產前全氟碳化物及抽菸暴露和早產兒對新生兒臍帶血去氧核醣核酸甲基化之影響

Influence of Preterm Birth and Prenatal Exposure to Perfluorinated Compounds and Tobacco Smoke on Methylation of Cord Blood DNA

連培真(Pei-Chen Lien)

指導教授 ： 劉貞佑

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10561

摘要

背景及目的: 去氧核醣核酸甲基化（DNA methylation）是表觀遺傳修飾（epigenetic modification）的其中一種調控方式，使得基因的表現可能受到環境變化而影響。產前暴露於全氟化合物（perfluorinated compounds）和抽菸有害物質都與不良出生結果及其他健康影響有所關聯，但潛在的機制仍所知甚少。越來越多的證據顯示，發生於胚胎發育早期的表觀基因重組現象（epigenetic reprogramming）使得胎兒在子宮內發育時期相對於其他時期更易受到環境暴露影響，且此影響可能增加日後罹患某些疾病的風險。此研究目的為探討DNA甲基化（LINE-1、Alu和IGF2）的改變是否可能為產前暴露全氟化合物與抽菸有害物質和出生結果相關的潛在調控因子之一。
方法: 本研究對象為486對在2004年4月至2005年1月期間參與台灣出生世代長期追蹤研究的母嬰配對。使用超高效液相層析串聯質譜法測得臍帶血中4種全氟碳化合物，包括全氟辛烷磺酸（perfluorooctyl sulfonate, PFOS）、全氟辛酸（perfluorooctanoic acid, PFOA）、全氟壬酸（perfluorononanoic acid, PFNA）及全氟癸酸（perfluoroundecanoic acid, PFUA）。量測古丁尼（cotinine）濃度作為抽菸有害物質暴露之生物標記。使用焦磷酸測序（pyrosequencing）方法量測臍帶血中DNA重複性片段以代表全基因體甲基化程度，包括長散置式重複性片段（LINE-1）及短散置式重複性片段（Alu）。其中一個與胎兒生長發育相關的基因，類胰島素生長因子Ⅱ（insulin-like growth factor 2）被進行測定。
結果: LINE-1、Alu和IGF2 平均（標準差）甲基化程度分別為80.4（4.0）、20.8（1.7）和47.1（7.2）％（範圍：60.3-81.9、16.0-29.3和14.2-72.3）。簡單與多變項線性回歸分析顯示LINE-1甲基化程度與臍帶血中全氟辛烷磺酸濃度有顯著負相關 (簡單線性回歸: β = -1.28, p<0.01; 多變項線性回歸: β = -1.11, p<0.05)。臍帶血中較高古丁尼濃度的嬰兒與較低的LINE-1及Alu甲基化有顯著相關 (LINE-1: β = -0.81, p<0.001; Alu: β = -0.17, p<0.05) 。在高濃度古丁尼組別中，較低的LINE-1甲基化程度與較高的全氟辛烷磺酸和全氟癸酸濃度有關 (PFOS: r = -0.2, p<0.05; PFUA: r = -0.25, p<0.01)。且較低的Alu甲基化程度也與較高的全氟辛烷磺酸濃度有關 (r = -0.2, p<0.05) 。研究結果沒有發現IGF2甲基化與產前4種全氟碳化合物與抽菸有害物暴露之相關性。在甲基化與出生結果的相關性部分，早產兒比非早產兒有顯著較低的Alu甲基化程度。
結論: 我們的研究指出產前致癌物質像是全氟辛烷磺酸和抽菸有害物的暴露可能對胎兒全基因體甲基化有負面影響。此外，我們的研究也發現早產兒有較低的Alu全基因體甲基化程度。綜合以上兩點，DNA甲基化的改變可能是部分調節產前全氟辛烷磺酸與抽菸有害物質暴露和不良出生結果相關的因子之一。

關鍵字

表基因 ； 去氧核醣核酸甲基化 ； 產前暴露 ； 全氟碳化物 ； 抽菸有害物 ； 出生結果

並列摘要

Background: DNA methylation, an essential epigenetic modification, allows gene expression to be modified in response to environmental changes. Prenatal exposures to perfluorinated compounds (PFCs) and smoking are linked to adverse birth outcomes and other health effects, but the potential mechanisms remain largely unknown. Increasing evidence suggests that during the critical period of prenatal development, fetus may be more vulnerable to environmental factors by affecting the epigenetic reprogramming and consequently increase the risk of disease development in later life. The objective of the study was to investigate whether the changes of DNA methylation (LINE-1, Alu and IGF2) may be one of the potential modulator behind the relationship between prenatal exposure (PFOS, PFOA, PFNA, PFUA and tobacco smoke) and birth outcomes.
Methods: The study subjects were from the Taiwan Birth Panel Study, which were recruited from April 2004 to January 2005. Four PFCs in cord blood, including perfluorooctyl sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluoroundecanoic acid (PFUA), were analyzed by ultra-high-performance liquid chromatography/tandem mass spectrometry. Cotinine were measured as a biomarker of smoking exposure. The global DNA methylation patterns were quantified in cord blood using DNA repetitive elements including long interspersed nucleotide element (LINE-1) and short interspersed nucleotide elements (Alu) by pyrosequencing. One specific gene (insulin-like growth factor 2) related to fetal growth was measured.
Results: Mean (SD) methylation levels of LINE-1, Alu and IGF2 were 80.4 (4.0), 20.8 (1.7) and 47.1 (7.2) % (range: 60.3-81.9, 16.0-29.3 and 14.2-72.3), respectively. Simple and multiple linear regression models shown a significant association between LINE-1 methylation level and cord blood PFOS concentration (crude model: β = -1.28, p<0.01; adjusted model: β= -1.11, p<0.05). Lower LINE-1 and Alu methylation level was associated with higher PFOS and PFUA concentrations in high cotinine group (LINE-1, PFOS: r = -0.2, p<0.05; PFUA: r = -0.25, p<0.01; Alu, PFOS: r = -0.2, p<0.05). The infants with higher cotinine level had a significantly lower LINE-1 and Alu methylation levels (LINE-1: β = -0.81, p<0.001; Alu: β = -0.17, p<0.05). No significant associations were found between prenatal 4 major PFCs and cotinine exposure and IGF2 gene methylation level. Infants had significant difference on Alu methylation level between preterm birth and not preterm birth, with preterm birth having a lower average methylation level of Alu than not preterm birth.
Conclusions: Our results suggest that prenatal exposure to potentially carcinogenic compounds such as PFOS and tobacco smoke may have adverse impact on fetal global DNA methylation patterns. Otherwise, our finding shown that lower global DNA methylation level of Alu was associated with preterm birth. Together, DNA methylation changes may be one of the possible modulator of the relationships between prenatal PFOS and tobacco exposure and adverse birth outcome.

並列關鍵字

epigenetic ； DNA methylation ； prenatal exposure ； perfluorinated compounds ； tobacco smoke ； birth outcomes

參考文獻

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