Abstract
Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (Na(V) channels) and blocking of voltage-gated potassium channels (K(V) channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using K(V) channel inhibitors as sensitizing reagents for N2a cells. The addition of the K(V) channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of K(V) channels on the toxicity of CTXs in a cell-based assay.