砷代謝相關酵素、尿液8-OHdG、血漿維生素、p53、p21與CCND1、砷代謝能力與泌尿上皮癌

Polymorphisms of AS3MT, PNP, GSTO1 and GSTO2, Urinary 8-OHdG, Plasma Vitamins, Polymorphisms of p53, p21 and CCND1, Arsenic Metabolism Capability and Urothelial Carcinoma

鍾季容(Chi-Jung Chung)

指導教授 ： 薛玉梅

臺北醫學大學/公共衛生暨營養學院/公共衛生學研究所/博士(2009年)

摘要

過去研究顯示，暴露含砷地下水與泌尿上皮癌有顯著劑量效應關係。然而過去研究獲得砷暴露指標大多經由環境測量，並未考慮每個人每日實際的喝水量，無法有效反應個體內實際的無機砷暴露量，且無法進一步窺視無機砷進入人體後生成之無機砷代謝物種所帶來之危害。此外，台灣2000年自來水含砷量標準已從原先的50 μg/L降至10 μg/L，而歐洲和美國也分別在2003年和2006年將飲用水含砷量規定為10 μg/L。本論文選擇台北作為研究地區（即非砷暴露區，自來水平均含砷量為0.7 μg/L），探討低砷暴露下，不同砷代謝物種濃度與泌尿上皮癌相關性。
人體內無機砷代謝過程包括一連串還原和氧化甲基化酵素反應。研究發現無機砷代謝過程會引發活性氧物種的產生、增加細胞氧化性傷害，並抑制p53和p21等細胞週期因子之作用，使受損細胞失去調控與過度增生，最終導致癌化現象。本論文第一個目的為探討不同砷代謝相關酵素基因多形性是否會影響尿液砷代謝型態。第二個目的為探討尿液砷物種型態、氧化壓力指標即尿液8-OH-dG與泌尿上皮癌的相關性。血漿維生素研究發現可減少細胞氧化性傷害，但是否可降低與砷相關之尿液8-OH-dG文獻甚少，本論文第三個目的為探討血漿維生素越高者，是否可降低由砷引發氧化性傷害造成之泌尿上皮癌風險。最後，氧化性傷害可能藉由抑制細胞週期因子作用，導致癌化。故本論文第四個目的為闡明細胞週期因子相關基因多形性與尿液砷物種型態影響泌尿上皮癌的危險性。
本論文為病例對照研究，泌尿上皮癌病例來自台大醫院泌尿科，對照個案來自台北醫學大學老人健檢與萬芳醫院自費健檢民眾。尿液中的三價無機砷、五價無機砷、單甲基砷酸與雙甲基砷酸濃度，由高效能液相層析儀，連結氫化器與原子吸收光譜儀測量。砷代謝相關酵素AS3MT、PNP、GSTO1和GSTO2基因多形性，則是利用MALDI-TOF質譜儀以及聚合酵素連鎖反應和限制片段長度多形性判定。尿液8-OH-dG含量為利用體外競爭型酵素連結免疫吸附分析法檢測。血漿維生素A（retinol）、維生素E（alpha-tocopherol）、番茄紅素（lycopene）和胡蘿蔔素（beta-carotene）利用高效能液相層析儀進行分離和測量。細胞週期因子p53、p21和CCND1基因多形性，則是利用聚合酵素連鎖反應和限制片段長度多形性判定。
本論文檢測砷代謝相關酵素基因型發現，帶有GSTO1 Ala140Asp一股以上基因變異者以及GSTO2 Asn142Asp二股基因變異者，其尿液單甲基砷酸百分比顯著較其他基因型者為低。帶有GSTO1 Glu208Lys一股基因變異者其尿液無機砷百分比則是顯著較二股基因野生型者為低。此外，藉由測量尿液8-OH-dG含量，發現尿液砷代謝物種與氧化壓力增加有關。尿中總砷、三價無機砷、單甲基砷酸與雙甲基砷酸濃度越高，尿液8-OH-dG含量也隨之增加。尿液8-OH-dG含量越高且總砷越高、或且單甲基砷酸百分比越高或且雙甲基砷酸百分比越低者，泌尿上皮癌危險對比值也隨之增加。血漿維生素E對於泌尿上皮癌具有顯著的保護作用。帶有p21基因多形性與泌尿上皮癌的危險對比值為1.53，95%信賴區間為1.02- 2.29。帶有p53、p21和CCND1細胞週期相關因子基因變異數越多者，以及同時有較高的累積抽菸量、總砷濃度、無機砷百分比、單甲基砷酸百分比或越低的雙甲基砷酸百分比，其泌尿上皮癌的危險對比值呈顯著劑量效應關係。
環境無機砷暴露引發多種健康危害。除環境降低無機砷暴露來源外，本論文結果提供篩檢先天基因易感性對泌尿上皮癌較高之族群，而後天則提供增加日常飲食蔬菜和水果的攝取，提高血漿維生素含量，以降低泌尿上皮癌風險之建議。

