Carcinogenic Metabolites Formed from Antiestrogen

描述（由申请人提供）：“完美的”选择性雌激素受体调节剂（SERM）的开发对绝经后妇女的健康至关重要。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，其机制涉及氧化代谢为氧化还原活性/亲电类醌。我们假设serm的不良反应也与它们对类黄酮的氧化代谢有关。这些类黄酮是否有害目前尚不清楚，本提案的重点是确定结构对这些机制的影响，以提供导致“完美”SERM发展的关键信息。具体目标是：1。结构对SERM类黄酮形成及反应性的影响。我们已经证明，经典的醌类化合物、二醌类化合物和邻醌类化合物是由三苯乙烯、苯并噻吩和其他serm合成的。我们计划研究新型SERMs如bazodoxifene和lasofoxifene以及Lilly萘酚类似物被氧化为类醌的相对能力。将在亚细胞部分和石川子宫内膜细胞中研究类醌的形成速率、类型以及它们的反应性。2. SERM quinoids的蛋白靶点是什么？在目前的提案中，我们计划使用COATag方法和“点击化学”或改进的Staudinger连接方法来检测大鼠乳腺亚细胞部分和石川细胞中的蛋白质共价修饰。目的蛋白将通过亲和层析分离，通过2D电泳分离，消化，并通过MALDI-TOF和LC-MS-MS分析。我们预测，SERM类醌的反应性将对哪些蛋白质被修饰以及目标蛋白质内的蛋白质烷基化位点有很大的影响。3. SERM类醌是否修饰DNA并诱导细胞转化？这一目标将集中于SERM类醌的类型和反应性如何决定DNA损伤的程度。脱氧核苷和DNA的初步模型研究将允许表征稳定的DNA加合物，分析去纯化加合物，以及确定DNA氧化。然后，我们将分析serm诱导的石川细胞系DNA损伤。无尿嘧啶位点（AP）将使用醛反应探针法直接定量，由这些诱变病变引起的翻转和转变将通过错配捕获方法检测。最后，将在MCF-10A细胞中进行转化研究，并将转化的克隆植入胸腺裸鼠体内，研究其诱导肿瘤形成的能力。这些研究将阐明每种SERM的类醌形成和细胞靶点的相对重要性，从而使反应性与结构的相关性成为可能，从而揭示影响细胞中SERM类醌行为的一般原理。公共卫生相关性：“完美”血清素的发展对绝经后妇女的健康至关重要，可以预防骨质疏松症、心血管疾病和潮热。然而，一些SERMs通过形成反应性化合物增加了某些癌症的风险。本提案的重点是研究这些潜在的致癌活性化合物，以努力提供导致“完美”SERM发展的关键信息。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Development of the "perfect" SERM is of paramount importance in postmenopausal women's health in order to prevent osteoporosis, cardiovascular disease, and hot flashes. However, some SERMs increases the risk of some cancers through formation of reactive compounds. It is the focus of this proposal to investigate these potentially carcinogenic reactive compounds in an effort to provide crucial information leading to the development of the "perfect" SERM.