Nuclear Oxidation of a Major Peroxidation DNA Adduct, M₁dG, in the Genome

Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M₁dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M₁dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system. Here, we report that M₁dG levels in intact DNA were increased from basal levels of 1 adduct per 10⁸ nucleotides to 2 adducts per 10⁶ nucleotides following adenine propenal treatment of RKO, HEK293, or HepG2 cells. We also found that M₁dG in genomic DNA was oxidized in a time-dependent fashion to a single product, 6-oxo-M₁dG (to-5 adducts per 10⁷ nucleotides), and that this oxidation correlated with a decline in M₁dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M₁dG (1 adduct per 10⁶ nucleotides) and the endogenous formation of 6-oxo-M₁dG. This is the first report of the production of 6-oxo-M₁dG in genomic DNA in intact cells, and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis, and repair.