Isomeric HRT estrogen-DNA adducts: Structure and Repair

描述(由申请人提供)：一种广泛使用的激素替代疗法(HRT)配方(仅在2003年就有约5700万张处方)含有马匹结合雌激素(En)、马匹林和8，9-脱氢雌酮(总成分~54%)，以及内源性雌酮和17β-雌二醇。虽然乳腺癌和其他激素敏感组织的长期风险已经被广泛宣传，但女性继续使用这种配方，因为与缓解绝经后系统相关的显著健康益处。马和内源性雌激素对遗传毒性代谢物的代谢激活与HRT相关癌症的病因学有关。众所周知，儿茶酚4-羟基喹啉(4-OHEN)是三种马雌激素中最重要的代谢物，它在体外、在大鼠乳腺组织模型系统和在人类乳腺组织中形成不寻常的循环、稳定的DNA加合物。DNA中的4-羟基乙酸乙酯与胞嘧啶、腺嘌呤和鸟嘌呤的加合物已被识别，每种加合物都有四种立体异构体。如果不被细胞DNA修复机制移除，这些加合物可能会被DNA聚合酶错误地复制，从而导致突变，最终可能导致肿瘤的发展。核苷酸切除修复(NER)是从人类细胞DNA中去除大量加合物的主要修复机制，其效率取决于加合物的结构属性及其在DNA结构中造成的扭曲。然而，目前还没有关于NER酶如何处理12种不同的4-OHEN-DNA加合物的信息。该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人类NER酶去除它们的效率。在目标1中，将构建具有单个4-OHEN-DNA损伤的位点特异性寡核苷酸。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进行分析，而在目标3中，将使用细胞中的无细胞提取物以及在经4-OHNE处理的几种不同类型的细胞中评估对DNA修复的抵抗力。在人类细胞中识别对DNA修复具有抵抗力的4-OHEN-DNA加合物具有重要意义，因为这些信息可以帮助(1)开发生物标记物，用于识别受HR7不利影响的女性，以及(2)在DNA加合物水平上刺激对核苷酸切除修复酶抵抗力较低的新型改良雌激素替代药物的设计，因此作为潜在的癌症启动剂的活性较低。

DESCRIPTION (provided by applicant): 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 17beta-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 stereoisomeric 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 excision repair enzymes at the DNA adduct level, and thus less active as potential cancer initiating agents.