Analysis of DNA adducts in mice treated with safrole 2'',3''-oxide

黃樟素為自然存在之物質，為黃樟油的主要成分，也存在於許多香料中，如：羅勒、荳蔻、肉桂、黑與白胡椒…等，一般人在日常生活中可能會暴露到黃樟素。黃樟素進入人體後會被代謝成四種主要代謝物，而其中2'',3''- 環氧黃樟素(SFO)為黃樟素的親電子代謝物質之一，前人研究嘗試以32P-後標籤法分析，無法在暴露SFO的小鼠肝臟組織內分析到SFO在體內生成的DNA共價鍵結物，因此在過去一直被認為不具有基因毒性，然而，其結構與已知的動物致癌物環氧苯乙烷(Styrene-7,8-oxide)相似，此外，根據先前研究顯示，暴露SFO會造成小鼠紅血球細胞中的微核(micronuclei)增加以及細胞的DNA鍊斷裂，而我們實驗室先前已證實SFO會在體外與DNA形成共價鍵結物，經分析尿液中鹼基共價鍵結物證明SFO會誘發小鼠形成該共價鍵結物，但動物體內組織的鹼基共價結物還有待研究。因此本論文的目的在於探討環氧黃樟素在動物體內組織產生DNA共價鍵結與劑量間的關係，此結果將有助了解環氧黃樟素在動物體內造成基因毒性的機制。 本研究利用腹腔注射CD-1品系之雌性小鼠進行持續四週的重覆暴露實驗，分成七組：控制組(olive oil)、黃樟素低高劑量組(150, 300 mg/kg)以及環氧黃樟素四個劑量組(30, 60, 90, 120 mg/kg)的環氧黃樟素劑量，接著取出器官組織進行DNA萃取，並使用液相層析搭配電噴灑離子化串聯式質譜儀進行分析。我們成功地在環氧黃樟素處理組小鼠的肝臟組織DNA與尿液中分析到N7γ-SFO-Gua，此共價鍵結物含量隨環氧黃樟素劑量增加而增加，但多數的鹼基共價鍵結物經尿液排出。 這些結果暗示了環氧黃樟素形成共價鍵結物後，經去嘌呤化的機制使DNA產生缺鹼基位置(apurinic/apyrimidinic site, AP site)而導致環氧黃樟素具有致突變性，這也是國際上首度有研究顯示SFO在活體內會產生DNA共價鍵結物。Safrole (1-allyl-3,4-methylenedioxybenzene), the main component of the sassafras oil, is commonly present in plants such as anise, cinnamon, basil, nutmeg, and pepper. People could be exposed to safrole in their daily life. Safrole-2’,3’- oxide (SFO), an active metabolite and an electrophile of safrole, is suspected to be responsible for genotoxicity and mutagenicity in Salmonella typhimurium strains TA1535 and TA100. In a previous study, scientists tried to analyze SFO-induced DNA adducts by using 32P-postlabeling method. Nevertheless, they failed to analyze any SFO-induced DNA adducts in liver tissue of mice. Therefore, SFO was not considered as a genotoxic carcinogen. However, the structure of SFO is similar with that of styrene-7,8-oxide, an animal carcinogen, and recent studies have shown that SFO can induce cytotoxicity, DNA strand breaks, micronuclei formation both in vitro and in vivo. Recently, our lab verified that SFO could cause in vivo formation of N7γ-SFO-Gua, which might then be rapidly depurinated from the DNA backbone and excreted through urine. In the present study, we aimed to further investigate SFO-induced DNA adducts in animal tissue and in HepG2 cells. Analysis of SFO-induced DNA adducts not only confirms SFO genotoxicity, but also serves as risk-associated biomarkers for cancers. Therefore, the objective of this study was to determine the SFO-induced DNA adducts in HepG2 cell and tissues of mice-treated with SFO by using an solid-phase extraction liquid chromatography/tandem mass spectrometry method. N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine(N7γ-SFO-Gua), N1-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)adenine(N1γ-SFO-dAdo) were first measured in HepG2 cells treated with 250 and 375 μM SFO. Female CD-1 mice were repeatedly treated with 150 and 300 mg/kg/day of safrole and 30, 60, 90, and 120 mg/kg/day of SFO through ip injection for continuous 28 days. N7γ-SFO-Gua in liver DNA and urine samples were further analyzed. Results show dose-dependent increases in N7γ-SFO-Gua contents in liver and urine and demonstrate that SFO indeed caused formation of DNA adducts, which probably depurinated from DNA backbone and suggested the genotoxicity of SFO in mice.口試委員會審定書 i 誌謝 ii 摘要 iii Abstract iv 目錄 vi 圖目錄 viii 表目錄 x 第一章 前言 1 1.1 黃樟素(Safrole)簡介 1 1.1.1物理化學特性 1 1.1.3黃樟素的代謝物 2 1.1.4環氧黃樟素以及前人動物實驗數據 4 1.2 去氧核醣核酸(DNA)與DNA共價鍵結物(DNA adducts)簡介 4 1.2.1 DNA共價鍵結物的修復與癌症的關係 4 1.2.2 環氧黃樟素致癌機制的相關研究 5 1.2.3 DNA共價鍵結物的分析方法 6 1.3 同位素稀釋質譜法 7 1.4 研究設計背景 8 1.5 研究目的 9 第二章 實驗材料與方法 10 2.1 樣本來源 10 2.1.1化學標準品 10 2.1.2實驗動物 10 2.2 實驗架構 11 2.3 動物實驗處理 14 2.4 DNA共價鍵結物實驗方法 15 2.4.1組織與細胞樣本前處理 15 2.4.2尿液樣本前處理 17 2.5 液相層析串聯式質譜儀分析方法 18 2.6 分析方法之驗證 18 2.6.1 DNA基質校正曲線配製與結果 18 2.6.2 尿液基質校正曲線配製與結果 19 2.6.3組織DNA基質效應、回收率、過程效率 19 2.6.4同日及異日檢量線之再現性 21 2.7 鳥糞嘌呤定量 22 2.7.1酸水解 22 2.7.2高效能液相層析儀分析方法 22 第三章 結果 24 3.1 動物體外細胞實驗 24 3.2 動物實驗 24 第四章 討論 26 4.1 綜合結果討論 26 4.2 研究限制 28 4.3 結論 28 參考文獻 2