Mutagenicity of 5-Hydroxymethyl-2′-deoxyuridine to Chinese Hamster Cells¹

5-Hydroxymethyluracil (HmUra) is formed from thymine in DNA through the action of ionizing radiation or reactive oxygen species generated by activated leukocytes. HmUra is removed from DNA by a specific DNA glycosylase, suggesting that it is also formed from endogenously generated reactive oxygen species and that its formation in DNA is potentially deleterious. To determine whether HmUra residues in DNA are mutagenic, hamster V79 cells were grown in the presence of 5-hydroxymethyl-2′-deoxyuridine (HmdUrd) which is incorporated into DNA, and mutagenicity at the ouabain- and thioguanine-resistant loci was determined. Levels of substitution ranged from 1/500 to 1/5,000 HmUra residues/thymine residues. There was slight mutagenicity at the thioguanine-resistant locus but none at the ouabain-resistant locus. The mutagenicity of HmdUrd, expressed as a function of HmUra substitution in DNA, was 1/30,000 in the hypoxanthine-guanine-phosphoribosyltransferase target gene. This low frequency indicates that the oxidation of thymine to HmUra in a preexisting AT base pair does not contribute significantly to the mutagenicity of ionizing radiation, because the yield of HmUra formed in DNA at mutagenic doses of radiation is too low. To determine whether repair of HmUra might be inhibited by ionizing radiation, cells were grown in medium containing HmdUrd and exposed to as much as 5 Gy of γ-irradiation, and the removal of HmUra from DNA was measured. No inhibition of repair was noted. Preirradiation of cells neither accelerated the rate of repair nor raised the level of HmUra-DNA glycosylase activity, indicating that repair of HmUra was not induced by this type of oxidative stress. Although the mutagenicity of HmUra residues in DNA is low, even a rare mutation might be sufficiently deleterious to higher organisms to promote the development of HmUra-DNA glycosylase activity.