Study the Inhibition Effects of Toona sinensis Extracts in Hepatocarcinogenesis in Rats and Human Hepatoma Cells (HepG2 cells) and Mutagenicity

香椿（Toona sinensis）屬楝科多年生落葉喬木，自古以來香椿的根、皮及葉普遍為民間常見的保健藥材。已知香椿具有抗發炎、止痛、抑制血小板凝集、改善生殖細胞活性、降低大鼠血糖等保健功效，目前更有研究指出香椿具抗肺癌及卵巢癌作用，或透過抗氧化的路徑誘導人類的白血病細胞（HL60）產生細胞凋亡的現象。本研究以香椿水煎煮液的乾燥物(TSL-1)為樣本，進一步瞭解香椿是否也具抗肝癌的作用，在In vivo方面，利用DEN及2AAF快速誘發大鼠肝癌的動物模式進行評估，試驗分成對照組、陽性對照組及TSL-1處理組，第0天時腹腔注射 200 mg/kg DEN，第2週時，陽性對照組於飼料中添加200 ppm 2AAF，TSL-1處理組除2AAF外，同時每日再以餵管餵食方式投予0.1、0.5及1 g/kg TSL-1，第3週施以1/3部分肝切除手術，經八週後，進行血清生化學檢查及組織病理學檢查、測定γ-GT、GST-P前癌酵素含量及抗氧化活性等分析，藉以評估TSL-1對於抑制大鼠肝腫瘤作用。結果顯示，TSL-1可有效減緩大鼠肝腫瘤的病變，血液中AST、ALT及γ-GT，及肝臟組織中γ-GT陽性面積及GST-P的活性均有顯著性降低 (p<0.05)。另外，TSL-1降低肝臟脂質過氧化物質MDA的產生及增加抗氧化物質GSH及SOD的活性(p<0.05)，推測可能與香椿具有強抗氧化特性有關。在In vitro方面，利用MTT assay及靛藍氏染色法探討香椿對肝癌細胞之細胞毒性作用。結果顯示，TSL-1對肝癌細胞具有明顯之細胞毒性， 12小時半致死濃度 (LC50) 為0.7 mg/ml。利用流式細胞儀及Hoechst stain等方法進行探討香椿水萃取液對HepG2細胞作用的機制路徑。結果顯示，TSL-1雖有增加HepG2 cell細胞凋亡的發生率，但仍以細胞壞死發生比率較高，推測可能為直接造成肝癌細胞死亡。另外，探討TSL-1是否具有抑制致癌劑或致基因突變劑的作用，而達到抑制肝細胞癌化的效果。以沙門氏菌回復突變測試法 (Ames test)進行TSL-1抗致突變性試驗，由實驗結果可知，TSL-1在2.5 mg/plate濃度下，不論有無經過肝酵素(S9)代謝，可抑制2AAF誘發Sal. typhimuriumTA98及TA100突變率達70%以上。綜合以上的結果，TSL-1具抑制2AAF誘發大鼠肝腫瘤作用，其作用可能與其具有抗氧化及抑制基因突變途徑有關，但其明確的作用機制，仍有待進一步探討。Toona sinensis Roem is a perennial deciduous tree of the family Meliaceae. Almost all part of T. sinensis, including seeds, bark, root bark, petioles, and leaves, show medicinal effect. Leaves of T. sinensis exhibite anti-inflammatory, anti-doting, lowing plasma glucose in diabetic rats. In addition, it also improves the activity of human sperm, and induces apoptosis of leukemia cells. The aim of this study was to evaluate the inhibition effect of TSL-1 in chemical-induced hepatoma in rats, and the cytotoxicity in HepG2 cells and the anti-mutagenicity in Ames test. In vivo assay, rats were intraperitoneally injected a single dose of diethylnitrosamine (DEN) (200 mg/kg) on the first day, then given acetylaminofluorene (2-AAF) 200 mg/kg in diet on week 2, and supplemented with TSL-1(0.1, 0.5 and 1 g/kg) for 8 weeks. A one-third hepatectocy was performed on the third week in all rats. Results revealed that TSL -1 significantly inhibited the degrees of 2AAF-induced hepatoma in rats, decreased the elevation of AST, ALT and GGT in serum, lessened the area of preneoplastic GGT foci and glutathion S-transferase placental (GST-P) in liver tissuse and lowered the lipid peroxidation of malondialdehyde (MDA), but increased the antioxidant activities of GSH and SOD in liver homogenous tissues. In vitro, the cytotoxicity of TSL-1 toward HepG2 cells was measured by using the MTT and Trypan blue assays. The results showed that TSL-1 had a dose-related cytotoicity effect on HepG2 cells, and the LC50 of TSL-1 was 0.7 mg/ml in the 24 hours incubation. Altnough TSL-1 could induce apoptotic effect on HepG2 cells but mainly via necrosis pathway. Furthermore, the antimutagenicity of TSL-1 was determined in the Ames test of TA98 and TA100. Results that TSL-1 exhibited more than 70% inhibition of AAF-induced mutagenicitiys in TA98 and TA100. In conclusion TSL-1 could inhibit the degree of 2AAF-induced hepatoma in rats, and might be related to its strong antioxidant and anti-genetic activities. Howerver, the real mechanism of TSL-1 needs to eluadate in the future目 錄 頁次 中文摘要 ---------------------------------------------------------------------------- Ⅰ 英文摘要 ---------------------------------------------------------------------------- Ⅱ 目錄 ---------------------------------------------------------------------------- Ⅲ 圖目錄 ---------------------------------------------------------------------------- Ⅳ 表目錄 ---------------------------------------------------------------------------- Ⅵ 第一章 前言 -------------------------------------------------------------------- 1 第二章 文獻探討 -------------------------------------------------------------- 2 第一節 香椿之文獻資料 ----------------------------------------------------- 2 第二節 肝癌之文獻資料 ----------------------------------------------------- 6 第三節 香椿抑制肝癌細胞生長之研究 ----------------------------------- 9 第四節 生物體內抗氧化防禦系統 ----------------------------------------- 14 第五節 抗致突變性及基因毒性 -------------------------------------------- 18 第三章 研究目的及實驗設計 ----------------------------------------------- 24 第四章 材料與方法 ----------------------------------------------------------- 26 第一部分 快速誘導肝癌模式探討香春對肝癌的抑制效果 -------------- 26 第二部分 香椿水萃取物對人類肝癌細胞株(HepG2 cell)的抑制效果 - 37 第三部分 抗致突變性試驗 ----------------------------------------------------- 44 第五章 結果 -------------------------------------------------------------------- 48 第六章 討論 -------------------------------------------------------------------- 84 第七章 結論 -------------------------------------------------------------------- 95 參考文獻 ---------------------------------------------------------------------------- 9

