Study On The Cytotoxicities And Detecting Method Of Sodium Ion-channel Toxins

Algal toxins widely exist in a variety of red-tide algae and other marine organisms. They not only endanger fishery and aquaculture industry, but also cause direct menace to the heath of human beings. Ion-channel toxin, produced by red-tide algae and also some marine organisms is one of the most poisonous algal toxins. In order to elucidate the cytotoxic mechanisms of ion-channel toxins, the toxic effect of four sorts of sodium ion-channel toxin—STX,GTX1,4,GTX2,3,TTX were determined by utilizing a cell strain from Neuro-2a. As a result, an approach for detecting the cytotoxicities of sodium ion-channel toxins was established by taking advantage of STX and TTX. The established method was utilized to detect toxin level of the shellfish and algae. The results were then compared with that obtained by mouse bioassay and HPLC method.Our studies showed that STX,GTX1,4,GTX2,3,TTX all caused negative effect on the growth of Neuro-2a cells in the long term. Within 24h, no significant inhibition of cell growth was observed. However, after 48h, cells growth were inhibited in the presence of either the four toxins and the extent of the inhibition was shown to be in a dose-dependent manner. The half inhibition concentrations (IC50) were 250ng/ml,1000ng/ml,1300ng/ml,700ng/ml for STX,GTX1,4,GTX2,3,TTX, respectively. The desmoenzyme active effect of four sorts of sodium ion-channel toxin—STX,GTX1,4,GTX2,3,TTX to Neuro-2a cells also studied in this paper for the first time. The studies showed that four toxins were also found to influence the Na+-K+-ATPase and TChE activity of Neuro-2a. After 24h, Na+-K+-ATPase and TChE activity were inhibited and decreased with the increased dose of toxins. Obviously, the sodium ion-channel toxins have certain influence to the desmoenzyme function, with blocking the sodium ion channel of cell membrane and resulting the ion stream imbalanced, thus have the poisonous effect to the cells. A later investigation on cell membrane permeability showed that regardless of their dosage, the LDH leakage rate of four toxin groups exhibited no significant difference compared with control group, which suggested the cytotoxicity of four toxins were not through their effect on cell membrane permeability.Additionally, based on method reported by Jellett (1992) and using the two standard toxins of STX,TTX and two biological toxins of Ouabain,Veritridine, we successfully established the standard curves for determining the cytotoxicities of STX and TTX,Y=0.266X+51.184 and Y=1.6068X+47.186 with a detection limit of 5ng/ml and 0.8ng/ml ,respectively. Moreover, the toxin level of 19 samples of Nassarius from Zhoushan and Ganyu and 5 samples of A.tamarense kept in our lab were tested using our established methods. Results showed good correlation to those obtained by mouse bioassay and HPLC. Accordingly, based this detecting method was proved to be high-throughput, time-saving, and low detection limit, this method has the potential of application in coastal environment.