臺灣博碩士論文加值系統

環境污染物交互作用對細胞色素P450 1A1基因表現影響之機轉研究
Benzo[a]pyrene (BaP), benzo[g,h,i]perylene (BghiP)和1-nitropyrene (1-NP)是廣泛分佈於環境中的污染物。BaP是人類可能之致癌物，許多研究結果顯示有些環境污染物對BaP所形成的動物致癌性具有促進或抑制作用，然而化合物間的交互作用對BaP致癌性表現影響的分子機轉，至今仍不清楚。已知多環芳香烴對細胞色素P450 1A1 (CYP1A1) 之誘發能力和形成DNA鍵結物以及引起腫瘤形成呈正相關性。因此本研究以人類細胞探討兩種混合物所產生之交互作用，進而瞭解其作用機轉。其中以BaP和BghiP或是BaP和1-NP兩種混合物分別處理人類肝癌細胞株HepG2，並分析其細胞基因毒性，結果發現以32P-postlabeling方法所測得之BaP-DNA鍵結物含量會隨著BghiP添加濃度而增加，但是當1-NP和BaP同時處理細胞時，BaP-DNA鍵結物的表現量卻顯著受到抑制。進一步以Western blot和Northern blot分析結果發現BghiP會促進BaP所誘發之CYP1A1蛋白及mRNA表現，而1-NP則會稍許減弱CYP1A1 mRNA的表現，但卻造成CYP1A1蛋白大量減少。再由gel retardation assay分析結果得知，BghiP會促進BaP所誘發之aryl hydrocarbon receptor (AhR)-Arnt複合體結合到DRE (dioxin receptor element)的能力。因此BghiP可經由活化AhR而促進BaP所誘發之CYP1A1基因表現，進而促進BaP的基因毒性表現，並暗示CYP1A1基因的誘發作用可能參與BghiP對BaP致癌性所表現的輔致癌作用(co-carcinogenesis)。而1-NP的處理結果則發現其對AhR活化型的表現量具有抑制作用，因此1-NP確實會經由AhR路徑而影響CYP1A1 mRNA的表現。然而，1-NP抑制CYP1A1蛋白表現量卻遠高於對CYP1A1 mRNA表現的抑制，由in vitro proteolysis 的分析結果發現，同時含有BaP和1-NP時，CYP1A1蛋白的分解速率比單獨含有BaP或1-NP的快，在加入自由基捕捉劑dimethyl sulfoxide (DMSO)和抗氧化劑dithiothreitol之後，CYP1A1蛋白分解作用則顯著減緩。由細胞存活率分析的結果發現，HepG2細胞之死亡率會隨著CYP1A1蛋白量降低而增高。因此，僅有少部分的CYP1A1蛋白下降是經由1-NP對CYP1A1基因轉錄層次的抑制作用所造成的，而與自由基有關的後轉譯作用(post-translation)可能才是抑制CYP1A1蛋白表現的可能主要機轉。
OGG1基因表現做為暴露烹調油煙之生物指標和肺腫瘤形成之相關性研究
人類8-oxoguanine DNA glycosylase 1 (hOGG1)基因產物具有移除8-OHdG的DNA glycosylase/AP lyase的酵素活性。有學者以hOGG1基因發生突變，推測hOGG1基因可能是一種抑癌基因且參與肺癌之形成，但其基因表現在肺腫瘤化過程中所扮演的角色並不清楚。因此，本研究以RT-PCR方法分析73位肺癌患者腫瘤組織中hOGG1基因的表現，並與所收集的臨床資料作一相關性分析，同時以22位非肺癌患者的正常肺組織作為研究對照組，以瞭解其在肺癌腫瘤過程中所扮演的角色。結果發現在非癌症患者之正常肺組織中都無法偵測到hOGG1基因的表現量，而肺癌患者腫瘤組織則有17位患者被偵測到(17/73, 23.3%)，這結果與肺細胞中hOGG1基因表現的結果相似。由統計分析結果發現，hOGG1 基因的表現與腫瘤期別 (tumor stage) 相關，具有顯著統計意義(p=0.001)，能偵測到hOGG1 mRNA之患者大多為三、四期 (13 /17, 76.5%)，同時發現hOGG1 mRNA表現與腫瘤分期的TNM值有關，當T值愈大時，則hOGG1基因表現之頻率則愈高(p=0.014)。雖然hOGG1基因表現與N值沒有達到統計意義的相關性 (p=0.054)，但當N值愈大時，hOGG1基因表現率則有較高之傾向，由此推測hOGG1基因可能也參與淋巴結轉移。因此，hOGG1基因的表現可能影響腫瘤的大小和淋巴結轉移的能力，進而影響腫瘤分期。另外，本研究發現hOGG1 mRNA表現與以免疫染色方法偵測到RB蛋白之間具有相關性(p=0.039)，在腫瘤組織中，當調控細胞週期的RB蛋白失去功能時，會促使癌細胞增生，腫瘤可能隨之增大，這與腫瘤組織中所發現hOGG1基因表現和腫瘤大小的相關性的發現似乎符合。同時分析肺腺癌細胞株H1299所得的結果證實hOGG1基因的表現會隨著細胞週期而改變，由流式細胞分析儀所測得的結果發現hOGG1基因表現達最高量在G0/G1期進入S期之間，而此結果可由H1299細胞中的RB蛋白的磷酸化時間得到證實。因此推測hOGG1基因的表現可能受到細胞週期的調控，而hOGG1基因會隨細胞的增生而大量表現。所以在腫瘤組織中可發現hOGG1基因的表現與腫瘤大小及RB蛋白的消失相關。而由存活率之分析結果發現，hOGG1基因表現的肺癌患者之存活率低於不表現的患者(p=0.0319)。綜合上述之結果推測，hOGG1 mRNA的表現與肺癌的腫瘤化過程可能有關，同時認為hOGG1基因之表現或許可作為腫瘤惡化程度之指標。另外，由過去研究顯示hOGG1是移除8-OHdG的DNA修補酵素，因此假設hOGG1基因表現可作為氧化性傷害的生物指標。本研究以烹調油煙和過氧化氫(H2O2) 分別處理肺細胞株CL-3，結果發現hOGG1 mRNA會被誘發表現，同時由本實驗室過去的研究結果顯示烹調油煙所處理之小牛胸腺DNA中8-OH dG的形成量具有線性濃度效應，而證實烹調油煙可能會引起氧化性傷害，但是hOGG1是否能作暴露油煙指標，另外實驗室也發現經常暴露油煙之廚師和較少暴露油煙之健康人的淋巴球中hOGG1 mRNA表現頻率呈現劑量反應關係，具有顯著的統計差異(p<0.05)。綜合上述之結果證實hOGG1基因表現或許可作為一種偵測細胞受到氧化性傷害的生物指標，同時暗示氧化性傷害在女性肺癌發生機轉上可能扮演某種角色。

Alteration of benzo[a]pyrene-induced cytochrome P450 1A1 gene expression by benzo[g,h,i]perylene or 1-nitropyrene in binary mixtures of mimic environmental pollutants
