Part I:氯化三丁錫與氯化三苯錫對正常大鼠肝臟上皮細胞間隙交流影響之探討/Part II:利用石墨爐式原子吸收光譜法及感應耦合電漿質譜分析法測定合金鑄造廠工人血液及尿液中金屬濃度暴露評估

Part I:Effect on Gap Junction Intercellular Communication by Tributyltin Chloride (TBTC) and Triphenyltin Chloride (TPTC) in WB-F344 Rat Liver Epithelial Cells./Part II:Determination of Metal in Blood and Urine of Alloy Casting Factory Workers by Graphite-Furnace Atomic Absorption Spectrometry (GF-AAS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

李家榮(Chia-Rong Lee)

指導教授 ： 蔡錦蓮

高雄醫學大學/健康科學院/職業安全衛生研究所/碩士(2008年)

摘要

Part I:氯化三丁錫與氯化三苯錫對正常大鼠肝臟上皮細胞間隙交流影響之探討

在過去的研究中指出細胞間隙交流功能被阻斷視為是細胞癌化的一個指標，同時有機錫已經被證實對生物體有害但目前尚無研究明確說明其在體內的作用機制，故希望藉由本研究探討氯化三丁錫及氯化三苯錫對正常大鼠肝臟上皮細胞（WB-F344）的細胞間隙交流影響及相關蛋白表現之間的關係，藉由細胞間隙交流觀察細胞功能性的變化；即時聚合酶連鎖反應（Real-time PCR）觀察RNA的表現以及利用西方墨點法（Western Blot）觀察蛋白質的表現。
研究結果顯示，氯化三丁錫及氯化三苯錫對於細胞的毒殺效應都相當強烈，僅需要相當微量的濃度極可能對細胞生長造成影響，隨著暴露濃度與時間的增加有時間與劑量效應的關係，且氯化三丁錫的毒性較氯化三苯錫大10倍左右。
氯化三丁錫及氯化三苯錫雖然同為有機錫化合物，但對於正常大鼠肝臟上皮細胞的影響不盡相同，氯化三苯錫在濃度達到3.9 μM時，暴露一小時後細胞間隙交流的訊息傳遞功能即被阻斷；但氯化三丁錫的濃度即便已高到暴露後會改變細胞外觀，對於細胞間隙交流的功能而言仍無顯著的影響。顯示氯化三丁錫與氯化三苯錫雖然同為有機錫化合物，但其對於WB-F344細胞的毒理作用，可能因其化學結構的不同，而有著不同的結果。
本研究實驗結果顯示不論是RNA或是蛋白質的表現上，WB-F344細胞在不同濃度的氯化三苯錫暴露下，Cx43的表現隨著濃度增加而被抑制，但若暴露在不同濃度的氯化三丁錫中，反而呈現被激發的狀態；而E-Cadherin的RNA及蛋白值表現則是隨著兩種藥物濃度的增加而逐漸被抑制；在β-Catenin方面，暴露氯化三苯錫的細胞則有隨著濃度增加而被抑制的現象，與RNA表現類似，但氯化三丁錫的暴露卻似乎不對β-Catenin造成影響；Occludin的蛋白質表現上，結果與RNA的表現相當吻合，都是隨著兩種藥物的暴露濃度增加而被抑制其表現量；最後，在Cx32的表現部分，在不同濃度的氯化三苯錫暴露下，Cx32的表現隨著濃度增加而被激發，但若暴露在不同濃度的氯化三丁錫中，反而呈現被抑制的狀態，但與RNA的結果比較，卻發現呈現完全相反的結果。
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Part II:利用石墨爐式原子吸收光譜法及感應耦合電漿質譜分析法測定合金鑄造廠工人血液及尿液中金屬濃度暴露評估