Subchronic Immunotoxicity Assessment of Genetically Modified Virus-Resistant Papaya in Rats

Papaya is an important fruit that provides a variety of vitamins with nutritional value and also holds some pharmacological properties, including immunomodulation. Genetically modified (GM) papaya plants resistant to Papaya ringspot virus (PRSV) infection have been generated by cloning the coat protein gene of the PRSV which can be used as a valuable strategy to fight PRSV infection and to increase papaya production. In order to assess the safety of GM papaya as a food, this subchronic study was conducted to assess the immunomodulatory responses of the GM papaya line 823-2210, when compared with its parent plant of non-GM papaya, Tainung-2 (TN-2), in Sprague-Dawley (SD) rats. Both non-GM and GM 823-2210 papaya fruits at low (1 g/kg bw) and high (2 g/kg bw) dosages were administered via daily oral gavage to male and female rats consecutively for 90 days. Immunophenotyping, mitogen-induced splenic cell proliferation, antigen-specific antibody response, and histopathology of the spleen and thymus were evaluated at the end of the experiment. Results of immunotoxicity assays revealed no consistent difference between rats fed for 90 days with GM 823-2210 papaya fruits, as opposed to those fed non-GM TN-2 papaya fruits, suggesting that with regard to immunomodulatory responses, GM 823-2210 papaya fruits maintain substantial equivalence to fruits of their non-GM TN-2 parent