腎細胞癌及移行性上皮癌與腎臟皮質、腎盂、輸尿管中金屬元素關係之探討-前驅研究

Abstract

流行病學研究中已經證實砷的暴露與移行性上皮癌之間有著顯著的劑量反應關係存在，但目前仍然相當缺乏重金屬與砷物種型態在泌尿道癌症病患生物檢體中之分布資料。因此，本次研究為探討多元素金屬在泌尿道癌症病患腎皮質、腎盂、輸尿管與尿液中之分布情形，以及了解泌尿道癌症病患體內甲基化代謝能力之情形。 在本次研究中，一共招募了17名來自於台大醫院泌尿部的移行性上皮癌與腎皮質癌個案，並依照TNM的癌症分類系統進行癌症病程的分級。同時並以問卷收集個案之年齡、身高、體重、運動習慣、抽菸、酗酒、飲茶、咖啡攝取、職業暴露史、飲食習慣、中草藥使用與家族及病史等資料。 在檢體的收樣上，以離心管收集個案手術當天早晨之第一泡尿液，並由醫師協助於腎臟摘除後進行組織檢體收集。正常與腫瘤組織樣本先經密閉式微波消化系統進行前處理，再以感應耦合電漿質譜儀進行砷、鎘、硒、錳與鍶等金屬之分析。同時並同步分析NIST 1577C牛肝標準參考品，用於分析方法之品質管制。尿液樣本則會先經過0.45 μm的濾膜過濾，其濾液以ICP-MS分析尿中之砷、鎘、硒、釩、鉻、錳、鎳、銅、鉛與鍶之濃度。尿液中砷物種的測定則是先以高效率液相層析儀層析出三價砷、雙甲基砷酸、單甲基砷酸與五價砷，其層析液再直接串聯至感應耦合電漿質譜儀定量。尿液分析的部分，以Seronorm尿液標準參考品之同步分析作為分析之品質管制步驟。 在本次研究結果中，可以看出隨著泌尿道癌症病患尿液中硒濃度的增加，尿液中的總砷與雙甲基砷酸也有顯著上升的趨勢(p<0.05)，另外，資料中也顯示出女性比起男性有較好的甲基化能力，但隨著年齡較增長之個案其甲基化能力則有降低的趨勢。而不同的金屬在各個癌症分期中，其濃度分布趨勢則各有不同。 在組織金屬濃度的分析上，砷與鎘在腎皮質正常組織中的濃度都比腎盂與輸尿管正常組織中高(p<0.05)，而鎘在正常組織中則是有比腫瘤組織還要高的情形(p<0.05)，砷在腫瘤與正常組織中則未看出差異。 本前驅研究的初步結果顯示，不同金屬在體內之累積，可能與體內砷物種型態之表現有相關。而各種金屬在不同組織器官上，確實也存在有濃度分布的差異。在後續的研究上，應進一步探討不同金屬元素與泌尿道癌症之間的交互作用，及其與癌症病程、年齡及性別對於體內甲基化能力的影響。針對不同部位之正常組織及腫瘤組織中金屬元素之分布，也需進一步比較，並確認在本研究中所看到的趨勢變化。

Background Epidemiological studies have demonstrated a significant dose-response relationship between arsenic exposure and the cancers of the urinary organs, especially transitional cell carcinoma (TCC). However, there was still lack of concentrations data of metal and arsenic species in the biological samples of urinary cancer subjects. Hence, the goals of this study were set: (1) to explore the relationship of renal cell carcinoma and transitional cell carcinoma, respectively, with the distributions of metal levels in renal cortex, pelvis and ureter, and (2) to study the association of the occurrence of renal cell carcinoma and transitional cell carcinoma, respectively, with the metabolic methylation in human body. Material and Methods Study subjects In this study, 17 urinary cancer subjects were recruited from the Urological Department of the National Taiwan University Hospital, with TCC or RCC at different stages. All subjects were histologically diagnosed, and categorized into different tumors stages based on the 1997 TNM classification system. Questionnaire Questionnaire was applied to collect study subject’s personal information on risk factors of urinary cancer, such as age, body height, weight, exercise habits, smoking history, occupational exposure history, alcohol drinking, tea consumption, coffee consumption, dietary habit, use of herbal medicine and family disease history. Collection and preparation of samples We use the 50 ml centrifuge tube to collect the morning void urine from subject, and through the assistant of surgen to obtain the tissue samples after surgery.Tissue samples were decomposed in closed vessels using a microwave-assisted method. Inductively coupled plasma mass spectrometry was used to analyze the contents of As, Cd, Se, Mn and Sr in normal and tumor kidney tissue. The accuracy of the ICP-MS (7500C, Agilent Technologies, Japan) analysis was validated with NIST SRM 1577c bovine liver. Urine sample was filtered through a 0.45-μm membrane prior to being subjected to ICP-MS and HPLC-ICPMS analysis. Trace metals including As, Cd, Se, V, Cr, Mn, Ni, Cu, Pb and Sr in urine were determined with ICP-MS, while As3+, DMA, MMA and As5+ were determined by HPLC-ICP-MS. Accuracy was checked with the SERO trace elements reference in urine. Results & Discussions Urinary concentration of total arsenic and the DMA percentage in total arsenic concentration significantly increased as the concentration of urinary selenium increased. Besides, results of the present study suggested that women presented greater capability in arsenic methylation than men, and the methylation capability diminished as the age increasing. This study also presented differences in the concentrations distributions of the selected trace metals in urine at different cancer stages. These findings indicated that some trace metals might play important roles in the pathogenesis of urinary cancer. Moreover, the concentrations of As and Cd in normal cortex were higher than in normal pelvis and normal urinary track (p<0.05). The Cd concentration in normal tissue was found to be higher than those in tumor tissue, whereas As levels in tumor tissue was not found to be different than those in normal tissue. Conclusions, the metal accumulation in human body might affect the concentration distribution of arsenic species. Furthermore, there were different metal concentration distributions in different human urinary organs. Futher research should study the interaction between the trends of different metals and urinary cancer, moreover, to realize the association among metals, cancer stage, age, gender, and methylation capability. Besides, it needs to explore the metal distributions in normal and tumor tissues. It is also warranted to involve more participants to obtain statistically significant data in order to confirm the metal distribution.