Effects of Genetic Polymorphisms of the Human PNP, CYT19 and GSTO1 on Individual Variation of Urinary Arsenic Metabolites after Oyster Ingestion

摘要 一般人暴露到砷的主要途徑為食物與飲用水。一般而言，無機砷的毒性較有機砷的毒性大；目前無機砷的代謝作用方面也有較多且完整的研究。有關人體對有機砷的代謝機制方面的研究並不多，其詳細的代謝機轉仍不清楚。然而，無機砷和有機砷的代謝作用在個體間確實有變異性的存在；影響這些變異性的因子包括年齡、性別、種族、砷物種的形式、環境因子和基因多形性等。本研究的目的主要是探討人體在食用牡蠣後，PNP, CYT19和GSTO1基因多形性對人體代謝有機砷的速率所可能產生的影響，以及和尿液中砷代謝物質濃度的變化情形之間的相關性。 本研究的51位受試者，分別來自過去進行的三個飲食控制實驗。受試者在這為期七天的飲食控制實驗中，除了第四天進食所提供的海產類食物之外，其餘時間皆限制其攝食任何水生類動植物及其相關製品。同時收集受試者連續七天早晨的第一泡尿液，分析尿中As5+、As3+、DMA、MMA濃度。進一步收集受試者的血液樣本，針對PNP, CYT19和GST1基因，進行單核甘酸多形性(Single nucleotide polymorphisms, SNPs)分析。 結果顯示，DMA是尿液中最主要的砷代謝物種，其平均濃度在食用牡蠣後的第一天顯著地上升到最高值(31.1±15.2μg/L)，之後則逐漸下降。As3+濃度的變化也有類似的情形發生。依據尿中DMA濃度在食用牡蠣後的第一天、第二天或第三天達到最高值，將受試者的代謝速率分成快、中、慢三組，各組分別有28人、19人及4人。由此可知，不同個體之間對有機砷的代謝速率有所差異。 本研究發現，DMA代謝速率最快的受試者，其平均年齡(28.3±1.0歲)顯著大於DMA代謝速率最慢的受試者(25.7±1.6歲)。另外，CYT19基因 intron6上點位發生的SNP (G > C)突變和DMA代謝速率之間，有顯著的相關(p=0.027)；經校正其他可能的干擾因子，包括性別、年齡、BMI、和單位體重食用牡蠣量之後，相較於基因型為一般型的受試者而言，在此點位上發生SNP的受試者，其DMA的代謝速率顯著較快(OR=0.26, 95% C.I=0.07-0.93)。進一步，利用線性回歸分析分別去探討受試者年齡和此點位的SNP和食用牡蠣後尿液中DMA增加的濃度以及DMA累積增加濃度之間的相關性。結果顯示，當受試者的年齡越大時，DMA累積增加濃度顯著地減少(β=-3.97)。CYT19基因多形性方面，有此SNP組別的受試者，其尿液中DMA增加濃度和累積增加濃度皆增加(β=4.68, β=1.67)，但沒有達到統計上的顯著水準。 由上述結果，推測年齡和CYT19基因intron6上點位的SNP (G > C)都可能是影響受試者在食用牡蠣後，其DMA代謝速率快慢的重要因素之ㄧ。也因為DMA代謝速率在個體上的差異性，進而造成不同個體之間對砷代謝作用有變異性存在。Abstract Food and drinking water are the major exposure routes of arsenic among the general population. In general, it was believed that the toxicity of inorganic arsenic was greater than organic arsenic. Up to now, inorganic arsenic is more thoroughly studied than organic arsenic. Consequently, the metabolism of organic arsenic has not been closely unveilded yet. However, there are considerable variations in the metabolism of inorganic and organic arsenic among individuals. Factors which have been shown to influence the methylation of arsenic include age, gender, ethnics, form of arsenic administered, environmental factors and genetic polymorphism, etc. The goal of the present study is to explore the effects of genetic polymorphisms of the human PNP, CYT19 and GSTO1 on the variation of arsenic metabolic rate among individuals, and the correlation between genetic polymorphism and the variation of urinary arsenic metabolites after oyster ingestion. Fifty one study subjects were recruited from the past three dietary control studies. During the one-week dietary control study, the study subjects ate the same food stuffs containing no seafood in all meals to exclude the unexpected seafood uptake, except for the ingestion of designated amount of oyster on the fourth day. First morning-voided urine samples of the study subjects were collected for 7 consecutive days and analyzed by HPLC-HG-AAS and HPLC-ICP-MS for urinary arsenic