關鍵字

砷代謝物種型態 ； 砷代謝相關酵素基因多形性 ； 8-OH-dG ； 血漿維生素 ； 細胞週期因子基因多形性 ； 泌尿上皮癌

並列摘要

Epidemiological studies have reported a dose–response relationship between long-term exposure to inorganic arsenic from drinking water and the risk of urothelial carcinoma (UC). Previous studies estimated the indices of inorganic arsenic exposure using environmental measurements, it could not actually reflect individual arsenic exposure in body. In addition, the standard level of arsenic in tap water was reduced from 50 μg/L to 10 μg/L since 2000 in Taiwan, and the same levels of Europeans and American were announced in 2003 and 2006, respectively. The purpose of this study was to explore the association between urinary arsenic profiles and UC under low arsenic exposure in Taipei.
Biotransformation of inorganic arsenic in human included the process of oxidative methylation and reduction. The generation of reactive oxygen species (ROS) in the intermediate arsenic species pathway induced cellular oxidative damage and inhibited the function of p53 and p21 in the checkpoint of cell cycle. Damaged cells resulted from ROS were out of control and finally resulted in hyperproliferation and carcinogenesis. The purpose of this study first was to evaluate whether single nucleotide polymorphisms (SNPs) in the arsenic-metabolizing genes affected urinary arsenic profile or not. Second, to evaluate the relationship among the levels of urinary 8-hydroxydeoxyguanosine (8-OH-dG), arsenic profile, and UC. Studies have found that elevated levels of plasma micronutrients would reduce the oxidative damage. However, the literatures of the association between micronutrients and arsenic-related 8-OH-dG were still few. The third purpose of this paper was to investigate whether plasma micronutrients can decrease UC risks through reducing arsenic-induced DNA damage. Finally, oxidative damage could result in carcinogenesis through inhibiting the function of cell cycle. Hence, the fourth purpose of this paper was to explore the relationships among the polymorphisms of cell cycle, urinary arsenic profile and UC risk.
A hospital-based case-contorl study was conducted in our works. UC cases were from the National Taiwan University Hospital and healthy controls were from Taipei Municipal Wan Fang Hospital and Taipei Medical Hospital. Urinary arsenic species, including arsenite (InAs3+), arsenate (InAs5+), monomethylarsinous acid (MMA5+) and dimethylarsinic acid (DMA5+) were measured by high-performance liquid chromatography (HPLC), equipped with a hydride generator and atomic absorption spectrometer (HG-AAS). Genotyping for SNPs in AS3MT Met287Thr (rs11191439), PNP His20His (rs1049562), PNP Gly51Ser (rs1049564), PNP Pro57Pro (rs1130650) and GSTO1 Glu208Lys (rs11509438) was performed using high-throughput matrix-assisted laser desorption and ionisation time-offlight (MALDI-TOF) mass spectrometry (SEQUENOM MassARRAY system； Sequenom, San Diego, CA, USA). In addition, genotyping for GSTO1 Ala140Asp (rs4925) and GSTO2 Asn142Asp (rs156697) was carried out by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Urinary 8-OH-dG measurement was analyzed with a competitive enzyme-linked immunosorbent assay (ELISA) kit in vitro (Japan Institute for the Control of Aging, Fukuroi, Japan). Plasma concentrations of retinol, alpha-tocopherol, lycopene and beta-carotene were analyzed by reversed-phase HPLC (Hitachi). Genotyping for SNPs in p53, p21 and CCND1 was carried out using PCR-RFLP.
Considering the urinary arsenic profiles in control groups, people with GSTO1 140 Ala/Asp or Asp/Asp or with GSTO2 142 Asp/Asp had significantly lower urinary MMA% than those with other genotypes. Further, people with GSTO1 208 Glu/Lys had significantly lower urinary InAs3+% than those with GSTO1 208 Glu/Glu. We found significant association between urinary arsenic profiles and increased oxidative damage. High urinary 8-OH-dG levels were associated with increased total arsenic concentrations, inorganic arsenite, MMA5+ and DMA5+. Significant dose–response relationships of UC risks were observed in people who had higher 8-OH-dG combined with higher total arsenic, or with higher MMA% or with lower DMA%. Alpha-tocopherols levels had significant protection from UC risk. The odds ratios of UC risk for people with p21 codon 31 Arg/Arg were 1.53 (95% CI: 1.02 – 2.29) than other genotype. There were significant dose–response relationships among those subjects carrying with greater numbers of gene variants and higher environmental exposure and UC risk Environmental exposure included cumulative exposure of cigarette smoking, total arsenic concentration, and MMA%. Furthermore, subjects with a lower DMA% and with greater gene variants had an increased UC risk.
Environmental inorganic arsenic exposure would induce healthy hazards in human. Now, the standard level of arsenic of drinking water is reduced to 10 μg/ L. Besides reducing the origins of environmental inorganic arsenic, these results in the present study provided effective screening markers in individual susceptibility against UC risk. Further, we recommend that increasing acquired intakes of daily vegetables and fruits to raise the levels of plasma micronutrients might reduce the UC risk.

並列關鍵字

urinary arsenic profiles ； polymorphisms in arsenic metabolism genes ； urinary 8-OH-dG ； plasma micronutrients ； polymorphisms in cell cycle regulatory genes ； urothelial carcinoma

參考文獻

Reference List

1. Ahmad, S., Kitchin, K. T., and Cullen, W. R. (2002). Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin. Toxicol. Lett. 133(1), 47-57.

2. Ahmad, S., Kitchin, K. T., and Cullen, W. R. (2000). Arsenic species that cause release of iron from ferritin and generation of activated oxygen. Arch. Biochem. Biophys. 382(2), 195-202.

3. Aposhian, H. V., and Aposhian, M. M. (2006). Arsenic toxicology: five questions. Chem Res Toxicol 19(1), 1-15.

4. Aposhian, H. V., Zakharyan, R. A., Avram, M. D., Sampayo-Reyes, A., and Wollenberg, M. L. (2004). A review of the enzymology of arsenic metabolism and a new potential role of hydrogen peroxide in the detoxication of the trivalent arsenic species. Toxicol. Appl. Pharmacol. 198(3), 327-335.