Uracil misincorporation in human DNA detected using single cell gel electrophoresis

Poor folate status may be important in the aetiology of several epithelialcell malignancies including cancer of the uterine cervix. Folic acid isessential in the synthesis of purine nucleotides and the pyrimidinenucleoside thymidine and it is probable that imbalances in these DNAprecursors negatively effect DNA stability and may ultimately lead tomalignant transformation. The development of a modified 'comet assay' usingthe bacterial DNA repair enzyme uracil DNA glycosylase, to detectmisincorporated uracil in human DNA is reported here. The effect ofperturbing folic acid and deoxyuridine levels on uracil misincorporation innormal human lymphocytes and cultured human tumour cells was investigatedusing this assay. HeLa cells and peripheral human lymphocytes incubated asagarose-embedded nucleoids, with 1 unit of uracil DNA glycosylase permicrog of DNA, contained low levels of uracil in their DNA. Both HeLa cellsand stimulated human lymphocytes cultured in folate-deficient medium weregrowth arrested. Incubating human lymphocytes in folate-deficient mediumsignificantly increased the level of uracil detected compared with controlcells. HeLa cells showed an increase in non-specific DNA damage (strandbreaks). Deoxyuridine (100 microM) significantly increased the level ofuracil detected in the DNA of both folate-deficient and control HeLa cells.It appears that this modified comet assay specifically detectsmisincorporated uracil in single human cells. It should, therefore, provevaluable in determining the role of folic acid status in DNA instabilityand cancer.