PEROXY RADICAL MEDIATED DNA DAMAGE

在人类中的流行病学数据以及在动物模型中的许多研究有力地表明，高多不饱和脂肪饮食会导致癌症发病率的显着增加。膳食脂肪直接影响细胞膜的不饱和脂肪含量。与饱和脂肪不同，多不饱和脂肪酸(PUFAs)可能会发生自氧化，产生亲电性醛和能够与DNA反应并破坏DNA的反应性过氧化自由基。到目前为止，人们的注意力主要集中在DNA中形成的醛基加合物对DNA的损伤上。在这项建议中，我们概述了一个化学原理，表明过氧基也可能通过形成DNA碱基氧化产物来破坏DNA。这项拟议研究的总体目标是确定过氧化自由基氧化损伤是否对多不饱和脂肪的遗传毒性效应有实质性影响。我们将研究由明确的化学方法产生的过氧基对核苷和质粒DNA的反应性。核苷损伤产物将通过一系列分析方法进行鉴定，包括LC-MS、GC-MS和荧光光谱。我们将利用损伤特异性糖基酶和聚合酶链式反应定位质粒和基因组DNA中的氧化和醛加合物碱基损伤产物。我们还将确定在正常和修复缺陷的人成纤维细胞中进行正向突变试验所产生的突变谱。介绍了一种测定完整细胞核膜过氧化所致DNA损伤的方法。将构建合成膜-DNA复合体，以系统地评估膜填充效应和不饱和程度对DNA损伤的影响。基于醛加合物形成途径或过氧化自由基氧化途径产生的DNA损伤产物的光谱，我们相信我们可以区分哪一种途径对多不饱和脂肪过氧化引起的整体遗传毒性损伤的贡献更大。对饮食多不饱和脂肪如何造成遗传毒性损害的了解的增加，可能有助于开发减少这类内源性DNA损害的策略。

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.