PEROXY RADICAL MEDIATED DNA DAMAGE

人类流行病学数据以及许多动物模型研究强烈表明，多不饱和脂肪含量高的饮食会导致癌症发病率显著增加。膳食脂肪直接影响细胞膜不饱和脂质的含量。与饱和脂肪不同，多不饱和脂肪酸（PUFAs）可以发生自氧化，产生亲电醛和活性过氧自由基，能够与DNA反应并破坏DNA。迄今为止，大多数注意力都集中在DNA中醛基加合物形成的DNA损伤上。在这个提议中，我们概述了一个化学原理，表明过氧自由基也可能通过形成DNA碱基氧化产物来损害DNA。这项拟议研究的总体目标是确定过氧自由基氧化损伤是否对多不饱和脂肪的遗传毒性作用有重大贡献。我们将研究通过明确的化学方法对核苷和质粒DNA产生的过氧自由基的反应性。核苷损伤产物将通过包括HPLC-MS， GC-MS和荧光光谱在内的一系列分析方法进行鉴定。我们将利用损伤特异性糖基酶和PCR绘制质粒和基因组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类成纤维细胞的正向突变测定中产生的突变谱。描述了一种在完整细胞中测定由核膜过氧化引起的DNA损伤的方法。将构建合成膜-DNA复合物，系统地评估膜填充效应和不饱和程度对DNA损伤谱的影响。根据醛加合物或过氧自由基氧化途径形成的DNA损伤产物的谱，我们相信我们可以区分哪种途径对多不饱和脂肪过氧化引起的整体遗传毒性损伤贡献更大。对膳食多不饱和脂肪如何引起基因毒性损伤的进一步了解，可能有助于制定减少这类内源性DNA损伤的策略。

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.