臺灣博碩士論文加值系統

中文摘要

根據流行病學報告指出，既抽煙又嚼檳榔的人得口腔癌的機率是一般人的89倍。因此，本研究欲探討檳榔中添加石灰所造成的鹼性環境是否能夠促進香菸致癌物質的吸收，進一步造成加成性的影響。4-(Methylnitrosamino)-1-
(3-pyridyl)-1- butanone (NNK)在動物模式中是個強力的致癌物，而且在體內快速代謝成代謝產物4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL)。另外，NNK經由cytochrome P450代謝活化後會對DNA造成傷害。因此，本論文將NNK塗抹在倉鼠口腔頰囊上，利用液相層析串聯式質譜儀定量尿液中
NNAL含量，藉此評估NNK在不同酸鹼環境下（pH 7.0與8.7）的吸收情形。此外，也會定量尿中氧化性傷害指標8-hydroxy-2’-deoxyguanosine (8-OHdG)及DNA甲基化產物7-methylguanine（N7-MeG）。NNAL、8-OHdG和N7-MeG首先經由固相萃取法萃取，再利用液相層析儀分離，最後由電灑游離原質譜儀進行定量。NNAL、8-OHdG和N7-MeG之回收率在94~116％之間，三者之線性濃度範圍分別為0.05~50 ngml-1、0.05~20 ngml-1、0.5~20 ngml-1。NNAL與8-OHdG的偵測極限為濃度0.01 ngml-1，定量極限則為濃度0.05 ngml-1；至於
N7-MeG的偵測極限及定量極限分別為濃度0.01 ngml-1及0.5 ngml-1。因此，本實驗可以準確的利用液相層析串聯式質譜儀去定量NNAL、8-OHdG和N7-MeG這三個生物標記。實驗結果顯示，鹼性環境下（pH 8.7）尿中NNAL含量明顯較正常酸鹼值下（pH 7.0）來的高，代表著鹼性環境可以幫助NNK的吸收。此外，在pH 8.7下尿中8-OHdG和N7-MeG增加量亦較pH 7.0來的高，這樣的結果顯示出NNK在鹼性環境下可能會造成較大的DNA氧化性傷害及甲基化損害。

Abstract

Epidemiological studies revealed that the incidence of oral cancer was 89-fold higher in people who smoked and chewed betel quid than those without such habits. Therefore, this study was designed to test whether alkaline environment generated by chewing betel quid containing lime facilitate the absorption of cigarette smoke containing carcinogens and consequently contribute to the synergistic potential. 4-(Methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potent tobacco-specific carcinogen in animals and is rapidly reduced to its main metabolite, 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL). NNK is also metabolically activated by cytochromes P450 to produce DNA damage. In this study, NNK was applied to hamster buccal pouch at different pH (pH 7.0 and 8.7) and utilized the liquid chromatography/ tandem mass spectrometry (LC/MS/MS) to quantify urinary NNAL and try to test the absorption of NNK. Urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) and 7-methylguanine（N7-MeG） were also determined, which are biomarkers for oxidative DNA damage and methylation lesion, respectively. NNAL and 8-OHdG were isolated by solid-phase extraction (SPE), separated by liquid chromatography and quantified by electrospray ionization mass spectrometry. The recovery of NNAL, 8-OHdG and N7-MeG were in the range of 94 to 116 ％. The linearity of these three analytes ranged from 0.05 to 50 ngml-1, 0.05 to 20 ngml-1 and 0.5 to 20 ngml-1, respectively . Limit of detection (LOD) was 0.01 ngml-1 and that of quantification (LOQ) was 0.05 ngml-1 for NNAL and 8-OHdG. LOD and LOQ of N7-MeG approached 0.01 and 0.5 ngml-1, respectively. This accurate LC/MS/MS method has potential to determine three biomarkers, such as NNAL, 8-OHdG and N7-MeG. These data implied that urinary NNAL was significantly higher under the alkaline condition (pH 8.7) than that in normal pH (pH 7.0), indicating that the absorption of NNK is facilitated in alkaline environment. Moreover, the level in urinary 8-OHdG and N7-MeG were also higher at pH 8.7 than at pH 7.0, demonstrating that NNK might induce more oxidative DNA damage and methylation lesion under the alkaline condition. Overall, these findings indicated that alkaline environment generated by chewing betel quid containing lime enhances the absorption of tobacco carcinogen NNK.

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