임신 중 BDE-47 및 BDE-209에 노출된 어미와 새끼 Sprague-Dawley 랫드의 Global DNA 메틸화 양상과 비만 감수성과 연관된 유전자 발현 Global DNA Methylation Patterns and Gene Expression Associated with Obesity-Susceptibility in Offspring of Pregnant Sprague-Dawley Rats Exposed to BDE-47 and BDE-209원문보기
잔류성 유기 오염 물질은 후성학적 메커니즘과 비만의 발달에 영향을 줄 수가 있다. 폴리브롬화 디페닐 에테르는 주요한 잔류성 유기 오염 물질 중 하나이며, 난연제로 널리 쓰인다. 출생전 잔류성 유기 오염 물질과 같은 내분비교란물질에 노출시 LINE-1 (long interspersed nuclear elements)의 global DNA 메틸화와 비만 위험도의 증가에 영향을 미칠 수 있다. 따라서, 이번 연구는 임신한 스프라그-돌리 백서를 이용하여 태반과 모유를 통하여 전달된 BDE-47, BDE-209가 LINE-1에서의 후성학적인 변화와 obesogen으로서 발달과정에 따른 유전적 비만 감수성의 증가에 영향을 줄 수 있는지에 대해서 보고자 하였다. 어미와 새끼에서 LINE-1의 광범위 DNA 메틸화와 비만과 관련된 유전자 발현은 methylation-sensitive high resolution melting analysis (MS-HRM), direct bisulfite sequencing와 quantitative real time polymerase chain reaction (qPCR)을 사용하여 각각 분석하였다. MS-HRM 결과는 출생 후 4일의 노출군 새끼에서 (4마리 중 2마리) LINE-1의 광범위 DNA 저메틸화 양상을 보여주었지만, bisulfite sequencing은 노출군과 비노출군에서 차이가 없었다. ${\beta}$-산화 경로와 adipokines과 관련된 어미의 유전자 발현은 두 그룹간 차이를 보였다. 반면에, 새끼의 유전자 발현은 비슷한 양상을 나타내었다. ${\beta}$-산화 경로와 비만과 관련된 유전자 발현 중 $PPAR-{\alpha}$를 제외하고는 출생 시에 유의하게 증가하였다. 결론적으로, 이번 연구는 BDE-47, BDE-209의 동시 노출이 태반과 모유를 통해서 새끼에서의 후성학적인 변화와 비만과 관련된 유전자 발현 변화에 영향을 미칠 수 있는 것을 보여주었다.
잔류성 유기 오염 물질은 후성학적 메커니즘과 비만의 발달에 영향을 줄 수가 있다. 폴리브롬화 디페닐 에테르는 주요한 잔류성 유기 오염 물질 중 하나이며, 난연제로 널리 쓰인다. 출생전 잔류성 유기 오염 물질과 같은 내분비교란물질에 노출시 LINE-1 (long interspersed nuclear elements)의 global DNA 메틸화와 비만 위험도의 증가에 영향을 미칠 수 있다. 따라서, 이번 연구는 임신한 스프라그-돌리 백서를 이용하여 태반과 모유를 통하여 전달된 BDE-47, BDE-209가 LINE-1에서의 후성학적인 변화와 obesogen으로서 발달과정에 따른 유전적 비만 감수성의 증가에 영향을 줄 수 있는지에 대해서 보고자 하였다. 어미와 새끼에서 LINE-1의 광범위 DNA 메틸화와 비만과 관련된 유전자 발현은 methylation-sensitive high resolution melting analysis (MS-HRM), direct bisulfite sequencing와 quantitative real time polymerase chain reaction (qPCR)을 사용하여 각각 분석하였다. MS-HRM 결과는 출생 후 4일의 노출군 새끼에서 (4마리 중 2마리) LINE-1의 광범위 DNA 저메틸화 양상을 보여주었지만, bisulfite sequencing은 노출군과 비노출군에서 차이가 없었다. ${\beta}$-산화 경로와 adipokines과 관련된 어미의 유전자 발현은 두 그룹간 차이를 보였다. 반면에, 새끼의 유전자 발현은 비슷한 양상을 나타내었다. ${\beta}$-산화 경로와 비만과 관련된 유전자 발현 중 $PPAR-{\alpha}$를 제외하고는 출생 시에 유의하게 증가하였다. 결론적으로, 이번 연구는 BDE-47, BDE-209의 동시 노출이 태반과 모유를 통해서 새끼에서의 후성학적인 변화와 비만과 관련된 유전자 발현 변화에 영향을 미칠 수 있는 것을 보여주었다.
