title: Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes.
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 0-3000 nM 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 100 pg of uracil yielded a mean uracil measurement of 110.1 pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12 nM, and minima at 0 and 3000 nM 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 3000 nM 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 3 microg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.
authors.affiliation: Department of Cell and Molecular Biology, University of California, Berkley, CA 94720, USA.
authors.forename: Susan T
authors.initials: ST
authors.lastname: Mashiyama
authors.forename: Chantal
authors.initials: C
authors.lastname: Courtemanche
authors.forename: Ilan
authors.initials: I
authors.lastname: Elson-Schwab
authors.forename: Jimmy
authors.initials: J
authors.lastname: Crott
authors.forename: Bee Lan
authors.initials: BL
authors.lastname: Lee
authors.forename: Choon Nam
authors.initials: CN
authors.lastname: Ong
authors.forename: Michael
authors.initials: M
authors.lastname: Fenech
authors.forename: Bruce N
authors.initials: BN
authors.lastname: Ames
mesh_terms: D002478:Cells, Cultured; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D005492:Folic Acid / Q000378:metabolism; D005494:Folic Acid Deficiency; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D009705:Nucleosides / Q000494:pharmacology; D012680:Sensitivity and Specificity; D014498:Uracil / Q000032:analysis* / Q000378:metabolism
chemical_list: D009705:Nucleosides; D014498:Uracil; D004247:DNA; D005492:Folic Acid