全氟辛烷磺酸的暴露與大鼠PPAR基因表現量及DNA甲基化的關聯性

Perfluorooctane Sulfonate Exposure in Relation to PPARs’ Gene Expressions and DNA Methylation in a Rat Model

洪浚傑(Chun-Chieh Hung)

指導教授 ： 劉貞佑

國立臺灣大學/公共衛生學院/食品安全與健康研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101797

摘要

背景
全氟辛烷磺酸(PFOS)為八碳鏈結構的化學物質，由於其結構特性，因此其具有防水、防油的效果。常運用於表面處理、半導體製程中的光微影術部分、金屬去污溶劑以及食品器具表面塗層等，主要暴露途徑為透過受污染的食物和飲用水、使用相關產品及生產相關產品的職業暴露。且由於屬於持久性有機汙染物，在環境中及進入到體內皆不易被分解及排出。目前發現對於人體及動物都有負面效應的發生。
過氧化物酶體增殖物活化受體(PPAR)為核受體超家族的一員。在細胞生長、發育、分化與新陳代謝均有重要作用。核受體與配體結合後才會被活化並負責引導轉錄，由於核受體都位於細胞內部，因此它們的配體為脂溶性，這樣才能穿越由脂肪構成的細胞膜。PFOS由於其結構類似於脂肪酸，且對於核受體的親和力更高，因此容易引發相關作用。
DNA甲基化現象可調控轉錄，進而影響基因表達。於胚胎發展、出生後發育、癌症或環境荷爾蒙之影響等領域都是熱門的研究標的。當DNA進行甲基化修飾時，會抑制啟動子及轉錄起始點的轉錄作用，使基因表達量下降或不表達。
研究目的
本研究之目的為觀察暴露PFOS與各個器官Ppara和Pparg表現量的關聯性，並以DNA甲基化作為機制探討。
方法
五週齡大的Sprague Dawley雄性大鼠隨機分派到三個濃度點，包括0、5、10 mg/kg·d PFOS，每組6隻，暴露3週後犧牲取得血液、心臟、肝臟、肺臟、腎臟、胰臟及睪丸。基因表現量由定量即時聚合酶連鎖反應來進行測定；DNA甲基化則透過焦磷酸測序來進行。收集到的資料會以簡單線性回歸來觀察暴露PFOS、基因表現量及DNA甲基化的關聯性。
結果
在基因表現量的部分，暴露PFOS與血液的Ppara基因表現量(由Hprt校正：β = 2.00，p = 0.01；由Sdha校正：β= 0.20，p = 0.05)和腎臟的 Pparg 基因表現量(由Sdha校正：β = 1.49，p = 0.03)呈現正相關，而胰臟的Ppara基因表現量(由Sdha校正：β = -0.10，p < 0.01)則呈現負相關。
在DNA甲基化的部分，暴露PFOS與Ppara在心臟(位點2：β = 0.09，p = 0.05)與胰臟(位點3：β = 0.09; p = 0.02)的甲基化程度呈現正相關；Pparg則是在胰臟(位點3：β = 0.22，p = 0.02) 與肺臟(位點6：β = 0.51，p < 0.01)的甲基化程度呈現正相關，而血液(位點4：β = -0.60，p = 0.03；位點5：β = -0.57，p = 0.05；位點6：β = -0.53，p = 0.04)則呈現負相關。
在DNA甲基化與基因表現量的關聯性中，我們發現Pparg在心臟(由Sdha校正；位點3：β = 0.61；95% confidence interval = 0.01 to 1.20；p = 0.05；位點5：β = 1.00；95% confidence interval = 0.40 to 1.60；p < 0.01)呈現正相關，而胰臟(由Sdha校正；位點1：β = -1.35，p = 0.05；位點5：β = -1.07，p = 0.05)則呈現負相關。
儘管沒有器官在三個路徑皆達到統計顯著，不過在胰臟Pparg可以觀察到暴露PFOS會影響到DNA甲基化，而影響到基因表現量，可推測DNA甲基化可能在這之中有一定的影響力。
結論
本研究指出，暴露PFOS對大鼠的Ppara和Pparg的DNA甲基化及基因表現量有影響，尤其是胰臟Pparg，後續研究可針對其他甲基化位點以及上下游基因表現量做更進一步的探討。

