Chemical properties and biological activities of three highly purified major tetrodotoxin analogues

Abstract

Tetrodotoxin (TTX), the pufferfish toxin, has the potential to cause fatal food poisoning because of its potent voltage-gated sodium channel (Na_v) blocking activity. 4-epiTTX, 11-norTTX-6(S)-ol, and 11-oxoTTX are the major TTX analogues found in marine animals; thus, their chemical properties and biological activities should be determined. In this study, these three TTX analogues were purified to a high level (purity >97%) from pufferfish and newts. The ratios of the hemilactal to the 10,7-lactone forms were determined using ¹H NMR, and ¹³C NMR data were also obtained. 4-epiTTX underwent considerable transformation to TTX in physiological conditions. The Na_v blocking activity of these analogues was evaluated by whole-cell patch-clamp recording using a human Na_v1.2, colorimetric cell-based assay and mouse bioassay. The toxicity equivalency factors (TEFs setting TTX 1) of the three analogues were estimated; 4-epiTTX (0.06), 11-norTTX-6(S)-ol (0.02), and 11-oxoTTX (1.2) using patch-clamp recording, and 11-norTTX-6(S)-ol (0.50) and 11-oxoTTX (0.42) using the mouse bioassay. These data confirmed the low Na_v blocking activity of 4-epiTTX and high activity of 11-oxoTTX.

The ratios of hemilactal and 10,7-lactone forms of tetrodotoxin (TTX) and its three major analogues, and their human Na_v1.2 blocking activity