The opposing effects of omega-3 vs. omega-6 fatty acids in colon carcinogenesis, i.e. the former are protective, and the latter are promoting, have been well-established in laboratory animals and in population studies. Current studies suggest that the protective effect of omega-3 fatty acids can be at least in part attributed to their ability to induce apoptosis as a response to oxidative DNA damage. Two types of DNA damage result from oxidation of fatty acids that are detected in vivo: the oxidized bases and the cyclic adducts derived from enals. In this proposal, we will investigate the roles of cyclic adducts in the induction of apoptosis by omega-3 fatty acids in the colon. We will focus on the cyclic deoxyguanosine adducts, the major sites of modification in DNA by enals: acrolein (Acr-dG), t-4-hydroxy-2-nonenal (HNE-dG), and malondialdehyde (MdG). These adducts represent a new class of endogenous DNA lesions from lipid peroxidation which are repaired by nuclear excision repair (NER). We found in vitro that Acr and HNE adducts are formed differentially from omega-3 and omega-6 fatty acids; omega-3 fatty acids are major sources of Acr adducts, whereas HNE adducts are formed exclusively from omega-6 fatty acids. Similar to the in vitro results, the study in vivo showed a significant increase in the formation of Acr-dG adducts in the colon of rats fed diets rich in omega-3 fatty acids (high fish oil diet). Hence, we hypothesize that omega-3 fatty acids, as major sources for cyclic adduct formation, increase the formation of cyclic dG adducts in colonic DNA, and if these adducts are not repaired efficiently, they will reach a threshold level to trigger apoptosis. We will carry out the following aims to examine this hypothesis:
Aim 1, we will determine, as an extension of our studies on Acr-dG, the relative contribution of omega-3 vs. omega-6 fatty acids in the formation of MdG in vitro and in vivo;
Aim 2, we will examine the relationships and kinetics of cyclic adduct formation with apoptosis in humans HT29 colon cells treated with omega-3 and omega-6 fatty acids;
Aim 3, we will determine the repair rates of the cyclic adducts in modified plasmid DNA by the NER pathway using HT29 cell extract;
Aim 4, we will investigate the roles of the cyclic adducts in apoptosis induced by fatty acids in NER(-) or NER (+) cells; and finally, Aim 5, we will examine, based on results from Aims 1 to 4, the roles of the cyclic adducts in modulating azoxymethane-induced colon carcinogenesis by dietary omega-3 vs. omega-6 fatty acids in F344 rats. These studies will provide insight into the significance of cyclic dG modifications as a class of endogenous DNA lesions in apoptosis and shed light on the mechanisms by which omega-3 fatty acids protect against colon carcinogenesis. ? ? ?
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