Isomeric HRT estrogen-DNA adducts: Structure and Repair

广泛使用的激素替代疗法(HRT)配方(仅2003年就开出约5700万张处方) 含有马类结合雌激素马精灵(En)、马琳和8，9-脱氢雌酮(总计~54%)。 成分)，以及内源性雌酮和1713-雌二醇。尽管罹患癌症的长期风险 乳房和其他对激素敏感的组织已经得到了很好的宣传，女性继续使用这一点 这是因为与缓解绝经后系统有关的重大健康益处。 马和内源性雌激素对遗传毒性代谢物的代谢激活已被牵连。 在与激素替代疗法相关的癌症病因学方面。已知邻苯二酚4-羟基喹啉(4-OHEN)是 在所有三种马雌激素中最重要的代谢物，它形成不寻常的循环，稳定的 DNA加合物在体外，在大鼠乳腺组织模型系统中，在人类乳腺组织中。4-的加合物 DNA中含有胞嘧啶、腺嘌呤和鸟嘌呤的化合物已被识别，每种类型的加合物都是 以四种立体异构体为特征的。如果不是通过细胞DNA修复机制移除，如 加合物可能被DNA聚合酶错误地复制，从而导致突变，最终可能导致 与肿瘤的发展有关。核苷酸切除修复(NER)是主要的修复机制 从人类细胞的DNA中去除笨重的加合物，其效率取决于 加合物及其在DNA结构中引起的扭曲。然而，目前还没有关于 NER酶处理12种不同的4-OHEN-DNA加合物。这个项目的目标是确定 这些加合物的不同立体异构体的构象如何影响效率 它们被人的NER酶去除。在目标1中，具有单个4-OHEN-DNA的位点特异性寡核苷酸 损伤将会被建造。在目标2中，它们的结构特征将通过高分辨率核磁共振和 计算技术，而在目标3中，将使用无细胞来评估对DNA修复的抵抗力 细胞中的提取物，以及经4-OHEN处理的几种不同类型细胞的提取物。 在人类细胞中抵抗DNA修复的4-OHEN-DNA加合物意义重大，因为这一信息 可以帮助(1)开发生物标记物，用于识别处于HR7不良影响风险中的妇女，以及(2) 刺激设计对核苷酸抗药性较低的新的、经修改的雌激素替代药物 _，DNA加合物水平的Xcision修复酶，因此作为潜在的癌症启动剂活性较低。

A widely used hormone replacement therapy (HRT) formulation (~ 57 million prescriptions in 2003 alone) contains the conjugated equine estrogens equilenin (EN), equilin, and 8,9-dehydro-estrone (total ~ 54% composition), and the endogenous estrone and 1713-estradiol. Although the long term risks of cancers of the breast and other hormone-sensitive tissues have been well publicized, women continue to use this formulation because of the significant health benefits associated with the relief of post-menopausal systems. The metabolic activation of equine and endogenous estrogens to genotoxic metabolites has been implicated in the etiology of cancers associated with HRT. It is known that the catechol 4-hydroxyequilenin (4-OHEN) is the most significant metabolite among all three equine estrogens, and that it forms unusual cyclic, stable DNA adducts in vitro, in rat mammary tissue model systems, and in human breast tissue. Adducts of 4- OHEN with cytosine, adenine, and guanine in DNA have been recognized, and each type of adduct is characterized by four stereoisomedc forms. If not removed by cellular DNA repair mechanisms, such adducts may be incorrectly replicated by DNA polymerases, thus causing mutations that may ultimately lead to the development of tumors. Nucleotide excision repair (NER) is the major repair mechanisms that removes bulky adducts from DNA in human cells with efficiencies that depend on the structural properties of the adducts and the distortions they cause in the DNA structure. However, there is no information on how the 12 different 4-OHEN-DNA adducts are processed by NER enzymes. The goal of this project is to determine how the conformations of the different stereoisomeric forms of these adducts influence the efficiencies of their removal by human NER enzymes. In aim 1, site-specific oligonucleotides with single 4-OHEN-DNA lesions will be constructed. In aim 2, their structural features will be analyzed by high resolution NMR and computational techniques, while in aim 3, the resistance to DNA repair will be evaluated using cell free extracts in cells, and in several different types of cells in culture treated with 4-OHEN.The identification of the 4-OHEN-DNA adducts that are resistant to DNA repair in human cells is significant because this information could help to (1) develop biomarkers for identifying women at risk to the adverse effects of HR7, and (2) stimulate the design of new, modified estrogen replacement drugs that are less resistant to nucleotide _,xcision repair enzymes at the DNA adduct level, and thus less active as potential cancer initiating agents.