Isomeric HRT estrogen-DNA adducts: Structure and Repair

一种广泛使用的激素替代疗法（HRT）制剂（仅2003年就有约5700万张处方） 含有共轭马雌激素马烯雌酮（EN）、马烯雌酮和8，9-脱氢雌酮（总量约54%） 组合物），以及内源性雌酮和1713-雌二醇。虽然癌症的长期风险 乳房和其他乳房敏感组织已经得到了很好的宣传，妇女继续使用这种方法， 由于与绝经后系统的缓解相关的显著健康益处， 马和内源性雌激素的代谢活化遗传毒性代谢产物已牵连 与激素替代疗法相关的癌症的病因学。已知儿茶酚4-羟基马钱宁（4-OHEN）是 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 体外、大鼠乳腺组织模型系统和人乳腺组织中的DNA加合物。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，并且每种类型的加合物都是 以四种立体异构形式为特征。如果不通过细胞DNA修复机制去除， 加合物可能会被DNA聚合酶错误地复制，从而导致突变，最终导致 肿瘤的发展。核苷酸切除修复（NER）是主要的修复机制， 从人类细胞中的DNA中去除大体积加合物，其效率取决于 加合物和它们在DNA结构中引起的扭曲。然而，没有资料说明 12种不同的4-OHEN-DNA加合物被NER酶加工。这个项目的目标是确定 这些加合物的不同立体异构形式的构象如何影响 它们被人类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 将构建损伤。在目标2中，将通过高分辨率NMR分析它们的结构特征， 在目标3中，将使用无细胞技术评估对DNA修复的抗性， 在细胞中的提取物，以及在用4-OHEN处理的培养物中的几种不同类型的细胞中。 4-OHEN-DNA加合物对人类细胞中的DNA修复具有抗性是重要的，因为这些信息 可以帮助（1）开发生物标志物，用于识别有风险的女性对HR 7的不良影响，和（2） 刺激设计新的、改良的雌激素替代药物， 在DNA加合物水平上，切割修复酶，因此作为潜在的癌症引发剂的活性较低。

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.