Aging alters hepatic DNA hydroxymethylation, as measured by liquid chromatography/mass spectrometry.
Aging alters hepatic DNA hydroxymethylation, as measured by liquid chromatography/mass spectrometry.
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DOI：
10.15430/jcp.2014.19.4.301
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发表时间：
2014-12
影响因子：
2.5
通讯作者：
Choi SW
中科院分区：
文献类型：
--
作者：
Tammen SA;Dolnikowski GG;Ausman LM;Liu Z;Kim KC;Friso S;Choi SW
衰老是癌症最重要的危险因素之一。异常的表观遗传变化似乎是衰老和癌症的常见驱动因素。其中包括DNA甲基化和DNA羟甲基化的变化。 DNA CpG 二核苷酸中胞嘧啶的 5' 碳可以被甲基化或羟甲基化。与 5'-甲基胞嘧啶一样，5'-羟甲基胞嘧啶可能因衰老而发生变化，可能导致转录和癌症发展的下游变化。我们建立了一种利用液相色谱/质谱（LC/MS-MS）测量DNA中5'-甲基-2'-脱氧胞苷和5'-羟甲基-2'-脱氧胞苷的方法，并用该方法测量了年轻和年老C57Bl/6雄性小鼠肝组织中甲基化或羟甲基化的总胞嘧啶的百分比。通过用核酸酶 P1、磷酸二酯酶 I 和碱性磷酸酶连续消化，对 DNA 进行酶水解。将同位素异构体 [15N3]-2'-脱氧胞苷和（甲基-d3，环-6-d1）-5-甲基-2'-脱氧胞苷添加到 DNA 水解产物中作为内标。 DNA 甲基化和羟甲基化计算为基因组 DNA 中总脱氧胞苷的百分比。 DNA 甲基化和羟甲基化的日内变化分别为 3.45% 和 8.40%，而日间变化分别为 6.14% 和 17.68%。使用该方法确定老年小鼠的肝脏DNA羟甲基化水平相对年轻小鼠有所增加（0.32±0.02% vs. 0.24±0.01%，P = 0.02），而5'-甲基胞嘧啶没有显着变化。通过 LC/MS-MS 方法测量的 DNA 羟甲基化可以成为一种新型的衰老生物标志物。研究 DNA 羟甲基化在与年龄相关的癌症的发生和预防中的潜在作用将是有用的。
Aging is one of the most important risk factors for cancer. It appears that aberrant epigenetic changes might be a common driver of aging and cancer. Among them are changes in DNA methylation and DNA hydroxymethylation. The 5′ carbon of cytosines in CpG dinucleotides of DNA can be either methylated or hydroxymethylated. Like 5′-methylcytosine, changes in 5′-hydroxymethylcytosine may occur due to aging, potentially leading to downstream changes in transcription and cancer development. We set up a method to measure 5′-methyl-2′-deoxycytidine and 5′-hydroxymethyl-2′-deoxycytidine in DNA using liquid chromatography/mass spectrometry (LC/MS-MS) and used this method to measure the percentage of total cytosine that was either methylated or hydroxymethylated in the liver tissues of young and old C57Bl/6 male mice. The DNA was enzymatically hydrolyzed by sequential digestion with nuclease P1, phosphodiesterase I and alkaline phosphatase. The isotopomers [15N3]-2′-deoxycytidine and (methyl-d3, ring-6-d1)-5-methyl-2′-deoxycytidine were added to the DNA hydrolysates as internal standards. DNA methylation and hydroxymethylation were calculated as a percentage of total deoxycytidine in genomic DNA. Within day variations for DNA methylation and hydroxymethylation were 3.45% and 8.40%, while day to day variations were 6.14% and 17.68%, respectively. Using this method it was determined that hepatic DNA of old mice had increased levels of hydroxymethylation relative to young (0.32 ± 0.02% vs. 0.24 ± 0.01%, P = 0.02), with no significant changes in 5′-methylcytosine. DNA hydroxymethylation measured by LC/MS-MS method can be a novel biomarker of aging. It will be useful to investigate the potential role of DNA hydroxymethylation in the development and prevention of age-associated cancer.