合金鑄造廠勞工在作業環境中較常接觸到砷、鎘、鉻、鉛、鎳、銅、鋅、鐵、鈹等金屬物質；本研究依據採樣工廠的特性，擬分析血液及尿液中銅、鋅、鐵、鉛、鈹等金屬元素，其中銅、鋅、鐵、鉛的分析方法已普遍進行；而鈹在血液及尿液中的分析方法在國內尚未被建立，因此，本研究欲建立鈹在血液及尿液中的分析方法。
在分析條件設定方面，以石墨爐式原子吸收光譜法測定鈹在尿液及血液中的濃度，石墨爐式原子吸收光譜法需要針對特定元素調整乾燥、灰化及原子化溫度還有原子化時間，經多次實驗調整，將尿液及血液分別稀釋2倍及5倍，並以基質匹配的方法模擬樣品基質，將基質干擾效應予以補正消除，取得最佳分析條件（血液部分：乾燥溫度110℃、灰化溫度1400℃、原子化溫度/時間為2400℃/3秒；尿液部分：乾燥溫度110℃、灰化溫度1500℃、原子化溫度/時間為2400℃/3秒），並選擇以0.7%的硝酸鎂做為鈹稀釋液中的基質修飾劑；在此條件下分別以標準品添加，測定方法偵測下限（尿液為0.2 ppb；血液為0.4 ppb），尿液部分，當標準品添加濃度在0.2~2.0 ppb時，回收率在93.33~105.5%之間，變異係數皆在15%以下；血液部份，當標準品添加濃度在0.4~2.0 ppb時，回收率為83.81~96.33%之間，變異係數皆在10%以下。
將原子吸收光譜法分析結果與感應耦合電漿質譜分析法做比較，測定結果顯示相同檢體各元素之間的相關性都不佳（R2值：血液中銅0.2481、鋅0.1332、鐵0.0229、鉛0.2029、鈹0.0034；尿液中鈹0.4529）。
本研究針對台灣中、北部三間合金鑄造廠勞工計127名之血液及尿液檢體作分析，結果顯示尿液及血液中鈹的含量極低，都在方法偵測下限之下，除了鈹以外，銅、鋅、的檢測值大部分皆落於一般正常範圍以內，血清中銅高於一般正常成人參考值上限140 μg/dL者占2.36%；血中鋅高於高於一般正常成人參考值上限140 μg/dL者占24.41%，其中並有2.36%高於200 μg/dL以上；唯鐵濃度超出一般範圍者較多，高於一般正常成人參考值上限200 μg/dL者占68.50%，甚至有3.15%高達700 μg/dL以上；而血中鉛含量高於高血鉛標準40 μg/dL者約占0.79%，1.57%介於30~40 μg/dL之間。

關鍵字

有機錫 ； 氯化三丁錫 ； 氯化三苯錫 ； 細胞間隙交流 ； 鈹 ； 原子吸收光譜 ； 血液 ； 尿液

並列摘要

Part I:Effect on Gap Junction Intercellular Communication by Tributyltin Chloride (TBTC) and Triphenyltin Chloride (TPTC) in WB-F344 Rat Liver Epithelial Cells.

Point out GJIC block into first indicator to regard as cell cancer among past research. Organic tin compound prove harmful to organism, so, this study probe into the effect on gap junction intercellular communication by Tributyltin Chloride (TBT) and Triphenyltin Chloride (TPTC) in Normal rat liver epitherial cell line (WB-F344). Observe the change of the cell functionality by GJIC, observe the behavior of RNA by Real-time PCR and observe the behavior of protein by Western Blot.
The result of this study, TPTC and TBT effects of poisoning on cells are all quite strong. Exposure concentration and increase of time have relation of Time & Dose dependent, and toxicity of TBT more an order than TPTC.
TPTC, and TBT though all organic tin compound, but the effect of WB-F344 cell was different. When the concentration of TPTC until 3.9 μM an hour, the GJIC will blocked. But even the concentration of TBT has been already high will change the cell appearance after exposing, still there is no apparent influence as to GJIC. Though TPTC and TBT are all the organic tin compound, but the toxicological function on WB-F344 cells was different, maybe the chemical structure not the same,and there are different results.
The result of this study, the effect on RNA and protein in WB-F344 cell, the behavior of Cx43 is inhibited as the concentration increases in TPTC, but the state stimulated in TBT of different concentration. RNA and protein value of E-Cadherin behave and is inhibited gradually with increase of thickness of TPTC and TBT. In part of β-Catenin, was inhibited as the concentration increases in TPTC as RNA, but disagree with TBT. For Occuldin, the result of protein as same as RNA is inhibited as the concentration increases in TPTC and TBT. Finally, the behavior of Cx32 is stimulated as the concentration increases in TPTC and inhibited in TBT, but compared with result of RNA, find that the totally opposite result appears.
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Part II:Determination of Metal in Blood and Urine of Alloy Casting Factory Workers by Graphite-Furnace Atomic Absorption Spectrometry (GF-AAS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

