and concentrations of diarrhetic shellfish toxin in the plankton community within a mussel farm area on the Swedish west coast

Oceanographic settings explain fluctuations in Dinophysis spp. and concentrations of diarrhetic shellfish toxin in the plankton community within a mussel farm area on the Swedish west coast

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors

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