| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 106 === Tetrodotoxin (TTX) is a non-protein toxin with strong toxicity. It can major exist in organisms through accumulation of food chains. TTX is also found in other species such as goby, crab, flatworm and so on. There are several cases of food poisoning caused by food containing TTX every year in Taiwan. In recent years, due to climate change and the "Lessepsian migration", cases of pufferfish poisoning also happened in European countries. Symptoms of poisoning include oral paresthesia, vomiting, diarrhea, dyspnea and even death. To ensure food safety and contol, the toxicity and toxin distribution data of new pufferfish should be perfectly established. In addition, since TTX has the property of blocking sodium channels and preventing action potentials from being generated, it has been attempted to be developed as an anti-pain agent in recent years. Therefore, the apoptosis pathway and cytotoxicity induced by TTX should be determined for medical purposes in the future.
The first chapter of this study intends to identify the species and analyze the toxicity of the pufferfish from the market and the poisoning cases. Two pufferfishes were sparately identified as tetraodontidae Arothron stellatus and Arothron hispidus. In order to understand the content and distribution of toxins in these species, the toxins from different organs were extracted. The C18 Sep-Pak cartridge column was used for purification and TTX with higher purity was obtained. Then the quantitative and qualitative analysis of the toxins were carried out by bioassay and LC-MS/MS method. The results showed that there were several analogues of TTX in both species. The highest toxicity (257.39 μg/g) was found in the ovaries of Arothron stellatus and the most toxic organ is muscle (1.25 μg/g) in Arothron hispidus. The ratio of the quantitative results of the bioassay to the LC-MS/MS test ranged between 60 and 150%, indicating that the quantitative results of the two methods were similar.
The second chapter of the study followed the method of the previous chapter. After the toxin extracted from the liver of Arothron stellatus, it was purified and treated to mouse hippocampal neuron cell, HT-22 cells, to investigate the toxicological effects. The results showed that toxins could induce reactive oxygen species (ROS) and cause oxidative stress after the cells were cultured for three days at each concentration. Meanwhile, mitochondrial membrane potential (MMP) decreases with dose-dependent and finally causes cell apoptosis. Finally It was found that cells exhibited apoptosis at low concentrations of toxin, whereas the proportion of cell necrosis increased at high concentrations.
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