Detection of Sodium Channel Toxins: Directed Cytotoxicity Assays of Purified Ciguatoxins, Brevetoxins, Saxitoxins, and Seafood Extracts

Abstract

Neuroblastoma cells in culture were used to detect sodium channel-specific marine toxins based on an end-point determination of mitochondrial dehydrogenase activity. The assay responds in a dose-dependent manner to ciguatoxins, brevetoxins, and saxitoxins, and delineates the toxic activity as either sodium channel enhancing or sodium channel blocking. The assay responds rapidly to sodium channel activating toxins, allowing dose dependent detection in 4 to 6 h. Brevetoxins can be detected at 250 pg, and purified ciguatoxins are detected in the low picogram and subpicogram levels. The results obtained from cell bioassay of ciguatoxic finfish extracts correlates with those obtained from mouse bioassays. Sodium channel blocking toxins can also be detected with an approximate sensitivity of 20 pg in 24 to 48 h. This cell-based technique is simple, sensitive, demonstrates potential as an alternative to animal testing for sodium channel activating and blocking toxins, and can be automated.

Scientific Opinion on marine biotoxins in shellfish – Emerging toxins: Ciguatoxin group

“…EFSA Journal 2010; 8 (6):1627 A membrane immunobead assay (MIA) was developed by Hokama et al (1998a), which uses a monoclonal antibody, prepared against purified moray eel, coated onto coloured polystyrene beads. Overall, the MIA showed a LOD of approximately 32 ng P-CTX-1/kg fish flesh (Manger et al, 1995;Hokama et al, 1998b;Campora et al, 2008a). However, the specificity of the MIA for individual CTX-group toxins was not described.…”

“…This method is based on the capability of the fluorescent dye bis-oxonol to detect changes in membrane potential in excitable cells. Cell viability was measured by using 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT assay) or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium in the presence of phenazine methosulfate (PMS) (MTS assay), where the absorbance of the reduction product formazan is measured (Manger et al, 1995;Dechraoui et al, 1999;Bottein Dechraoui et al, 2005a. Manger et al (1995) reported that results obtained from the assay using neuroblastoma cells to detect CTX contaminated finfish extracts correlated with results obtained from MBA.…”

Scientific Opinion on marine biotoxins in shellfish - Emerging toxins: Brevetoxin group

“…Several cytotoxicity assays using several endpoints and detection methods have been developed for the determination of BTX-group toxins in shellfish (Manger et al, 1993(Manger et al, , 1994(Manger et al, , 1995Trainer et al, 1995;Fairey et al, 1997;Dickey et al, 1999;Louzao et al, 2004;Bottein Dechraoui et al, 2007;Dechraoui Bottein et al, 2010). The cytotoxicity assay is based on the action of BTX-group toxins on voltage-gated sodium channels.…”

Evaluating the Risk of Ciguatera Fish Poisoning from Reef Fish Grown at Marine Aquaculture Facilities in Hawai'i

“…Lipid extracts from each fish were assessed for the presence of CTX using the NB assay for the detection of CTX, which was modified after the method of Manger et al (1995), and detailed in Campora et al (2008a).…”

“…A sandwich enzyme linked immunoassay (ELISA) recently developed to detect CTX in partially purified fish extract is semiquantifiable and shows promise in eliminating some of the problems observed when using other methods (Campora et al 2008a). In addition, a directed cytotoxicity assay that records sensitive changes in the Na + channel of neuroblastoma cells in the presence of sodium channel activators or inhibitors has been established for the detection of ciguatoxins in crude seafood extracts (Manger et al 1995). Using a microplate high-throughput format, the neuroblastoma assay serves as a valuable tool for marine toxin studies, capable of reliably detecting ciguatoxins via Na + channel activity at subpicogram levels.…”