Benzo[a]pyrene (BaP), benzo[g,h,i]perylene (BghiP) and 1-nitropyrene (1-NP) are all widespread environmental pollutants. BaP is thought to be a probable carcinogen in human cancer. Numerous studies have shown that BaP-induced carcinogenesis in rodents can be attenuated or gained by certain chemicals. However, the detailed molecular mechanisms through which these complex mixtures alter chemical carcinogenesis caused by BaP are not completely understood. The DNA adduct formation and cytochrome P450 1A1 (CYP1A1) induction have been suggested to be the major determinant of their carcinogenic activity. Therefore, a permanent human hepatoma cell lines HepG2 was used to explore the genotoxic action of BaP in combination with either BghiP or 1-NP. Results from 32P-postlabeling assay showed that the BaP-DNA adduct levels were increased by BghiP; whereas, those were reduced by 1-NP. In addition, the data from Western blot and Northern blot analyses showed that BghiP enhanced BaP-induced CYP 1A1 protein and its mRNA levels. However, 1-NP alleviated BaP-induced CYP1A1 mRNA expression, and caused a marked decrease in the protein levels. Furthermore, gel retardation assay was performed to elucidate the involvement of BghiP in CYP1A1 gene expression. The results showed that BghiP increased nuclear accumulation of aryl hydrocarbon receptor (AhR) in cells and/or activation of AhR to a DNA-binding form. There was a concordant increase in the transcription activation of CYP1A1 gene mediating through AhR signaling pathway. These findings demonstrate that BghiP enhanced BaP-induced CYP1A1 transcription by AhR activation, and suggest that the induction mechanism of CYP1A1 contributes to the co-carcinogenic potential of BghiP in BaP-induced carcinogenesis. On the other hand, the cotreatment with BaP and 1-NP decreased the AhR binding capacity to dioxin responsive element (DRE). This confirmed that the suppression of BaP-induced CYP1A1 mRNA expression through AhR signal pathway. However, the decrease in CYP1A1 protein levels was significantly larger than that in CYP1A1 mRNA levels. This inconsistent result was confirmed by in vitro proteolysis assay. The result revealed that an enhanced degradation rate of CYP1A1 protein was observed by adding the binary mixture of BaP and 1-NP. Administrating free radical scavengers, dimethyl sulfoxide (DMSO), or antioxidant, dithiothreitol, was added in the reaction mixture protected CYP1A1 protein degradation. Moreover, in cell viability assay, we found that mass cells death was accompanied with the decrease of CYP1A1 protein by 1-NP. These findings suggest that the transcriptional suppression by 1-NP may be partly involved in the regulation of CYP1A1 protein expression; however, free radical-mediated post-translational mechanism may play a dominated role in the loss of CYP1A1 protein content.
