臺灣博碩士論文加值系統

黃樟素 (safrole) 是自然界之植物精油，常用於化妝品和食品之芳香添加劑，然而它已被證實為動物肝臟之致癌劑。其致癌的能力經由主要代謝活化物 1’-hydroxysafrole，於sulfotransferase存在下，形成不穩定的sulfuric acid ester，進一步和去氧核醣核酸 (DNA) 共價結合，形成穩定的鍵結物(safrole-DNA adduct)。研究証實DNA adduct是致突變與致癌的起始者 (initiator)。除此之外，在台灣，每克荖花中含有15 mg的 safrole，因此於嚼食含荖花檳榔塊者的唾液中可以偵測到高達420 microM的safrole濃度。本實驗室亦證明safrole-DNA adduct的存在與口腔癌的致癌機轉有關係。縱然如此，safrole-DNA adduct 是否直接或藉由其他途徑參與癌化的過程目前仍然不清楚。為了更深入的瞭解safrole的致突變機轉，本研究利用內源性酵素基因系統-次黃嘌呤-鳥糞嘌呤磷酸核醣基轉化基因(hypoxanthine-guanine phosphoribosyltransferase gene, hprt)，探討1’-hydroxysafrole對 hprt 基因核酸序列的改變有何特異性，藉以瞭解safrole對基因突變的機制。
本研究以0~1000 microM之1’-hydroxysafrole處理中國倉鼠卵巢細胞株 (CHO-K1) 和人類纖維母細胞株 (HFW) 24小時。首先利用磷32後標定法(32P-postlabeling)分析發現1’-hydroxysafrole在CHO-K1細胞株與HFW細胞株會產生鍵結物N2-(trans-isosafrol-3’-yl)deoxyguanosine；也發現1’-hydroxysafrole在CHO-K1及HFW細胞株可誘發不同程度之細胞毒性，其IC50分別為16.1與43.2 microM。而1’-hydroxysafrole 誘發CHO-K1細胞株hprt 基因突變之能力與產生safrole-DNA adduct之能力有相關性；且1’-hydroxysafrole處理CHO-K1細胞株篩選hprt基因突變株作RT-PCR，結果得知1’-hydroxysafrole誘發的23株獨立突變株，有19株為單一的鹼基取代。以G:C 鹼基對發生突變機率最高(16/19)，其中G to A轉換突變(transition)為9/19。顯示safrole對CHO-K1細胞株之hprt基因所造成的突變有特異性。綜合以上結果得知：safrole引起的DNA鍵結物會誘發基因突變。

Safrole, an essential oil that has been used in cosmetics and as a food flavoring agents, is classified as a rodent hepatocarcinogen. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, sulfonated to an unstable sulfuric acid ester, and consequently forming stable safrole-DNA adducts. DNA adduct is known to be an initial step of mutagenesis and carcinogenesis. In Taiwan, Piper betle inflorescence contains high concentration of safrole (15 mg/g fresh weight), and may contribute to human exposure (420 microM of safrole in saliva) while chewing betel quid with Piper betle inflorescence. We have documented that safrole has potential to form stable safrole-dG adducts in humans oral cancer tissue following betel quid chewing, which may contribute to oral carcinogenesis. However, the role of safrole-DNA adduct in cancer is not known. To gain insight into the mechanisms of safrole-induced mutations, this study has determined the types and location (spectrum) of mutations induced in the coding region of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene by 1'-hydroxysafrole, the reactive metabolite of safrole.
CHO-K1 and HFW cells were exposed to 1'-hydroxysafrole(0~1000 microM) for 24 h. Using 32P-postlabeling technique, this study demonstrated that safrole forms the major premutagenic lesion, N2-(trans-isosafrol-3’-yl)deoxyguanosine. The IC50 of 1'-hydroxysafrole in CHO-K1 and HFW cells were 16.1 and 43.2 microM, respectively. The 1'-hydroxysafrole induced safrole-DNA adduct level is significantly correlated to hprt mutation frequency induced by 1'-hydroxysafrole. hprt mRNA from mutant clones were reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. The result indicated 19/23 safrole-induced mutants contained a single base pair substitution. The majority of the mutations at G: C base pair occurred at sites with G in the nontranscribed strand (16/19). G:C to A:T transitions were the most common type of base pair substitution (9/19). The result reflected the specific types of DNA damage produced by safrole. Results from this study further demonstrated the relationship between safrole-DNA adduct and safrole-induced mutagenesis.

壹、 中文摘要………………………………………….……1
貳、 英文摘要………………………………………….……3
參、 背景介紹………………………………………….……5
肆、 研究動機與目的…………………………………...…16
伍、 實驗材料…………………………………………...…17
陸、 實驗方法………………………………………...……19
柒、 實驗結果…………………………………………...…26
捌、 討論……………………………………………...……30
玖、 參考文獻……………………………………...………36
壹拾、 圖表…………………………………………….……47

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