Carcinogenic Metabolites Formed from Antiestrogens

开发“完美的”选择性雌激素受体调节剂(SERM)在 绝经后妇女的健康。SERM的有益和不良副作用直接 类似于雌激素，雌激素是已知的致癌物质，其机制涉及氧化代谢以 氧化还原活性/亲电性藜麦。我们假设，SERM的不利影响也与 他们的氧化新陈代谢成藜芦醇。这些藜麦类化合物是否有害目前尚不清楚，它 本提案的重点是确定结构对这些机制的影响，以努力提供 导致“完美的”SERM发展的关键信息。具体目标是：1.效果 SERM藜麦类化合物的形成和反应活性的结构。我们已经证明了经典的甲基对苯二酚， 甲基二苯二酚和邻苯二酚是由三苯基乙烯、苯并硫苯和 其他SERM。我们计划研究新类别的小型企业风险管理人员的相对能力，例如 将苯并呋喃和拉索福昔芬以及礼来公司的萘酚类似物氧化成藜芦醇。这一速度 将在亚细胞组分和石川组分中研究奎宁的形成、类型以及它们的反应活性 子宫内膜细胞。2.SERM藜麦类化合物的蛋白质靶标是什么？在目前的提案中，我们计划使用 用于检测蛋白质的COATAG方法学和“点击化学”或改良的Staudinger连接方法 大鼠乳腺亚细胞部分和Ishikawa细胞的共价修饰。目标蛋白将是 用亲和素亲和层析分离，2D电泳法分离，消化，分析 MALDI-TOF和LC-MS-MS。我们预测，SERM藜麦的反应性将对 哪些蛋白质被修饰，以及目标蛋白质中的蛋白质烷基化位置。3.做SERM 藜麦能修饰DNA并诱导细胞转化吗？这个目标将集中在如何类型和反应性 形成的血清奎宁将决定DNA损伤的程度。脱氧核苷类药物的初步模型研究 DNA将能够表征稳定的DNA加合物，分析净化加合物，以及 DNA氧化的测定。然后，我们将分析SERM诱导的石川细胞系的DNA损伤。 将使用醛反应探针分析和颠倒来直接定量阿嘌呤位点(AP) 这些突变损伤引起的转变将通过错配捕获方法检测到。 最后，将在MCF-10A细胞中进行转化研究，转化的克隆将被 移植到裸鼠体内，研究其诱导肿瘤形成的能力。这些研究将 阐明每种SERM的奎宁形成和细胞靶标的相对重要性，从而使 反应性与影响SERM藜麦类化合物行为的结构的相关性 就会出现在细胞中。

Development of the "perfect" selective estrogen receptor modulator (SERM) is of paramount importance in postmenopausal women's health. The beneficial and undesirable side effects of SERMs are directly analogous to estrogens, which are known carcinogens through a mechanism involving oxidative metabolism to redox active/electrophilic quinoids. We hypothesize that the adverse effects of SERMs are also related to their oxidative metabolism to quinoids. Whether these quinoids are detrimental are unknown at this time and it is the focus of this proposal to determine the effect of structure on these mechanisms in an effort to provide crucial information leading to the development of the "perfect" SERM. The specific aims are: 1. Effect of structure on the formation and reactivity of SERM quinoids. We have shown that classical quinone methides, di-quinone methides, and o-quinones are produced from triphenylethylene, benzothiophene, and miscellaneous SERMs. We plan to examine the relative abilities of new classes of SERMs such as bazodoxifene and lasofoxifene as well as the Lilly naphthol analogs to be oxidized to quinoids. The rate of formation, type of quinoid, as well as their reactivity will be studied in subcellular fractions and Ishikawa endometrial cells. 2. What are the protein targets of SERM quinoids? In the current proposal, we plan to use the COATag methodology and "click chemistry" or modified Staudinger ligation approaches to examine protein covalent modification in rat mammary subcellular fractions and Ishikawa cells. The targeted proteins will be isolated using avidin affinity chromatography, separated by 2D electrophoresis, digested, and analyzed by MALDI-TOF and LC-MS-MS. We predict that the reactivity of SERM quinoids will have a strong influence on which proteins are modified as well as sites of protein alkylation within the target proteins. 3. Do SERM quinoids modify DNA and induce cellular transformation? This aim will focus on how the type and reactivity of SERM quinoid formed will dictate the extent of DNA damage. Initial model studies with deoxynucleosides and DNA will allow characterization of stable DNA adducts, analysis of depurinating adducts, as well as determination of DNA oxidation. We will then analyze SERM-induced DNA damage in Ishikawa cell lines. Apurinic sites (AP) will be directly quantified using the aldehyde reactive probe assay and the transversions and transitions resulting from these mutagenic lesions will be detected by mismatch-capture methodology. Finally, transformation studies will be performed in MCF-10A cells and the transformed clones will be implanted into athymic nude mice to investigate their ability to induce tumor formation. These studies will elucidate the relative importance of quinoid formation and cellular targets for each SERM, thereby enabling correlations of reactivity with structure from which general principles influencing the behavior of SERM quinoids in cells will emerge.