| 摘要: | 癌症位居國人死亡原因首位，尋求預防與治療癌症的藥物乃是當代醫學重要的課題。近年來的研究已證實化學致癌物或其衍生物可與DNA反應形成DNA鍵結物(DNA adducts)，未即時被修補的DNA adducts在細胞複製時可能會誘發基因突變，進而引發癌症。此DNA損傷與基因突變已被公認為化學致癌的起始因子，基於化學預防DNA損傷與基因突變在減緩癌症發生的重要性，化學預防中藥的篩選與及機制的探討為一重要課題。本計畫建立了化學致癌物誘發DNA損傷與基因突變之動物模式，以便應用於研究可降低DNA損傷與基因突變進而用於抗癌防癌的中藥。首先採用中藥萃取出來的多酚類化合物單離成分茶多酚Epigallocatechin gallate (EGCG)和并沒食子酸Ellagic acid (EA)為試驗樣品進行體內測試，採用化學致癌物乙基亞硝酸基尿素(N-Ethyl N-nitrosourea, ENU)誘發老鼠為實驗模式，以致癌機轉中早期的DNA損傷與基因突變為指標，探討不同濃度的EGCG與EA對化學致癌劑ENU誘發老鼠組織DNA傷害與基因突變是否具有保護作用。小鼠經腹腔注射化學致癌物ENU作為陽性對照組，另外兩組小鼠經胃管餵食不同劑量的EGCG或EA，連續給于七天後再投于 ENU。一批老鼠在投于ENU之二小時後犧牲，取出肝臟以萃取DNA，用新發展完成之高敏感度與專屬性的氣相層析儀加質譜儀(GC/MS)方法定量分析 ENU誘發的DNA adduct 7-ethylguanine (7-EG)。另外一批老鼠在投于ENU之一個月後犧牲，取出脾臟分離淋巴細胞，以T-cell cloning方法培養在選擇性試劑(6-Thioguanine)中十天，以計算Hprt基因的突變機率。結果顯示陰性對照組(只餵水)老鼠肝臟組織的 7-EG的量很低，低於我們方法偵測的極限(<1.0 pmol/μmol guanine)。30 mg ENU/kg處理組老鼠肝臟組織的7-EG為53±9 pmol/μmol guanine，顯著高於陰性對照組老鼠。若以200 mg/kg與100 mg/kg EGCG前處理老鼠，其肝臟組織的7-EG分別為19±4與21±19 pmol/μmol guanine，明顯低於ENU處理組老鼠(P<0.05)。以200mg/kg與100 mg/kg EA前處理組老鼠肝臟組織的7-EG分別為31±10 pmol/μmol guanine(P<0.05)與54±9 pmol/μmol guanine (P>0.05)。此結果顯示EGCG與EA對ENU誘發的DNA傷害有拮抗的能力，但EGCG的抗DNA損傷的效果明顯比EA好。在基因傷害方面，ENU(30,60 and 90 mg/kg)處理老鼠脾臟淋巴細胞之Hprt突變率分別為36±15*10/sup -6/, 63±12*10/sup -6/與114±20x10/sup -6/，顯著高於陰性對照組老鼠的2.0±0.3*10/sup -6/ (P<0.001)，同時呈現劑量反應關係。而以100 mg/kg和200 mg/kg EA或EGCG連續餵食老鼠七天，平均Hprt突變率為2.0±1.0*10/sup -6/ (P>0.05)，與陰性對照組老鼠比較並無顯著差異，顯示EA和EGCG於本實驗所使用之劑量對老鼠並不具致突變性。以25 mg/kg、100 mg/kg或200 mg/kg EGCG前處理的老鼠可明顯降低ENU (60 or 30 mg/kg)誘發的脾臟淋巴細胞的hprt突變機率，對ENU致突變性的抑制率分別為38% (P<0.05)、53% (P<0.05)與62% (P<0.05)。而100 mg/kg或200 mg/kg EA前處理組老鼠亦有顯著降低Hprt基因傷害的趨勢，hprt突變機率的抑制率分別為34% (P<0.05)與44% (P<0.05)。綜合以上的結果，EGCG與EA對ENU所誘發的DNA損傷與hprt基因突變有明顯的拮抗作用，即有抗基因毒性的功能，目前正著手研究EGCG與EA是否藉著增強老鼠體內去毒能力、增加抗氧化能力或者強化DNA修補的能力而產生抗DNA損傷與基因突變的作用。本計畫利用中藥抽提物之單離成分以驗證此基因傷害與突變的動物模式，未來將繼續利用這個動物模式來探討各種中藥單劑與複方之抗癌效果與其作用機制。
Cancer is the leading death cause in Taiwan. It becomes an urgent issue to search for medicines against cancer. Recent studies have demonstrated that genotoxic chemicals or their derivatives can interact with DNA to form DNA adducts, which may cause mutations during DNA replication and subsequently lead to cancer development. DNA adducts and mutations are considered as early indicators in the pathogenesis of carcinogenesis. Medicines that could inhibit the formation of DNA adducts and mutations might suggest their potential in cancer prevention. In this study, we developed an animal model to investigate the protective effects of the ingredients of Chinese herbal medicines on genotoxicant-induced DNA damage and mutations. Ellagic acid (EA) or (-)-epigallocatechin gallate (EGCG), isolated ingredients from Chinese herbal medicines, are known to possess anti-carcinogenic effects in rodents. However, more studies are needed to understand their underlined mechanisms. Mice were orally fed with EA or (-)-epigallocatechin gallate (EGCG) for seven days before challenging with genotoxicant N-ethyl nitrosourea (ENU). Two hours after ENU administration, mice were sacrificed, and the liver DNA was extracted for analysis of DNA adduct (7-ethylguanine) by using highly sensitive and specific gas chromatography/mass spectrometry (GC/MS). Another sets of mice were sacrificed 4 weeks after ENU administration; the splenic lymphocytes were isolated and cultured in the presence of the selective agents 6-thioguanine. The mutant colonies were counted to determined hprt mutation frequency. The level of 7-EG in the liver tissues of 30 mg/kg ENU-exposed mice was 53±9 pmol/μmol guanine, much higher than that in control mice (<1.0 pmol/μmol guanine). Pretreatment with EGCG (100 and 200 mg/kg) or EA (200 mg/kg) significantly reduced ENU-induced 7-EG in mouse liver tissues (P<0.05). Treatment with 30, 60, and 90 mg/kg ENU in mice induced the hprt mutation frequencies of 36±15*10/sup -6/, 63±12*10/sup -6/ and 114±20*10/sup -6/, respectively, as compared to the control (2.0±0.3x10/sup -6/). Pretreatment with EGCG (25, 100 and 200 mg/kg) or EA (100 and 200 mg/kg) significantly reduced ENU-induced hprt mutations in mice (P<0.05). Our data showed that EA and EGCG could reduce ENU-induced DNA damage and hprt mutations, suggesting that the anti-carcinogenic effects of EA and EGCG might at least partly result from this mechanism. We successfully used the isolated ingredients of Chinese herbal medicines (EA or EGCG) to establish this animal model. Future studies will be extended to the anti-carcinogenic effects of single or mixed Chinese herbal medicines. The development of this animal model will provide a great opportunity to study the anti-genotoxic mechanisms of Chinese herbal medicines in animal. |
