臺灣博碩士論文加值系統

鉻(chromium,Cr)是一種常見的重金屬，在地殼中的含量為0.01％位居第17位，由於鉻或鉻化物具有高熔點、高沸點、高硬度、高耐磨及高耐腐蝕等特性。因此，鉻被廣泛使用於各種工業製程。鉻主要以兩種價態穩定的存在於自然界中，分別是三價鉻以及六價鉻，六價鉻被認為是具有較強的生物毒性。六價鉻暴露會產生反應性含氧物種(reactive oxygen species, ROS),而ROS可能與粒線體去氧核醣核酸（mitochondrial DNA, mtDNA）功能缺陷相關，如mtDNA 4,977-bp斷損突變（ΔmtDNA4977）及mtDNA複製套數。然而至今未有六價鉻造成mtDNA損害之相關研究，因此本研究目的為探討六價鉻暴露組與控制組間之粒線體DNA 4977-bp斷損突變（ΔmtDNA4977）與粒線體去氧核醣核酸（mtDNA）複製套數變化情形。本研究選取職業暴露六價鉻勞工與年齡、吸煙與喝酒配對之高暴露及低暴露組各13名勞工，分析每一位受試者之尿液中鉻濃度以及血液中ΔmtDNA4977與mtDNA複製套數(mtDNA copy number)。結果發現高鉻暴露組之相對ΔmtDNA4977量較低暴露組顯著為高( p＝0.020），而mtDNA copy number量則呈現低暴露組高於暴露組，但未呈現統計差異(p=0.054)。本研究結論為長期職業暴露六價鉻之勞工會造成其ΔmtDNA4977增加、mtDNA copy number減少，但受限於樣本數量，導致於copy number部分未能呈現統計上顯著。由於影響ΔmtDNA4977(4834)與mtDNA之因子甚多，未來則需較多的研究對象方能作為做進一步結論之依據。
另一方面也進行動物實驗去探討ROS濃度及粒線體DNA傷害和粒線體膜電位異常之相關性，當中採用8-12週之Sprague-Dawley 公鼠共25 隻，腹腔注射(intraperitoneal)方式暴露六價鉻鹽類重鉻酸鉀(potassium dichromate)溶於磷酸鹽緩衝液(PBS, Phosphate Buffered Saline)的水溶液，於連續暴露14天後犧牲採血液、尿液及精液樣本，檢測其ROS濃度、膜電位異常比例及大鼠的粒線體DNA 4834-bp斷損突變與粒線體複製套數變化情形。結果於膜電位方面依暴露濃度有一上升趨勢，但於DNA傷害及ROS濃度方面則無此趨勢，由於相較於勞工樣本，動物暴露時間太短可能導致其反應不明顯，以及樣本數不足也可能導致此一現象，未來需採較多的樣本數並採長期觀測方能作為做進一步結論之依據。

Chromium (Cr) has been widely used in industries because of its high melting point, hardness, and corrosion-resistant. Hexavalent chromium (Cr6+) has been considered with strong biotoxicity.
It is documented that Cr6+ could generate reactive oxygene species (ROS) to cause interior damages for bioorganisms. In many reports ROS has been associated with mitochondria DNA (mtDNA) alterations (including ΔmtDNA4977 and mtDNA copy number). The relationship between mtDNA alterations and Cr6+ exposure, however, has not been postulated yet. The purpose of this study was to investigate the association of ΔmtDNA4977 and mtDNA copy number with samples that exposed to Cr6+. 13 workers occupationally exposed to high levels of Cr6+and 13 age, seniority, drinking and smoking matched low-exposed workers were monitored on their urinary chromium concentrations by graphite atomic absorption spectrophotometer. We found the frequencies of relative ΔmtDNA4977 in high Cr6+ exposure group were significantly higher than those in the low Cr6+ exposure group (p＝0.020). On the other hand, the mtDNA copy number in high Cr6+ exposure group were marginally lower than those in the low Cr6+ exposure group (p＝0.054). We concluded that long-term exposure to hexavalent chromium could cause mitochondria alterations. Further study with more subjects involved is warranted.
Alternately, animal experiments were also conducted to study the relationships between the concentration of ROS and mitochondria membrane potential. Twenty-five male rats of 8-12 weeks old Sprague-Dawley were exposed to potassium dichromate by intraperitoneal. Sacrificing and quarrying their blood, urine and sperm samples after exposing for fourteen days of different concentrations of Cr6+. According to the increasing exposure concentration, the results of the membrane potential showed the same tendency, but in DNA lesion and ROS concentration were not have this trend. Relatively, the human samples show the significant correlation to exposure concentration. It might due to first, the fourteen days exposure period of animal study is too short to cause significant response to exposure concentration, and secondly sample size insufficient to lead to the fact of this phenomenon too. Further study with more subjects involved and long exposure time is warranted.

摘要 i
Abstract iii
致謝 iv
目錄 v
表目錄 vii
圖目錄 viii
第一章 序論 1
1-1 前言及研究動機 1
1-2 研究目的 2
1-3 研究架構 3
1-3-1.職業暴露部份 4
1-3-2.動物實驗部份 5
第二章 文獻探討 7
2-1 鉻之基本資料 7
2-1-1 鉻之物化特性 7
2-1-2 鉻之健康效應 7
2-1-3 鉻之應用及使用情形 10
2-2 粒線體之介紹 11
2-2-1 粒線體結構與其生理功能 11
2-2-2 粒線體膜電位及其變化 12
2-2-3 粒線體DNA及其損害 13
2-3 反應性含氧物種之探討 16
2-3-1 反應性含氧物種之簡介 16
2-3-2 反應性含氧物種之常見分析方法探討 17
第三章 材料與方法 19
3-1 樣本來源 19
3-1-1 職業現場之六價鉻暴露勞工 19
3-1-2 動物實驗 19
3-2 淋巴球離心法 20
3-3 粒線體DNA損害分析 21
3-3-1 血液及精液DNA萃取 21
3-3-2 mtDNA 損害測定 22
3-3-3 勞工樣本之ΔmtDNA 4977相對定量 24
3-3-4 勞工樣本之Copy number相對定量 25
3-3-5 動物樣本之ΔmtDNA 4834相對定量 26
3-3-6 動物樣本之Copy number相對定量 27
3-4 膜電位之測定 28
3-5 ROS之測定 29
3-5-1 流式細胞儀部分 29
3-5-2 化學發光儀部分 29
3-5-3 分析比較部份 30
第四章 結果 31
4-1 勞工樣本部份 31
4-2 動物實驗部份 38
4-2-1 膜電位部份 39
4-2-2 ROS部分 41
4-2-3 DNA傷害部份 45
第五章 討論 53
5-1 泥水業勞工部份 53
5-2 動物實驗部份 53
第六章 未來展望 55
第七章 參考文獻 57

【1】Baruthio F, Toxic effects of chromium and its compounds. Biol. Trace Elem. Res., 32: 145-153: