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Title: Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes
Authors: Mashiyama, S.T.
Courtemanche, C.
Elson-Schwab, I.
Ames, B.N.
Crott, J.
Fenech, M.
Lee, B.L.
Ong, C.N. 
Keywords: Cultured human lymphocytes
DNA damage
DNA-uracil detection
Folate deficiency
Gas chromatography-mass spectrometry
Issue Date: 2004
Citation: Mashiyama, S.T., Courtemanche, C., Elson-Schwab, I., Ames, B.N., Crott, J., Fenech, M., Lee, B.L., Ong, C.N. (2004). Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes. Analytical Biochemistry 330 (1) : 58-69. ScholarBank@NUS Repository.
Abstract: Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100pg of uracil yielded a mean uracil measurement of 110.1pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12nM, and minima at 0 and 3000nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000nM folate for 10 days, P<0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3μg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans. © 2004 Elsevier Inc. All rights reserved.
Source Title: Analytical Biochemistry
ISSN: 00032697
DOI: 10.1016/j.ab.2004.03.065
Appears in Collections:Staff Publications

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