Abstract
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.
Highlights
The bio-accumulation of marine biotoxins produced by phytoplankton in filter-feeding invertebrates and finfish poses significant health threats to consumers and has detrimental effects on the economies of nations highly dependent on seafood consumption for their subsistence [1,2,3].Among these potent marine biotoxins are neurotoxins acting on the voltage gated sodium channels (VGSCs) of excitable cells, namely VGSC activators such as brevetoxins (PbTxs) and ciguatoxins (CTXs), and VGSC inhibitors such as saxitoxins (STXs) and tetrodotoxins (TTXs) [1,2]
In the absence of duly validated reference methods, many marine biotoxins are still unregulated [5]. This is the case for instance for CTXs, neurotoxins active on VGSCs that are implicated in ciguatera poisoning [5]
The present work aimed at revisiting several key parameters of the cell-based assay (CBA)-N2a as a step towards the standardized and reliable detection of two groups of toxins frequently involved in toxic outbreaks, namely VGSC activators (e.g., CTXs, PbTxs) and inhibitors (e.g., STX, dc-STX)
Summary
The bio-accumulation of marine biotoxins produced by phytoplankton in filter-feeding invertebrates and finfish poses significant health threats to consumers and has detrimental effects on the economies of nations highly dependent on seafood consumption for their subsistence [1,2,3]. Among these potent marine biotoxins are neurotoxins acting on the voltage gated sodium channels (VGSCs) of excitable cells, namely VGSC activators such as brevetoxins (PbTxs) and ciguatoxins (CTXs), and VGSC inhibitors such as saxitoxins (STXs) and tetrodotoxins (TTXs) [1,2]. Among the readily available and most widely used in vitro methods, the functional assay known as the cell-based assay (CBA) that uses a neuroblastoma (N2a) cell line appears as the most promising one [2,12]
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