關鍵字

全氟辛烷磺酸(PFOS) ； Ppara ； Pparg ； 相對基因表現量 ； DNA甲基化

並列摘要

Background
Perfluorooctane sulfonate (PFOS) is a chemical substance with eight carbon chain structure. Due to its structural characteristics, it has the effects of water and oil resistance. It is often used in surface treatment, photolithography in the semiconductor process, metal decontamination solvents, and surface coating of food appliances, etc. The main exposure routes are through contaminated food and drinking water, use of related products and occupational exposure that produce related products. Because it is a persistent organic pollutant, it is not easy to be decomposed in the environment. It has been found that there are negative effects on humans and animals.
Peroxisome proliferator-activated receptors are a member of the nuclear receptor superfamily. It plays an important role in cell growth, development, differentiation and metabolism. Nuclear receptors are activated after binding to ligands and are responsible for transcription. Because nuclear receptors are located inside the cell, their ligands are fat-soluble to cross the cell membrane made of fat. PFOS has a structure like fatty acids and has a higher affinity for nuclear receptors, so it is easy to trigger related effects.
DNA methylation can regulate transcription, which in turn affects gene expression. The fields of embryonic development, post-natal development, cancer or the influence of environmental hormones are all popular research subjects. When DNA undergoes methylation modification, it will inhibit the transcription of the promoter and the transcription start point, resulting in decreased or no gene expression.
Objective
The purpose of this study is to observe the correlation between exposure to PFOS and gene expression of Ppara and Pparg in various organs, and DNA methylation was used as the mechanism to discuss.
Methods
Five-week-old Sprague Dawley male rats were randomly assigned to three concentration points, including 0, 5, and 10 mg/kg·d PFOS, 6 rats in each group. Blood, heart, liver, lung, kidney, pancreas and testis were collected after 3 weeks of exposure. Gene expression is measured by quantitative real-time polymerase chain reaction; DNA methylation is measured by pyrosequencing. Data was processed by simple linear regression.
Results
In the part of gene expression, exposure of PFOS and Ppara gene expression in blood (normalized by Hprt: β = 2.00, p = 0.01; normalized by Sdha: β = 0.20, p = 0.05) and Pparg gene expression in kidney (normalized by Sdha: β = 1.49, p = 0.03) showed a positive correlation, while Ppara gene expression in pancreas (normalized by Sdha: β = -0.10, p <0.01) showed a negative correlation.
In the part of DNA methylation, exposure of PFOS and Ppara in the heart (position 2: β = 0.09, p = 0.05) and pancreas (position 3: β = 0.09; p = 0.02) showed a positive correlation with the degree of methylation; in Pparg, we found that there was a positive correlation in pancreas (position 3: β = 0.22, p = 0.02) and lung (position 6: β = 0.51, p <0.01), while a negative correlation in blood (position 4: β = -0.60, p = 0.03; position 5: β = -0.57, p = 0.05; position 6: β = -0.53, p = 0.04).
In the association between DNA methylation and relative gene expression, there was a positive correlation in heart Pparg expression (normalized by Sdha; position 3: β = 0.61; p = 0.05; position 5: β = 1.00; p < 0.01), whereas a negative correlation in pancreas Pparg expression (normalized by Sdha; position 1: β = -1.35; p = 0.05; position 5: β = -1.07; p = 0.05).
Although no organ was statistically significant in all three pathways, we can find that exposure to PFOS in pancreas Pparg affect DNA methylation, which in turn affect gene expression. It can be speculated that DNA methylation may influence in this.
Conclusion
This study points out that exposure to PFOS has an impact on the DNA methylation and gene expression of Ppara and Pparg in rats, especially pancreas Pparg. Follow-up studies can target other methylation sites and upstream or downstream genes.

並列關鍵字

PFOS ； Ppara ； Pparg ； Relative gene expression ； DNA methylation

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