Epidemiological data in humans as well as many studies in animal models strongly suggests that diets high in polyunsaturated fats leads to a significant increase in the incidence of cancer. Dietary fats directly influence the unsaturated lipid content of cellular membranes. Unlike saturated fats, polyunsaturated fatty acids (PUFAs) may undergo autooxidation generating both electrophilic aldehydes and reactive peroxyl radicals capable of reacting with and damaging DNA. To date, most attention has been focused on DNA damage which results from the formation of aldehyde-base adducts in DNA. In this proposal, we outline a chemical rationale which suggests that peroxyl radicals may also damage DNA by forming DNA base oxidation products. The overall goal of this proposed research is to determine if peroxyl radical oxidative damage contributes substantially to the genotoxic effects of polyunsaturated fats. We will examine the reactivity of peroxyl radicals generated by unambiguous chemical methods towards both nucleosides and plasmid DNA. Nucleoside damage products will be identified by a battery of analytical methods including HPLC-MS, GC-MS and fluorescence spectroscopy. We will map oxidation and aldehyde adduct base damage products in plasmid and genomic DNA using damage specific glycosylases and PCR. We will also determine the spectrum of mutations generated in forward mutation assays in normal and repair deficient human fibroblasts. A method for determining the DNA damage resulting from nuclear membrane peroxidation in intact cells is described. Synthetic membrane-DNA complexes will be constructed to systematically evaluate membrane packing effects and degree of unsaturation on the DNA damage profile. Based upon the spectrum of DNA damage products expected from either the formation of aldehyde adducts or peroxyl radical oxidation pathways, we believe we can distinguish which pathway contributes more significantly to the overall genotoxic damage induced by the peroxidation of polyunsaturated fats. An increased understanding of how dietary polyunsaturated fats cause genotoxic damage may allow for the development of strategies for reducing this class of endogenous DNA damage.
