Carcinogenic Metabolites Formed from Antiestrogen

描述(申请人提供)：开发“完美的”选择性雌激素受体调节剂(SERM)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，雌激素是一种已知的致癌物质，其机制涉及氧化代谢以氧化还原活性/亲电性藜麦。我们推测，SERM的不利影响也与它们对藜芦醇的氧化代谢有关。目前尚不清楚这些藜麦类化合物是否有害，本提案的重点是确定结构对这些机制的影响，以努力提供关键信息，从而开发出“完美的”SERM。具体目标是：1.结构对SERM藜芦醇类化合物形成和活性的影响。我们已经证明，经典的甲基对苯二酚、甲基二对苯二酚和邻苯二酚都是由三苯乙基、苯并噻吩类和各种SERM生成的。我们计划研究新的类SERM的相对能力，如Bazodoxifene和lasofoxifene以及礼来公司的萘酚类似物被氧化为藜芦醇的能力。将在亚细胞部分和石川子宫内膜细胞中研究奎宁的形成速度、类型以及它们的反应性。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COATag方法和“点击化学”或改进的Staudinger连接方法来检查大鼠乳腺亚细胞部分和Ishikawa细胞中的蛋白质共价修饰。目的蛋白用亲和层析法分离，2D电泳法分离，酶切后用MALDI-TOF和LC-MS-MS进行分析。我们预测，SERM藜麦类化合物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产生强烈影响。3.SERM藜麦能修饰DNA并诱导细胞转化吗？这一目标将集中在SERM奎宁形成的类型和活性如何决定DNA损伤的程度。对脱氧核苷和DNA的初步模型研究将允许对稳定的DNA加合物进行表征，对净化加合物进行分析，以及确定DNA氧化。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直接对无嘌呤位点(AP)进行定量，并将通过错配捕获方法检测这些突变损伤所导致的颠换和转变。最后，将在MCF-10A细胞中进行转化研究，并将转化的克隆植入裸鼠体内，以观察其诱导肿瘤形成的能力。这些研究将阐明奎诺类化合物的形成和每个SERM的细胞靶标的相对重要性，从而使反应性与结构相关联，从而揭示影响SERM奎诺类化合物在细胞中行为的一般原理。公共卫生相关性：为了预防骨质疏松症、心血管疾病和潮热，发展“完美的”SERM对绝经后妇女的健康至关重要。然而，一些SERM通过形成活性化合物增加了某些癌症的风险。这项提案的重点是调查这些潜在的致癌活性化合物，以努力提供关键信息，从而开发出“完美的”SERM。

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.