Induction of OGG1 gene expression as an exposure biomarker of cooking oil fumes and the association with tumor progression in human lung cancer
The human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene encodes a DNA glycosylase/AP lyase that excises 8-hydroxydeoxyguanosine (8-OHdG) from DNA. Although hOGG1 has been suggested to be a tumor suppressor gene, infrequent mutations limit its impact in the development of tumor. In addition, little is known about the role of hOGG1 mRNA expression in lung tumorigenesis. Thus, in this study, 73 lung cancer patients and 22 non-cancer control subjects were enrolled to investigate the association of hOGG1 mRNA expression with lung cancer. The results from RT-PCR showed that hOGG1 mRNA expression was detected in 17 of 73 lung cancer patients (23.3%) and in none of the non-cancer controls. The expression of hOGG1 mRNA showed a good correlation with tumor size (p=0.014), tumor stage (p=0.001), and pRB negative immunostaining (p=0.039). In addition, the survivals of patients with hOGG1 mRNA expression were shorter than those without hOGG1 mRNA, indicating that hOGG1 mRNA expression may be associated with lung tumor progression. Furthermore, the data from H1299 lung cancer cells showed that hOGG1 mRNA expression reached the peak at G1-S transition, coinciding with phosphorylation of pRB. These results suggest that up-regulation of hOGG1 transcription may act as a predictor of lung tumor progression. On the other hand, it has been proposed that hOGG1 mRNA is a biomarker of cellular oxidative stress. Thus, in this study, hOGG1 mRNA expression was determined to assess the oxidative DNA damage induced by cooking oil fumes from frying fish (COF). The data from RT-PCR showed that the expression of hOGG1 mRNA was induced by hydrogen peroxide (H2O2) and COF in human lung adenocarcinoma CL-3 cells. It is consistent with other coworkers'' findings, showing that 8-OHdG levels were increased in a dose dependent manner when calf thymus DNA reacted with various concentrations of COF. Moreover, the results from a cross-sectional study showed that the hOGG1 mRNA expression frequencies of COF-exposed cooks and housewives were significantly higher than those of control subjects. These findings confirmed that expression of hOGG1 may be adequate to act as an exposure biomarker to assess the oxidative DNA damage induced by COF. This indicated that oxidative stress involved in the exposure risk of COF may play a role in lung cancer development among Chinese women.

中文摘要 ------------------------------------------------ I
Abstract -------------------------------------------------- IV
Contents ------------------------------------------------- VII
Abbreviations ------------------------------------------- IX
Chapter 1: Study background ----------------------- 1
1-1. BaP, BghiP and 1-NP ---------------------- 2
1-2. Chemical interactions ---------------------- 7
1-3. CYP1A1 ------------------------------------- 10
1-4. OGG1 -------------------------------------- 13
1-5. Study motivation -------------------------- 18
Tables and Figures ------------------------------ 21
Chapter 2: BghiP synergistically transactivates BaP-induced CYP1A1 gene expression by AhR pathway
Abstract ----------------------------------------- 31
2-1. Introduction ------------------------------- 32
2-2. Materials and Methods ------------------ 34
2-3. Results ------------------------------------- 37
2-4. Discussion --------------------------------- 39
Figures ------------------------------------------ 42
Chapter 3: 1-NP post-translationally suppresses BaP-induced CYP1A1 protein expression
Abstract ----------------------------------------- 46
3-1. Introduction ------------------------------ 47
3-2. Materials and Methods ----------------- 49
3-3. Results ------------------------------------ 52
3-4. Discussion -------------------------------- 54
Figures ------------------------------------------ 59
Chapter 4: hOGG1 mRNA expression as an oxidative stress exposure biomarker of COF and the association with tumor progression in non-small cell lung cancer
Abstract ------------------------------------------- 64
4-1. Introduction --------------------------------- 65
4-2. Materials and Methods -------------------- 68
4-3. Results --------------------------------------- 71
4-4. Discussion ---------------------------------- 75
Tables and Figures ------------------------------ 81
References ---------------------------------------------- 90
Appendix ----------------------------------------------- 117

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