Journal Article

Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)

Viallon, Jérôme, Chinain, Mireille, Darius, Hélène Taiana

2020

Overview

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

• Publisher: MDPI AG,MDPI
• Subject: absorbance data / Animals / Assaying / Automation / Bioassays / Biological Assay / Brevetoxins / CBA-N2a / Cell growth / Cell Line, Tumor / Cell Proliferation / Cell Survival - drug effects / Chromatography / Ciguatoxin / ciguatoxins / Ciguatoxins - analysis / Ciguatoxins - toxicity / Coefficient of variation / Consumption / Cytotoxicity / Dose-Response Relationship, Drug / Fish / Fishes - metabolism / High-throughput screening / Limit of Detection / Marine Toxins - analysis / Marine Toxins - toxicity / matrix effects / Mice / Neuroblastoma / Neurons - drug effects / Neurons - metabolism / Neurons - pathology / Ouabain / Ouabain - pharmacology / Oxocins - analysis / Oxocins - toxicity / Protocol / Reproducibility of Results / Saxitoxin / Saxitoxin - analysis / Saxitoxin - toxicity / Seafood / Shellfish / Sodium / Sodium channels / Sodium channels (voltage-gated) / Standardization / Time Factors / Toxicity / Toxins / Veratridine / Veratridine - pharmacology / Viability / Voltage-Gated Sodium Channel Agonists - analysis / Voltage-Gated Sodium Channel Agonists - toxicity / Voltage-Gated Sodium Channels - drug effects / Voltage-Gated Sodium Channels - metabolism