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

• Published in: Toxins 2020-04, Vol.12 (5), p.281
• Main Authors: Viallon, Jérôme, Chinain, Mireille, Darius, Hélène Taiana
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 2072-6651; 2072-6651
• DOI: 10.3390/toxins12050281

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 EC 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.