Persistent organic pollutants (POPs) can affect epigenetic mechanisms and obesity development. Polybrominated diphenyl ethers (PBDEs)-widely used to make flames-are one of the important POPs. Prenatal exposure to endocrine disrupting chemicals (EDCs), such as POPs, may affect global DNA methylation ...
Persistent organic pollutants (POPs) can affect epigenetic mechanisms and obesity development. Polybrominated diphenyl ethers (PBDEs)-widely used to make flames-are one of the important POPs. Prenatal exposure to endocrine disrupting chemicals (EDCs), such as POPs, may affect global DNA methylation in long interspersed nuclear elements (LINE-1), increasing the risk of obesity later in life. Therefore, pregnant Sprague-Dawley (SD) rats were used to elucidate whether BDE-47 and BDE-209 transferred through placenta and breast milk cause epigenetic changes in LINE-1 and increase genetic susceptibility to obesity as obesogen during the developmental periods. Global DNA methylation in LINE-1 and gene expression related to obesity were measured in dams and offspring, using a methylation-sensitive high resolution melting analysis (MS-HRM) and direct bisulfite sequencing and quantitative real time polymerase chain reaction (qPCR), respectively. The results of MS-HRM showed global DNA hypomethylation patterns in LINE-1 of exposed offspring (2 of total 4) at PND 4, but bisulfite sequencing showed no difference in both the exposed and non-exposed groups. Gene expression in dams related to ${\beta}$-oxidation pathway and those related to adipokines showed different patterns between the two groups. On the contrary, gene expressions of offspring showed a similar pattern. Gene expressions related to ${\beta}$-oxidation pathway and obesity were significantly increased when compared with 'at birth', but not $PPAR-{\alpha}$. In conclusion, this study demonstrated the possibility that co-exposure to BDE-47 and BDE-209-via the placenta and breast milk-may affect epigenetic changes and modulate gene expression levels related to obesity.
Persistent organic pollutants (POPs) can affect epigenetic mechanisms and obesity development. Polybrominated diphenyl ethers (PBDEs)-widely used to make flames-are one of the important POPs. Prenatal exposure to endocrine disrupting chemicals (EDCs), such as POPs, may affect global DNA methylation in long interspersed nuclear elements (LINE-1), increasing the risk of obesity later in life. Therefore, pregnant Sprague-Dawley (SD) rats were used to elucidate whether BDE-47 and BDE-209 transferred through placenta and breast milk cause epigenetic changes in LINE-1 and increase genetic susceptibility to obesity as obesogen during the developmental periods. Global DNA methylation in LINE-1 and gene expression related to obesity were measured in dams and offspring, using a methylation-sensitive high resolution melting analysis (MS-HRM) and direct bisulfite sequencing and quantitative real time polymerase chain reaction (qPCR), respectively. The results of MS-HRM showed global DNA hypomethylation patterns in LINE-1 of exposed offspring (2 of total 4) at PND 4, but bisulfite sequencing showed no difference in both the exposed and non-exposed groups. Gene expression in dams related to ${\beta}$-oxidation pathway and those related to adipokines showed different patterns between the two groups. On the contrary, gene expressions of offspring showed a similar pattern. Gene expressions related to ${\beta}$-oxidation pathway and obesity were significantly increased when compared with 'at birth', but not $PPAR-{\alpha}$. In conclusion, this study demonstrated the possibility that co-exposure to BDE-47 and BDE-209-via the placenta and breast milk-may affect epigenetic changes and modulate gene expression levels related to obesity.
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문제 정의
This study was performed to characterize global DNA methylation patterns of LINE-1 repetitive elements in exposed offspring of pregnant SD rats. The results showed exposed offspring at PND 4 day (N=2, total 4) have decreased methylation curves compared to standard and other samples curves.
가설 설정
The one non-exposed group was treated with corn oil in the same fashion and sacrificed at PND 4. We analyzed whether gene expression levels of obesity makers are different from the period of exposure. To do so, the results of the gene expression between the exposed and the non-exposed groups were compared with PND 4 and the changes of gene expression with different period of exposure were compared among the exposed groups.
제안 방법
Bisulfite sequencing to determine methylation status at CpG dinucleotides was carried out. Interestingly, no difference in sequence was observed between the two groups of samples in exposed and non-exposed offspring at PND 4 compared to reference sequence, even though methylated DNA standards as control were changed according to the degree of methylation (Figure 4).
The experiment in present study was carried out by using all samples wrapped with aluminum foil and kept at −80°C deep freezer until analysis.