species, i.e. As3+, As5+, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Then the blood samples of study subjects were collected for analyzing the genetic polymorphisms of the human PNP, CYT19 and GSTO1. In general, DMA was the major urine metabolite, and its concentration reached the highest level on the first day after oyster ingestion and gradually decreased latter. The daily fluctuation of As3+ was similar to that of DMA. The DMA metabolic rate was categorized into three levels, i.e. fast (n=28), medium (n=19) and slow (n=4) groups, respectively, according to the day on which the urinary DMA concentrations reached the highest levels among these days after oyster ingestion. Based on this, it was concluded that there was difference in the metabolic rate of organic arsenic among study subjects. The mean age of the fast metabolic group, 28.3±1.0 years, was significantly greater than that of the slow metabolic group, 25.7±1.6 years. SNP (G > C) in intron6 of CYT19 gene was positively associated with DMA metabolic rate (p=0.027) as compared to wild-type gene. The relationships of age and SNP (G > C) in intron6 of CYT19 with either maximum or cumulative DMA increased concentration were explored by multiple linear regression. Data showed that the older the subjects are, the less the cumulative DMA increased concentration are (β=-3.97). In addition, the maximum and the cumulative DMA increased concentrations of study subjects who had SNP (G > C) in intron6 of CYT19 gene were higher than subjects whose genes were wild-type (β=4.68, β=1.67, respectively,) although both of them were not significant statistically. According to the above, age and SNP (G > C) in intron6 of CYT19 were the factors that had influence on the variation of DMA metabolic rate and then caused the difference in urinary DMA metabolite among study subjects.目錄 摘要 2 Abstract 4 目錄 7 第一章 前言 9 1.1 研究背景 9 1.2 研究目的 10 第二章 文獻探討 11 2.1 砷在環境中分佈的情形 11 2.1.1 自然界的來源 11 2.1.2 人為的來源 11 2.2 砷在生物體中分佈的情形 11 2.2.1 無機砷的代謝作用 13 2.2.2 有機砷的代謝作用 14 2.3 砷對人體的影響 15 2.3.1 砷的毒性 15 2.3.2 砷對人體的健康效應 17 2.4 砷代謝作用的變異性 17 2.5 砷代謝作用中的基因多形性 19 2.5.1 Purine nucleoside phosphorylase (PNP) 20 2.5.2 Arsenic(III) methyltransferase (CYT19) 21 2.5.3 Glutathione-S-transferase omega1 (GSTO1) 22 第三章 材料與方法 24 3.1研究對象 24 3.2研究設計 24 3.3樣本的收集和分析 24 3.3.1尿液中砷物種的分析 24 3.3.2 PNP, CYT19和GSTO1 基因SNPs的分析 25 3.4統計分析 28 第四章 結果 30 4.1 受試者的基本資料 30 4.2 牡蠣中砷代謝物種分佈的情形 30 4.3 尿中砷代謝物種分佈的情形 31 4.4 以DMA代謝速率分組比較各相關因子之間的差異 32 4.5 受試者PNP, CYT19和GSTO1基因多形性分布的情形 34 4.6 PNP, CYT19和GSTO1基因多形性和食用牡蠣後尿中砷物種代謝速 率之間的相關性 35 4.7 年齡、CYT19基因intron6上點位的SNP (G > C)突變和食用牡蠣後受試者尿液中代謝DMA濃度變化之間的相關性 37 第五章 討論 43 5.1 食用牡蠣後尿液中砷物種濃度變化情形的探討 43 5.2 食用牡蠣後受試者代謝砷物種速率差異的探討 45 5.3 PNP, CYT19和GSTO1基因多形性分布情形的探討 46 5.4 基因多形性和砷物種代謝速率之間相關性的探討 47 5.5 影響食用牡蠣後尿中DMA濃度分布變異性的因子之探討 47 5.6 研究限制 49 第六章 結論 51 參考文獻 5