Risk Assessment of Seafood Safety on Tetrodotoxin by Using Animal Cells Model

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基本資料

Project title

Code/計畫編號

MOST107-2314-B019-002

Translated Name/計畫中文名

以動物細胞模式探討河魨毒之食品安全風險評估

Project Coordinator/計畫主持人

Deng-Fwu Hwang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=12670827

Year

2018

Start date/計畫起

01-08-2018

Expected Completion/計畫迄

01-07-2019

Bugetid/研究經費

1030千元

ResearchField/研究領域

食品科技(農)

河魨毒 (tetrodotoxin; TTX) 是種毒性強烈的非蛋白毒素，可藉由食物鏈累積的方式存在於生物中。國內外每年均有食用含 TTX 所引發之食物中毒案例發生，為確保食品安全管控，各物種河魨之毒性和毒素分布資料應予以建立。另外，由於 TTX 具有阻塞鈉離子通道之特性，近年來嘗試被開發成抗痛藥劑，因此不同細胞暴露於 TTX 後所引發之凋亡路徑和細胞毒性也應被確立，以助於日後做為醫療用途參考。本研究擬自宜蘭大溪外海捕獲之河魨進行毒素萃取，經外觀形態學與基因鑑定後，確認此河魨屬於四齒魨科魚種星斑叉鼻魨 Arothron stellatus，此物種河魨之毒性至今未明，為了解其體內毒素的含量與分布，將其解剖取不同器官，利用 C18 Sep-Pak cartridge column 進行 TTX純化，得到較高純度之 TTX，然後以生物試驗法檢測各部位之毒性，接著以 LC-MS/MS 做毒素之定量與定性及毒成分分析，並且比對兩種方法之差異性。自河魨肝臟萃取毒素後純化，並以此進行 TTX 對於小鼠神經纖維母細胞株 (mouse neuroblastoma cell line, Neuro-2a) 毒性作用之探討。將細胞暴露於 TTX 後，測定其氧化壓力、粒線體損傷情形以及內質網壓力變化，亦測定凋亡路徑中 caspase 活性及凋亡相關蛋白的表現量，期望建立 TTX 毒素之細胞死亡路徑相關特徵表現。由於日前鮮少有 TTX 對於腸道細胞毒性作用的研究，本研究最後一部分將探討 TTX 對於人類結腸癌上皮細胞 (human colonic epithelial cell; Caco-2) 之毒性作用，以不同濃度的 TTX 暴露於 Caco-2 細胞，利用 MTT 檢測細胞存活率，並測定其乳酸脫氫酶 (lactate dehydrogenase; LDH) 活性，另外，測定暴露毒素後細胞內氧化壓力、粒線體損傷情形等，以及凋亡相關蛋白表現量，比較 TTX 對於神經與腸道細胞作用機制的異同。期望藉由了解 TTX 作用於細胞所引發的凋亡途徑和細胞毒性，能有助於日後 TTX 作為治療神經傷害之疾病或抗痛劑等醫療用途參考。 Tetrodotoxin (TTX) is a marine toxin in Taiwan, which caused many poisoning accidents by ingesting seafoods in recent years. In this study, the toxicity and toxic components of puffer Arothron Stellatus is not established until now. It needs investigation to prevent from food poisoning cases. The species from the eastern coast of Taiwan has been collected and now is analyzing their toxicity and to establish the DNA data base for further comparison with other puffer data. The toxicity is determined by using mouse bioassay and HPLC-MS/MS. The reliable standard TTX from this puffer is gotten from purification and used for cell toxicity test. Furthermore, TTX is considered to use as medicine to heal the pain recently. Hence, the toxic mechanism of low dose of toxin on different human cells are necessary to study early. In this year, the different human or animal cells such as neuron cell and intestine cell are used to examine the cell toxicity of low dose of TTX. These results are useful to make sure the safety of seafood and those toxins applied as medicinal drug. These cell toxicity tests low TTX exposure whether to disrupt neuronal and intestinal cell differentiation process throught increasing intracellular oxidative stress, protein carbonyl level and redicing total thiol content, superoxide dismutase (SOD), and catalase activity. Apoptotic and inflammatory effects of TTX on neuronal and intstinal cells will be established and these results are useful to make sure the safety of seafood and toxin could be applied as medicinal drug.

Keyword(s)

河魨毒
LC-MS/MS
細胞毒性
凋亡機制
Tetrodotoxin
LC-MS/MS
Cytotoxicity
Apoptosis

DSpace CRIS

Risk Assessment of Seafood Safety on Tetrodotoxin by Using Animal Cells Model

基本資料

Description