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2003年就有约5700万张处方）包含共轭的马雌激素equilenin （EN）、equilin和8,9-脱氢雌酮（总成分约54%），以及内源性雌酮和17 -雌二醇。尽管乳腺癌和其他激素敏感组织癌症的长期风险已经广为人知，但女性仍然继续使用这种配方，因为它对绝经后系统的缓解有显著的健康益处。马和内源性雌激素对遗传毒性代谢物的代谢激活与激素替代疗法相关癌症的病因有关。众所周知，儿茶酚4-羟基喹啉（4-OHEN）是所有三种马雌激素中最重要的代谢物，它在体外、大鼠乳腺组织模型系统和人类乳腺组织中形成不寻常的环状、稳定的DNA加合物。4-OHEN与DNA中的胞嘧啶、腺嘌呤和鸟嘌呤的加合物已经被识别，每种加合物都有四种立体异构体形式。如果不通过细胞DNA修复机制去除，这些加合物可能会被DNA聚合酶错误地复制，从而引起突变，最终导致肿瘤的发展。核苷酸切除修复（NER）是一种主要的修复机制，它可以从人类细胞的DNA中去除大块加合物，其效率取决于加合物的结构特性及其在DNA结构中引起的扭曲。然而，没有关于12种不同的4-OHEN-DNA加合物如何被NER酶处理的信息。该项目的目标是确定这些加合物的不同立体异构体形式的构象如何影响它们被人类NER酶去除的效率。在Aim 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Aim 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Aim 3中，将使用细胞中的无细胞提取物和用4-OHEN处理的几种不同类型的培养细胞来评估DNA修复的抗性。鉴定人类细胞中对DNA修复具有抗性的4-OHEN-DNA加合物具有重要意义，因为这一信息可以帮助(1)开发生物标志物，用于识别有HR7不良反应风险的女性，以及(2)刺激设计新的改良雌激素替代药物，这些药物在DNA加合物水平上对核苷酸切除修复酶的抗性较低，因此作为潜在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.