2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38645It has been estimated that, world wide, ~600000000 people chew areca nut. IARC (2004) has declared betel quid without tobacco as causing oral cancer and has stated that there is sufficient evidence in experimental animals for the carcinogenicity of areca nut. Arecoline and arecaidine are reported to be the most abundant alkaloids in the areca nut. However, little is known about the metabolism and disposition of arecoline and arecaidine in humans or in animals. Until very recently, Giri et al. (2006 and 2007) reported the metabolic map of arecoline and arecaidine in the mouse. The major metabolite of both arecoline and arecaidine appear to be N-methylnipecotic acid. In the meantime, endogenous nitrosation of arecoline leads to the formation of areca-specific N-nitrosamines (ASNAs) such as 3-(methyl-N-nitrosamino)propionitrile (MNPN), N-nitrosoguvacoline and N-nitrosoguvacine. It has been also reported that a major urinary metabolite of N-nitrosoguvacoline and N-nitrosoguvacine was identified as N-nitrosonipecotic acid, which could be used as a marker of ASNAs exposure. NMPN has been previously detected in the saliva of betel quid chewers (without tobacco) and shown to be the most powerful carcinogen in rats. The α-hydroxylation of MNPN is an important activation pathway mediated mainly by P450s, among which CYP2A6 was the most efficient activators of NMPN. This pathway yields methanediazonium ions which methylate DNA bases. As the N7 position of guanine is the predominant reaction site, N7-methylguanine (N7-MeG) is potentially the most useful marker of exposure to endogenous and exogenous methylating agents. In addition to areca nut alkaloids, areca nut polyphenols under alkaline conditions can release reactive oxygen species (ROS), which are capable of inducing nucleotides modification and the formation of cellular 8-oxo-7,8-dihydro-2’-deoxyguanosin (8-oxodGuo). This lesion could be repaired either by base excision repair (BER) pathway and nucleotide excision repair (NER) pathway, and the resulting repair products were 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxodGuo, respectively. Taken together, although these biomarkers play important roles for assessing exposures and risk from areca nut chewing, little effort has been made to develop analytical methods. Therefore, the aims of this project are to firstly develop a serial “on-line SPE LC-MS/MS methods” for sensitive and specific analysis of (i) arecoline, arecaidine and N-methylnipecotic acid (biomarkers for areca alkaloids exposure); (ii) 3-(N-nitrosomethylamino)propionitrile and N-nitrosonipecotic acid (biomarkers for ASNAs exposure); (iii) cotinine (biomarker for tobacco smoke exposure); (iv) N7-MeG, 8-oxodGuo and 8-oxoGua (biomarkers of effect) in body fluids. Subsequently, we are going to apply our newly developed biomarkers by measuring body fluids of betel quid chewers, and further validate the associations of these biomarkers with betel quid exposure. In addition, we will also examine whether habitual chewers with inherited metabolic genotypes (CYP2A6, GSTT1 and GSTM1) may experience increased alkylation and oxidative DNA damage.嚼食檳榔者在世界各地約有 6 億人口，它是繼吸煙、酒精及咖啡因之後全球第四大的 生活習性。2004 年IARC 已將單獨嚼食檳榔的行為(Betel quid without tobacco)及檳榔子 (Areca nut)皆列為人類致癌物Group 1 。儘管全球檳榔嚼塊的組成變化很大，檳榔子(areca nut)依然它們主要的共通點，其主成分中檳榔鹼(areca nut alkaloids)及多酚類化合物 (polyphenolic compounds)被視為重要致癌因子。儘管檳榔鹼的代謝對於研究檳榔暴露及其潛 在的致癌性十分重要；在過去的三十年來，人類對於檳榔鹼的代謝所知不多。直到最近Giri et al. (2006 and 2007)方才運用小鼠建立較完整的代謝地圖並證實N-methylnipecotic acid 是 arecoline 及arecaidine 兩者共同且最主要的代謝產物。檳榔鹼在體內可經亞硝化反應 (nitrosation)形成檳榔特有亞硝胺(Areca-specific N-nitrosamines, ASNAs)，檳榔特有亞硝胺的 主要代謝物係為N-nitrosonipecotic acid 。在檳榔特有亞硝胺(ASNAs) 中以 3-(methyl-N-nitrosamino)propionitrile (MNPN)的致癌性最強，CYP2A6 是活化MNPN 最主要 的P450 酵素；NMPN 經活化後可造成DNA 甲基化 (methylation)，而N7-methylguanine 是 最主要的甲基化鍵結產物。此外，多酚類成份在添加石灰的鹼性環境中(pH＞9.5)，很容易 產生反應性含氧物質(Reactive oxygen species, ROS)；這會在體內造成基因氧化傷害，而 8-oxo-7,8-dihydro-2’-deoxyguanosin (8-oxodGuo)為主要的細胞內基因氧化性傷害產物。細胞 內基因氧化傷害可被適時修復，其主要的修復路徑有二：一為鹼基移除修復(Base Excision Repair, BER)，其修復產物為8-oxo-7,8-dihydroguanine (8-oxoGua)；二為核酸切除修復 (Nucleotide Excision Repair, NER)，其修復產物為8-oxodGuo。 儘管上述生物偵測指標對於檳榔子成分的暴露或致癌機制研究深具意義但生物偵測方 法缺乏，尤其是針對檳榔鹼及檳榔特有亞硝胺的相關研究。本研究的目的在於運用連線固 相萃取(on-line solid phase extraction)搭配LC-MS/MS 及同位素稀釋法(isotope-dilution) 全面 且完整地建立「檳榔暴露及效應的生物標記分析方法」。接著運用於嚼食檳榔者之各式體液 分析，期以分子的角度探究檳榔子成分對人體的潛在危害，並瞭解「代謝基因多型性」所 扮演的角色。 96 年度國科會初步核給一年計畫進行「檳榔子之生物偵測研究(I)」，以建立「檳榔鹼暴 露」的生物標記分析方法。本計畫「檳榔子之生物偵測研究(II)」將接續完成「檳榔特有亞 硝胺暴露」及「效應型」生物標記的分析方法建立，並全面運用於嚼食檳榔者族群體液分 析。本計畫(II)執行時間共需三年：第一年著重於運用on-line SPE LC-MS/MS 搭配 isotope-dilution 完整開發檳榔特有亞硝胺暴露(MNPN 及N-methylnipecotic acid)之各式生物 偵測方法。第二年著重於運用on-line SPE LC-MS/MS 搭配isotope-dilution 完整開發各式效 應指標(N7-methylguanine、8-oxodGuo 及8-oxoGua)，並展開嚼食檳榔族群人體樣本收樣(含 控制組共300 位)。此外，在台灣大部分的嚼食檳榔者都有抽煙，為了釐清吸煙的共暴露問 題本研究也將建立cotinine 之on-line SPE LC-MS/MS 分析法。第三年將所有開發出的生物 偵測方法全面運用於嚼食檳榔者樣本分析，以期比較各種暴露及效應指標的相關性及適用 性，同時探究與嚼食檳榔罹癌風險有關之代謝基因多型性(CYP2A6、GSTT1 及GSTM1)對嚼 食檳榔者體內DNA 甲基化與氧化傷害的影響。液相層析串聯質譜儀連線固相萃取檳榔鹼檳榔特有亞硝胺DNA 甲基化DNA 氧化傷害代謝基因多型性LC-MSMSon-line SPEareca nut alkaloidsareca-specific N-nitrosaminesDNAmethylationoxidative DNA damagemetabolic polymorphismsProject