There are many element of metal in the environment that the laborer worked in alloy casting factory, such as arsenic, cadmium, chromium, lead, nickel, copper, zinc, iron and beryllium, etc. This study according the factory characteristic of sampling source, plan to analysis copper, zinc, iron, lead and beryllium in urine and blood. The analytical method of copper, zinc, iron, lead have already generally used, but the method of beryllium in urine and blood has been not set up. Thus, this research wants to set up the analysis method of beryllium in urine and blood.
The condition set up of analytical that determination of beryllium in urine and blood by graphic-furnace atomic absorption spectrometry (GF-AAS). GF-AAS needs to set up the condition: temperature of drying, ashing and atomization, and the time of atomization. Using matrix matching to imitate the effect of matrix, samples dilute 2x for urine and 5x for whole blood, and addition matrix modifier (0.7% magnesium nitrate hexahydrate). The condition of beryllium in urine and blood by experimental tests (the condition of drying temperature @ 110℃, ashing temperature @ 1400℃, atomization temperature @ 2400℃ and time for 3 second in blood; drying temperature @ 110℃, ashing temperature @ 1500℃, atomization temperature @ 2400℃ and time for 3 second in urine.). The method detection limit was 0.2 ppb in urine and 0.4 ppb im blood. The concentration of addition in urine between 0.2~2.0 ppb, the recovery between 93.33~105.5%, coefficient of variation under than 15%; For blood, the concentration of addition between 0.4~2.0 ppb, recovery between 83.81~96.33%, coefficient of variation under than 10%.
The result was undesirable of GF-AAS compare with ICP-MS (R2 values in blood was 0.2481 in copper, 0.1332 in zinc, 0.0229 in iron, 0.2029 in lead and 0.0034 in beryllium; 0.4529 in beryllium of urine).
For this study, there are 127 samples from laborers of blood and urine whose worked at three alloy casting factories in Taiwan. The result of beryllium in urine and blood was low under the method detection limit. In addition to beryllium, the value of copper and zinc in plasma were normal, there are 2.36% of copper in plasma upper than the consultation normal range with adults’ (140 μg/dL); 24.41% of zinc upper than 140 μg/dL in the consultation normal range with adults’, there are 2.36% higher than 200 μg/dL; there are more persons who goes beyond the normal range of iron, the person who higher than general normal adults' consultation value upper limit 200 μg/dL accounts for 68.50%, even 3.15% are up to more than of 700 μg/dL; And the person who higher than lead standard of high blood lead 40 μg/dL accounts for 0.79% and 1.57% are between 30~40 μg/dL.

並列關鍵字

Organic Tin ； TBTC ； TPTC ； GJIC ； Beryllium ； GF-AAS ； Blood ； Urine

參考文獻

Part I:氯化三丁錫與氯化三苯錫對正常大鼠肝臟上皮細胞間隙交流影響之探討

1. Hoch M. Organotin compounds in the environment – an overview. Applied Geochemistry 2001;16:719-43.

2. Kumar NM, Gilula NB. The gap junction communication channel. Cell 1996;84:381-8.

3. Yamasaki H, Krutovskikh V, Mesnil M, et al. Role of connexin (gap junction) genes in cell growth control and carcinogenesis. C R Acad Sci III 1999;322:151-9.

4. Tin and Inorganoc Tin compounds. World Health Organization (WHO) 2005;Concise International Chemical Assessment Document 65 (CICAD 65).