Base propenals as a source of M1G in isolated DNA and cells: Deoxyribose 4’-oxidation produces either malondialdehyde or base propenals. Free

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The oxidation of cellular macromolecules yields a variety of electrophilic toxins that can react with DNA to form adducts. An example of this is the pyrimidopurinone adduct of guanine, M1G, that can arise in reactions of DNA with malondialdeyde (MDA), a product of the peroxidation of polyunsaturated fatty acids (PUFA). We have shown that M1G can also arise from reactions of dG in DNA with base propenals, products of 4’-oxidation of deoxyribose by bleomycin, peroxynitrite and other oxidants. We have undertaken a series of in vitro and cellular studies aimed at defining the source of M1G and the role of deoxyribose 4’-oxidation in its formation. Using both HPLC with post-column TBA derivatization for base propenals and MDA and an immunoblot assay for M1G, we have determined that the generation of base propenals correlates directly with M1G formation for peroxynitrite and bleomycin. However, we determined that gamma-radiation and Fe(II)-EDTA, both of which cause 4’-oxidation of deoxyribose in DNA, produce malondialdehyde instead of base propenals and that there was no M1G formed in DNA treated with either of these oxidants. These in vitro studies reveal the complexity of 4’-oxidation in deoxyribose and set the stage for cellular work with E. coli and human TK6 cells. The premise here was that by varying the PUFA content of cellular membranes, we could control the formation of MDA and thus assess its role in M1G formation. E. coli were grown in defined medium containing a wild-type mixture of fatty acids (WT), stearic acid (18:0) or linoleic acid (18:2). GC analysis revealed a complete absence of PUFA in the 18:0 cells, 5% PUFA in the WT cells and 50% PUFA in the 18:2 cells. Subsequent treatment of these cells with peroxynitrite revealed an inverse correlation of M1G with the membrane PUFA content, with no detectable MDA formation in the 18:0 cells that also experienced the highest levels of M1G. Similar results were obtained with TK6 cells exposed to SIN-1, a peroxynitrite generator, though the level of PUFA variation was limited by a cellular requirement for PUFA. Further studies are underway to quantify other lipid peroxidation-derived DNA adducts and to definitively define the contribution of MDA to M1G formation in cells.