Glutathione S-transferase (GST)是mammalian細胞中的一重要解毒(detoxification)酵素，而GST亦為dimeric cytosolic isoenzyme:包含了alpha、pi、mu、theta、zeta與sigma等6種。之前文獻指出，GST會將xenobiotics與glutathione (GSH)進行conjugation，使得這些偏非極性的xenobiotics的水溶性上升，而能順利排出體外；另一方面，GST也具有抗氧化功能故能避免lipid或核酸受到攻擊。因此GST在細胞中的解毒或防止癌症發生的機制中應扮演一重要的角色。之前關於GST mu的研究有許多方面都是探討mu class與癌症發生機率的關係，更有人認為GST mu可以列為膀胱癌的預測指標(marker)。又因為GST mu在肝細胞受致癌物質刺激下其表現會增加，故我們想觀察當GST mu overexpression時，對肝細胞抵抗致癌物所產生的肝毒性、基因毒性的情形以及對於肝細胞抵抗癌化的指標意義。
本次實驗是利用大白鼠初代培養之肝細胞為實驗工具，並建立短暫轉殖gst系統(Transient transfection system)，來觀察在肝細胞受Aflatoxin B1攻擊下rGST mu (Yb1)對肝細胞的保護情形。結果初步發現當轉殖入rGST mu (Yb1)後，Aflatoxin B1對肝細胞的傷害的確受到抑制。其中在GST activity的表現上，不論是轉殖rGST mu或是空的載體，其GST activity皆隨Aflatoxin B1處理濃度升高而呈現dose-dependant的提升；只是有轉殖rGST mu的組別不論做何種情況處理，其GST activity皆高於只轉殖空的載體的組別之外，GST activity還出現可能被Aflatoxin B1所induced的情況。在AST (aspartate amino transferase) activity的表現情況，當轉殖了rGST mu後其AST activity會隨著Aflatoxin B1的濃度升高而逐漸降低；而只轉殖空的載體(pcDNA3)時，AST activity反而逐漸升高(以0.01mM Aflatoxin B1處理12小時後達到最高)。以0.01mM的Aflatoxin B1處理12小時後，轉殖了rGST mu與轉殖空的載體其AST activity的差距最大。最後在LDH (lactate dehydrogenase) activity方面也是如此，轉殖了rGST mu並處理Aflatoxin B1處理12小時後，LDH會呈現一dose-dependant的下降趨勢而轉殖空的載體反而呈現了一上升的局面；又以0.01mM Aflatoxin B1處理12小時後LDH activity達到最高。另外以0.01mM的Aflatoxin B1處理12小時後，轉殖了rGST mu與轉殖空的載體其LDH activity的差距最大。
因此經Transient transfection rGST mu的大白鼠初代肝細胞對Aflatoxin B1的抵抗性的確有提升的表現，至於對Aflatoxin B1的基因毒性的影響、轉殖的rGST mu與Aflatoxin B1間更詳細的交互關係
與其影響癌化能力的則尚待進一步的探討。
Glutathione S-transferase (GST), an important detoxification dimeric enzyme in mammalian cells, includes six isoforms: alpha, pi, mu, theta, zeta and sigma classes. Previous investigations showed that GST would conjugate xenobiotics with the sulfhydry group of GSH (glutathione) to increase the solubility of these xenobiotics. On the other hand, GST has anti-oxidation function, which protect lipid or nucleic acid from the attack of toxins. Therefore, GST plays an important role in the mechanism of detoxification and anti-carcinogenesis. Among the isoforms, GST mu is thought to be related to cancer susceptibility. It is also suggested that GSTM1 could serve as a predictive marker for invasive bladder cancer. After the treatment of carcinogens, the expression of GST mu would increase in the hepatocytes. In order to clarify the role of GST mu, a transient expression system of GST mu was set up to study how the overexpression of GST mu affects the hepatotoxicity and genotoxicity of carcinogen.
We used the primary cultured hepatocytes of SD rat to establish the transient transfection system of rGST mu (Yb1), and challenged the cells with Aflatoxin B1. We found that the transfected rGST mu would attenuate the damage of hepatocytes caused by Aflatoxin B1. The treatment of Aflatoxin B1 for 12hr increased the cytosolic GST activity in both GST and control vector-transfected cells in a dose-dependent manner. The GST activity in the cells transfected with rGST mu was higher than those transfected with pcDNA3. The releases of AST and LDH activity, from the Aflatoxin B1-treated cells were inhibited by the transfection of rGST mu as compared to the control.
