4.4.2. Quantitation of CTX by In Vitro N2a Cytotoxicity Assay

The ciguatoxin content of each sample was measured as composite toxicity using an ouabain-veratridine (O/V) dependent in vitro neuroblastoma cytotoxicity assay (N2a assay) [74]. These assays utilized mouse Neuro-2a cells (ATCC, CL131; N2a), which were propagated and maintained under continuous growth as previously described [27,75]. Cells were harvested at 85–90% confluency and seeded to sterile 96-well polystyrene plates at a density of 4 × 10⁴ cells well⁻¹. The N2a assay measures sample toxicity as a loss in viability of N2a cells that have been sensitized with O/V, making these cell responses highly specific to sodium channel toxins (e.g., CTX) and thus adds a line of evidence for CTX (or a composite of CTXs) being present in samples when loss in viability is observed. Within each assay, the response of untreated N2a cells (serving as negative control) is assessed at the same sample doses provided to O/V-treated cells to determine if the sample contains other toxic substances that are not sodium channel toxins and could affect viability of O/V-treated cells.

For quantitative assays of CTX, triplicate dose-response curves were determined for both O/V-treated and untreated N2a cells exposed to eight concentrations of sample CTX residues redissolved by high-speed vortex in 100 µL of 5%-FBS-RPMI media spanning a 128-fold range. After 24 h of exposure to sample extracts, N2a cell viability is assessed as the reduction of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by metabolically active cells to a purple formazan product that is measured by absorbance at 570 nm. The concentration of sample extract at which 50% of N2a cells lost viability (IC₅₀) was compared to the IC₅₀ of a purified C-CTX-1 standard (50 pg starting dose) that was measured in a concurrent N2a assay seeded with the same batch of N2a cells seeded to sample assays. These analyses were possible due to an aliquot of purified C-CTX-1 stock that was purified from toxic Sphyraena barracuda harvested from the Virgin Islands and is the same FDA stock reported in several prior studies [e.g., 27, 75, and others]. Impurities were assessed by LC-MS/MS analyses prior to use and original stocks quantified via NMR and gravimetric analysis (data not available). The toxin content of samples is expressed as the mass of C-CTX-1 equivalents in Gambierdiscus cells (pg C-CTX-1 eq. cell⁻¹) based on the toxin sample cell counts described above. Samples in which the amount of extract required to cause a 50% loss in viability of O/V-treated N2a cells, and also caused significant loss in viability in untreated N2a cells, were considered below the limit of detection. Samples like these, in which there is clear CTX-specific activity, but quantitation criteria are not met, were considered positive detections of CTX, but their CTX content is described as “trace”.