The experiment was performed in 96-well plates (LightCyclerⓇ 480, Roche Diagnostics, Indianapolis, IN, USA) using a mix reaction (LightCyclerⓇ 480 High Resolution Melting Master Mix, Roche Diagnostics, Indianapolis, IN, USA), which contains a DNA intercalating dye, in a final volume of 20 uL, on a thermocycler instrument (LightCyclerⓇ 480II, Roche Diagnostics, Indianapolis, IN, USA). The following primer sets were designed by MethPrimer (http://www.
The exposed groups on GD 8 were treated by gavage with the mixture of BDE-209 (2.5 µmol/kg b.w) and 13C-BDE-47 (6 nmol/kg b.w) for 18 consecutive days from GD 8 to postnatal day (PND) 4.
This study was conducted to investigate the genes associated with obesity, whether they are affected by maternal BDE-47, BDE-209 transferred to offspring. The mRNA expressions of PPAR-α, ACOX 1 and PPAR-α increased in the exposed dams at birth compared to GD 14 but they showed significant increase only in PPAR-y.
w) for 18 consecutive days from GD 8 to postnatal day (PND) 4. Three exposed groups were serially sacrificed at GD 14, birth and PND 4, respectively. The one non-exposed group was treated with corn oil in the same fashion and sacrificed at PND 4.
대상 데이터
Sixteen pregnant Sprague-Dawley rats on gestation day (GD) 0 were purchased from Orient Bio (Seongnam, Korea). Sixteen pregnant Sprague-Dawley (SD) rats were used and randomly assigned to three different conditions of exposed (N=4 dams per group) and one non-exposed groups (N=4 dams). We used the PBDEs as mixture with BDE-47, BDE-209 because they are present in commercial products as mixture form and the most domestically predominant congeners are BDE-47, BDE-209.
Sixteen pregnant Sprague-Dawley rats on gestation day (GD) 0 were purchased from Orient Bio (Seongnam, Korea). Sixteen pregnant Sprague-Dawley (SD) rats were used and randomly assigned to three different conditions of exposed (N=4 dams per group) and one non-exposed groups (N=4 dams).
Representative standard melting curves for the determination of methylated DNA. Standard samples, from top to bottom, were 100%, 75%, 50%, 25%, 10%, 5% and 0% methylated.
데이터처리
Statistical analysis was performed using STATA version 12 (StataCorp, College Station, TX, USA). Statistical differences between exposed and non-exposed groups were evaluated by the Wilcoxon rank-sum test (non-parametric) to test for significant differences between independent 2 groups. A p-values less than 0.
성능/효과
The results of methylation-sensitive high-resolution melting (MS-HRM). In standard melting curves, (A) the melting curves of all samples and (B), (C) the melting curves of exposed offspring (2 of total 4) at PND 4. The arrow indicates more hypomethylated (approximately 25% to 50%) melting curve than other melting curves, respectively. (A) The blue circle means all samples of melting curves except exposed offspring (2 of total 4) at PND 4.
In this study, the gene expression of the offspring was significantly expressed compared with ‘at birth’ in exposed group.
The gene expression patterns of offspring exposed through placenta and breast milk were compared and it showed that PPAR-α and ACOX 1 which is related to -oxidation pathway have significantly increasing tendency in the exposed group but not PPAR-α.
This study was performed to characterize global DNA methylation patterns of LINE-1 repetitive elements in exposed offspring of pregnant SD rats. The results showed exposed offspring at PND 4 day (N=2, total 4) have decreased methylation curves compared to standard and other samples curves. Although bisulfite sequencing reactions were performed in order to confirm MS-HRM results, there were no changes in sequencing.
This study showed the possibility of global DNA methylation in the offspring exposed BDE-47, BDE-209 through placenta and breast milk, even though results from MS-HRM and direct bisulfite sequencing did not show the expected results. It also found that the co-exposure to BDE-47, BDE-209 in dams and offspring SD rat induced different gene expression levels associated β-oxidation and obesity.
To determine if PBDEs exposure through placenta and breast milk alters global DNA methylation, LINE-1 methylation patterns were measured in dams and offspring following exposure to mixture of BDE-47, BDE-209 by gavage for 18 days. The LINE-1 structure has a 5’ untranslated region (UTR) with internal promoter activity, two open reading frames (ORFs), a 3’UTR [16].
We analyzed whether gene expression levels of obesity makers are different from the period of exposure. To do so, the results of the gene expression between the exposed and the non-exposed groups were compared with PND 4 and the changes of gene expression with different period of exposure were compared among the exposed groups.
후속연구
For further study, it should include the epigenetic changes, β-oxidation and obesity gene expression by PBDEs exposure before pregnancy and increasing breast-feeding period.
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