Author/Authors :
Chango, Abalo Institut Polytechnique LaSalle Beauvais - Unit EGEAL and Laboratory of Nutritional Genomics, France , Chango, Abalo INSERM, France , Abdel Nour, Afif M. Institut Polytechnique LaSalle Beauvais - Unit EGEAL and Laboratory of Nutritional Genomics, France , Niquet, Celine Platform of Analytical Chemistry, France , Tessier, Frederic J. Platform of Analytical Chemistry, France
Abstract :
Objective: To develop a method for the simultaneous measurement of 5-methylcytosine (5-metC) and 2’-deoxyuridine monophosphate (dU). Materials and Methods: Genomic DNA was extracted from the HepG2 cell line grown in experimental complete medium or in folate-depleted medium. Samples were treated with RNAse A and RNAse T1 to avoid any RNA contamination. High-performance liquidchromatography (HPLC)/electrospray ionization mass spectrometric (ESI-MS) method was used to separate nucleotides after enzymatic hydrolysis of DNA with nuclease P1, phosphodiesterase I and alkaline phosphatase. Results: Using this sensitive new methodology, we were able to quantifysimultaneously the concentration of DNA-5-metC and DNAuracil in DNA. The linear correlation coefficient (R 2 ) between the MS signal and the concentration of 5-metC in a range of 0.5–5 muM or dU in a range of 10–100 mu M was 0.9954 and 0.9999, respectively. The coefficient of variation was 16.94 and 14.77%, respectively. The applicability of this assay is demonstrated by detection of a decrease in 5-metC% and elevation of dU/thymidylate (dT) into genomic DNA extracted from the HepG2 cell line grown in a folate-depleted me-dium. Conclusion: Our results confirm that the HPLC/ESI-MS method reported earlier for measuring 5-metC allows measurement of uracil misincorporation into DNA.
