DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro

Susan Joyce Duthie, A Hawdon

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    Abstract

    Folic acid is essential for the synthesis and repair of DNA. We report the effects of folate depletion on DNA stability in normal human lymphocytes in vitro. DNA strand breakage, uracil misincorporation, oxidative DNA base damage, and DNA repair capability were determined using variants of the comet assay (single cell gel electrophoresis), Lymphocyte proliferation was measured as an indicator of normal replication. Lymphocytes isolated from human venous blood were stimulated to grow in either complete medium containing folic acid (1 ng/ml-2 mu g/ml) or medium deficient in folic acid for up to 10 days. Cells prepared for comet analysis were treated either with the bacterial DNA repair enzyme endonuclease III to determine the level of oxidized pyrimidines in lymphocyte DNA or with uracil DNA glycosylase, which detects misincorporated uracil, Cell number and viability were measured. Normal human lymphocyte DNA contained detectable amounts of misincorporated uracil (estimated as approximately 1000 per cell). DNA strand breakage and uracil misincorporation increased in a time- and concentration-dependent manner after lymphocytes were cultured with decreasing amounts: of folic acid. DNA damage was induced at folic acid concentrations routinely observed in plasma from the human population (1-10 ng/ml), Lymphocytes cultured under folate-deficient conditions failed to grow normally compared with control cells. However, all lymphocytes remained viable as measured by Trypan blue exclusion. Cells deprived of folate were unable to efficiently repair oxidative DNA damage induced by hydrogen peroxide. Inhibition of repair was maximal after 8 days in culture. Folate supply had no effect on the level of oxidized pyrimidines in lymphocyte DNA, even after 10 days in culture, suggesting that folate deficiency increases uracil misincorporation relatively specifically. These in vitro results: help to determine the mechanism(s) through which folic acid maintains DNA stability.

    Original languageEnglish
    Pages (from-to)1491-1497
    Number of pages7
    JournalThe FASEB Journal
    Volume12
    Issue number14
    Publication statusPublished - Nov 1998

    Keywords

    • DNA breaks
    • misincorporated uracil
    • DNA repair
    • oxidized pyrimidines
    • comet assay
    • FACTORS INFLUENCING MUTATION
    • HAMSTER OVARY CELLS
    • FOLATE-DEFICIENCY
    • ULCERATIVE-COLITIS
    • HPRT LOCUS
    • VITAMIN-C
    • DAMAGE
    • CANCER
    • SUPPLEMENTATION
    • DYSPLASIA
    • DNA breaks
    • misincorporated uracil
    • DNA repair
    • oxidized pyrimidines

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