- 學位論文
非砷暴露地區病患的尿液砷物種、腎絲球過濾率、氧化壓力及細胞週期對於腎細胞癌的危險性
Urinary Arsenic Profile, eGFR, Oxidative Stress and Cell Cycle on the Risk of Renal Cell Carcinoma in a Non-Arsenic Exposure Area
摘要
腎細胞癌（renal cell carcinoma, RCC）是腎臟實質部分（renal parenchyma）最常發生的惡性腫瘤，近幾年來的發生率在全世界的多數國家均呈現逐年增加的趨勢，包括台灣在內。砷是環境中重要的致癌因子之一，已知皮膚癌、肺癌、肝癌、膀胱癌等均可能與砷的暴露有關。國內外有關腎細胞癌與砷的相關性研究相對缺乏。無機砷暴露會導致8-OHdG的氧化性傷害，而調控細胞週期的三個最常被檢視的基因的重要位點p53 codon72、p21 codon31及MDM2 SNP309，是否會修飾砷物種對腎細胞癌的風險，值得進一步探討。本研究分三部分進行病例對照研究，探討在非砷暴露台灣北部地區中，國人腎細胞癌的相關危險因子。 第一部分：探討非砷暴露地區中，尿液砷物種對於腎細胞癌的危險性 在2006年11月至2009年5月期間，我們從台大醫學院附設醫院收集了132位病理診斷確定為腎細胞癌的病患，另外從台北醫學院附設醫院以及台北市立萬芳醫院的成人健檢挑選260位年齡性別配對對照組。所有的研究個案均在告知研究目的並簽署受試者同意書之後，首先由訓練有素的研究人員以結構式問卷調查其社會人口學特徵、生活型態、飲食習慣、環境暴露等因子，並收集白天的一次中段尿液儲存於-20℃之中，以便進行尿液砷物種的分析，收集血液的肌酸酐資料並計算其腎絲球過濾率；對於預備接受腎臟部分或是全切除的腎細胞癌病患，其小便以及血清肌酸酐的檢測均是在手術進行之前收集的。在以多變項邏輯迴歸分析比較病例組與健康對照組後，發現顯著的保護因子為偶而喝酒、偶而喝咖啡以及偶而喝茶等，危險因子為吸菸者、高血壓以及較低的腎絲球過濾率等。加入尿液砷物種分析時，發現尿液中的總砷與腎細胞癌的危險性具有劑量效應的關係。而腎絲球過濾率與尿液的總砷具有負相關性。而尿液的總砷與高血壓對於腎細胞癌的危險性存在有意義的交互作用。當個案同時具有高血壓、較低的腎絲球過濾率以及較高的尿液總砷時，其腎細胞癌的風險可以達到對照組的6.01倍。而此三項危險因子的累積對於腎細胞癌的風險具有趨勢上的顯著意義。因此，此研究結果所得到的結論，證實即使是在非砷暴露的環境中，尿液中的總砷仍然是腎細胞癌的重要危險因子，而病患本身的低腎絲球過濾率或是高血壓則會增加此風險。 第二部分：探討非砷暴露地區中，尿液砷物種以及8-OHdG對於腎細胞癌的危險性 本研究偵測尿液中8-OHdG的含量來推測個體所承受的氧化壓力。此部份的研究個案為台大醫學院附設醫院收集132位病理診斷確定為腎細胞癌的病患，另外收集245位年齡與性別配對的對照組。所有的研究均在告知研究目的並簽署受試者同意書之後，收集白天的一次中段尿液以便進行尿液砷物種以及8-OHdG的分析，並由經過訓練過的醫護人員做完整的問卷詢問。同時，尿液的檢測是在腎臟部分或是全切除手術進行之前。8-OHdG的偵測是使用isotope-dilution liquid chromatography-Tandem Mass spectrometry with on-line solid-phase extraction，該偵測方式具有極佳的敏感度，其偵測下限為5.7ng/L。 研究結果發現腎細胞癌病患相對於對照組有較高的尿液8-OHdG濃度（4.81比上3.68 ng/mg creatinine, p < 0.001），同時在個案組及對照組之中，女性的尿液8-OHdG濃度均較男性為高，但未達統計上的差異性。同時，尿液8-OHdG濃度也未被尿液總砷的高低、抽菸或是喝酒所影響。 經過多變數的調整後，發現尿液中8-OHdG的濃度與腎細胞癌的風險性具有劑量反應的效應。當個案具有較高的尿液總砷（??15.85?慊/g creatinine）合併較高的尿液8-OHdG（??3.34 ng/mg creatinine）時，其腎細胞癌的風險值相對於對照組可以達到3.5倍之高。而此兩項危險因子的存在對於腎細胞癌的風險具有趨勢上的檢定意義（p = 0.002）。 第三部分：探討非砷暴露地區中，尿液砷物種以及細胞週期調控基因的基因多形性對於腎細胞癌的危險性 從台大醫學院附設醫院收集127位病理診斷確定為腎細胞癌的病患，另外從台北醫學院附設醫院以及台北市立萬芳醫院的成人健檢所收集254位年齡與性別配對的對照組。除了以結構式問卷調查其生活飲食習慣並收集白天的一次中段尿液以便進行尿液砷物種的分析，同時收集受試者的血液並萃取其DNA，進行基因多形性的檢測。使用TaqMan螢光定量技術（TaqMan allelic discrimination）來確定p53 codon 72: rs1042522, p21 codon 31: rs1801270及MDM2 SNP309: rs2279744位點基因表型；利用ABI Prism 7500HT sequence detection system來完成PCR cycling；利用The ABI Prism 7500HT sequence detection system and the ABI sequence detection system software version 2.2來完成genotype analysis。 研究結果發現帶有p53 Pro/Pro及Arg/Pro基因型的個案相較於p53 Arg/Arg 基因型，具有較高的腎細胞癌風險，且達到統計上的顯著性（OR 2.16, CI 1.06-4.38; OR 2.47, CI 1.15-5.29）。而帶有MDM2 SNP309 TG及GG 基因型的個案相較於MDM2 SNP309 TT基因型的個案，亦有較高的腎細胞癌風險，但未達到統計上的顯著性。 進行各基因型對於尿液中砷物種的差異性比較，僅發現帶有p53 Arg/Pro 及 Pro/Pro 基因型的腎細胞癌患者相較於p53 Arg/Arg 基因型的腎細胞癌患者具有較高的總砷以及較高的五價MMA百分比以及較低的五價DMA百分比。 進一步探討三個基因多形性彼此之間的交互作用，發現帶有p53 Arg/Pro及 Pro/Pro基因型以及MDM2 SNP309 TG及 GG 基因型個案其腎細胞癌的風險值為對照組的2.51倍； 帶有p21 Ser/Arg 及Arg/Arg 基因型以及MDM2 SNP309 TG及 GG基因型的個案其腎細胞癌的風險值為對照組的2.28倍。 最後探討尿液總坤對於各個位點的基因多形性所造成的腎細胞癌的風險效應。發現帶有p53 Arg/Pro and p53 Pro/Pro 基因型的個體當合併有較高的總砷時，其腎細胞癌的風險值為對照組的5.70倍；帶有MDM2 SNP309 TG及 GG基因型的個體當合併有較高的總砷時，其腎細胞癌的風險值為對照組的5.08倍。 結綸 根據一系列的研究發現，即使是處於非砷暴露的環境之中，個體仍然會因為其他的危險因子的存在，包括高血壓、低腎絲球過濾率的低下、氧化壓力過高、以及許多重要的細胞週期調控基因多形性的不同，藉由彼此之交互作用，因而提高了腎細胞癌發生的危險性。
