Chemical and biological evidence for base propenals as the major source of the endogenous M1dG adduct in cellular DNA

The endogenous DNA adduct, M(1)dG, has been shown to arise in vitro in reactions of dG with malondialdehyde (MDA), a product of both lipid peroxidation and 4'-oxidation of deoxyribose in DNA, and with base propenals also derived from deoxyribose 4'-oxidation. We now report the results of cellular studies consistent with base propenals, and not MDA, as the major source of M(1)dG under biological conditions. As a foundation for cellular studies, M(1)dG, base propenals, and MDA were quantified in purified DNA treated with oxidizing agents known to produce deoxyribose 4'-oxidation. The results revealed a consistent pattern; Fe2+-EDTA and gamma-radiation generated MDA but not base propenals or M(1)dG, whereas bleomycin and peroxynitrite (ONOO-) both produced M(1)dG as well as base propenals with no detectable MDA. These observations were then assessed in Escherichia coli with controlled membrane levels of polyunsaturated fatty acids (PUFA). ONOO- treatment (2 mM) of cells containing no PUFA (defined medium with 18:0/stearic acid) produced 6.5 M(1)dG/10(7) deoxynucleotides and no detectable lipid peroxidation products, including MDA, as compared with 3.8 M(1)dG/10(7) deoxynucleotides and 0.07 mu g/ ml lipid peroxidation products with control cells grown in a mixture of fatty acids (0.5% PUFA) mimicking Luria-Bertani medium. In cells grown with linoleic acid (18:2), the level of PUFA rose to 54% and the level of MDA rose to 0.14 mu g/ml, whereas M(1)dG fell to 1.4/10(7) deoxynucleotides. Parallel studies with gamma- radiation revealed levels of MDA similar to those produced by ONOO- but no detectable M(1)dG. These results are consistent with base propenals as the major source of M(1)dG in this model cell system.