Aging Alters Hepatic DNA Hydroxymethylation, as Measured by Liquid Chromatography/Mass Spectrometry

BACKGROUND: 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. METHODS: 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 [(15)N(3)]-2′-deoxycytidine and (methyl-d(3), ring-6-d(1))-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. RESULTS: 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. CONCLUSIONS: 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.