並列摘要
Renal cell carcinoma (RCC) is the most common malignant tumors from renal parenchyma. The incidence around the world is showing a rising trend, including Taiwan. Arsenic is a very important environment risk factor for cancer, including skin cancer, lung cancer, liver cancer, and bladder cancer. However, the majority of patients with renal cell carcinoma live in non-arsenic exposed and does arsenic still play a certain role in kidney cancer? Arsenic can induce 8-OHdG. On the other hand, wherther the cell cycle regulated three gene p53 codon72、p21 codon31及MDM2 SNP309 can modify arsenic induce RCC risk remains investigation. This study contains three parts to conduct a case-control study to explore the etiologic factor of RCC in non-arsenic exposure Taipei area. Part 1 Urinary arsenic profile over the RCC risk in a non-arsenic exposure area The case-control study was conducted between November 2006 and May 2009 with 132 patients with renal cell carcinoma, and 260 sex and age matched controls from a hospital based pool. Pathological verification of renal cell carcinoma was completed by image guided biopsy or surgical resection of renal tumors. Urinary arsenic species, including inorganic arsenic, monomethylarsonic acid and dimethylarsinic acid, were determined with a high performance liquid chromatography linked hydride generator and atomic absorption spectrometry. Estimated glomerular filtration rate was calculated using the Modification of Diet in Renal Disease Study equation. Urinary total arsenic was significantly associated with renal cell carcinoma risk in a dose-response relationship after multivariate adjustment. Low estimated glomerular filtration rate or hypertension was significantly related to renal cell carcinoma risk. Estimated glomerular filtration rate was significantly negatively related with urinary total arsenic. A significant interaction was seen between the urinary total arsenic and hypertension on renal cell carcinoma risk. The greatest odds ratio (6.01) was seen in the subjects with hypertension, low estimated glomerular filtration rate and high urinary total arsenic. A trend test indicated that the risk of renal cell carcinoma increased along with the accumulating number of these 3 risk factors (p < 0.0001). Part 2. Urinary arsenic profile and 8-OHdG over the RCC risk in a non- arsenic exposure area This case-control study was conducted with 132 RCC patients and 245 age- and sex-matched controls from a hospital-based pool between November 2006 and May 2009. Pathological verification of RCC was completed by image-guided biopsy or surgical resection of renal tumors. Urinary 8-OHdG levels were determined using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Concentrations of urinary arsenic species, including arsenite (As3+), arsenate (As5+), monomethylarsonic acid (MMA5+) and dimethylarsinic acid (DMA5+), were determined by a high performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. Level of urinary 8-OHdG was significantly associated with RCC risk in a dose-response relationship after multivariate adjustment. Urinary 8-OHdG was significantly related to urinary total arsenic. The greatest odds ratio (3.50) was seen in the individuals with high urinary 8-OHdG and high urinary total arsenic. A trend test indicated that the risk of RCC was increased with one of these factors and was further increased with both (p = 0.002). Part 3. Urinary arsenic profile and cell cycle-regulated genes polymorphism over the RCC risk in a non-arsenic exposure area This study involved 132 RCC patients and 257 age- and gender-matched controls from a hospital-based pool. Concentrations of urinary arsenic species, including inorganic arsenic, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) were determined by a high-performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. Polymorphisms of p53 Arg72Pro, p21 Ser31Arg, and MDM2 SNP309 were examined using polymerase chain reaction and restriction fragment length polymerase. The p53 Pro/Pro genotype and MDM2 SNP309 GG genotype significantly increased RCC risk compared to the p53 Arg/Arg genotype and MDM2 SNP309 TT genotype. Subjects with the p53 codon 72 Arg/Arg genotype had a significantly low percentage of inorganic arsenic, a low percentage of MMA, and a high percentage of DMA, which indicates efficient arsenic methylation capacity. Subjects with the p53 Arg/Pro+Pro/Pro genotype or MDM2 SNP309 TG+GG genotype, in conjunction with high urinary total arsenic (>15.95 μg/g creatinine), had a significantly higher RCC risk than those with the p53 Arg/Arg or MDM2 SNP309 TT genotypes and low urinary total arsenic. Conclusion This is the first study to demonstrate that higher urinary total arsenic was associated with an increased risk of RCC in a non-arsenic exposure area and other important factors were associated with RCC risk, including: low eGFR, hypertension, high urine 8-OHdG, cell-cycle regulatory gene polymorphism. Further studies are necessary to clarify the possible mechanisms in this issue.
並列關鍵字
arsenic ； methylation ； kidney ； glomerular filtration rate ； renal cell carcinoma ； 8-hydroxydeoxyguanosine ； P53 ； MDM2參考文獻
延伸閱讀
- 王德麟（2012）。尿液中無機砷代謝物種及腎臟皮質、腎盂、輸尿管金屬分布與腎臟癌關係之探討〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2012.03032
- 蔡承軒（2014）。血漿硒濃度、尿中砷濃度及硒蛋白基因多形性與攝護腺癌〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-3107201400094700
- 簡郁鍾（2006）。砷暴露途徑及其與居民尿液砷物種之探討〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2006.00057
- 林冠菻（2019）。腫瘤壞死因子的單一核苷酸多型性與血漿砷之交互作用對腎絲球過濾率之影響〔碩士論文，高雄醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0011-1208201900080700
- Peng, Y. L. (2016). The effect of febuxostat and other urate-lowering agents on uric acid management and renal outcome in patients with chronic kidney disease [master's thesis, Kaohsiung Medical University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0011-2607201613361500