Bioactivation of Dietary Phenols by Hemoproteins

描述(由申请人提供)：一种成熟的小鼠肺部两阶段致癌实验模型，包括单剂量的引发剂和多剂量的酚类抗氧化剂BHT。在这个系统中，肿瘤发生的戏剧性增强是由于肺细胞色素P450形成了对苯二酚的甲基。这些代谢物是亲电性的，并与细胞蛋白质结合，产生一系列最终导致增强肿瘤形成的效应。我们这个项目下一阶段的目标是确定BHT衍生的苯二酚甲基化合物的重要目标，并确定这些加合物在推广中的作用。由此产生的数据将解决我们的工作假设，即亲电启动子通过与参与细胞信号传递的蛋白质形成共价加合物来发挥作用。我们将利用一些有效的工具来检测和鉴定重要的加合物，包括具有不同促进活性的BHT的结构类似物，来自促进敏感(B)和抗促进(B-)小鼠品系的肺的Clara和2型细胞，来自小鼠肺上皮和致瘤兄弟姐妹的非致瘤细胞系，识别苯酚基的多克隆抗体，以及各种质谱学技术。这项工作的结果将使人们能够更深入地了解亲电代谢产物促进肿瘤的机制。具体目标如下。目的：从促进剂敏感(B)和抗促进剂(B-)品系的小鼠肺细胞以及致瘤和非致瘤的上皮细胞系中鉴定甲基喹酮的蛋白靶点，并评价其在促进启动细胞选择性克隆性增殖中的作用。在比较细胞组中形成的不同数量的蛋白质加合物将被标记以供识别。目的：研究上皮细胞系和小鼠肺分离细胞中代谢产生的苯醌甲醚对促进剂的生物化学效应。将在表达细胞色素P450的细胞系以及从B/B-小鼠分离的Clara和2型细胞中确定氧化应激和对抗氧化酶的影响。将确定甲基苯醌对缝隙连接细胞间通讯和细胞凋亡的影响；将使用同位素编码的亲和标签和质谱学来检测细胞系和分离细胞中的蛋白质表达和蛋白质磷酸化。

DESCRIPTION (provided by applicant): A well-developed experimental model for two-stage carcinogenesis in mouse lung involves a single dose of an initiator followed by multiple doses of the phenolic antioxidant BHT. The dramatic enhancement of tumorigenesis in this system is due to the formation of quinone methides by pulmonary cytochromes P450. These metabolites are electrophilic and bind to cellular proteins producing a number of effects that eventually lead to enhanced tumor formation. Our goals for the next phase of this project are to identify the important targets of BHT-derived quinone methides and determine the roles such adducts in promotion. The resulting data will address our working hypothesis that electrophilic promoters function through the formation of covalent adducts with proteins involved in cell signaling. We will utilize a number of effective tools to detect and identify important adducts, including structural analogs of BHT with differing promotion potencies, Clara and type 2 cells from the lungs of promotion-sensitive (B+) and promotion-resistant (B-) mouse strains, non-tumorigenic cell lines derived from mouse lung epithelia and the tumorigenic siblings, polyclonal antibodies that recognize the phenol group, and a variety of mass spectrometric techniques. The results from this work will enable improved insights into the mechanisms of tumor promotion by electrophilic metabolites. The specific aims are as follows. Aim 1: Identify selected protein targets of quinone methides in lung cells from promotion-sensitive (B+) and promotion-resistant (B-) strains of mice and in tumorigenic and non-tumorigenic epithelial cell lines, and assess their roles in promoting the selective clonal expansion of initiated cells. Protein adducts formed in differing amounts in the comparison cell groups will be flagged for identification. Aim 2: Investigate biochemical effects of relevance to promotion resulting from metabolically-generated quinone methides produced in epithelial cell lines and cells isolated from mouse lung. Oxidative stress and effects on antioxidant enzymes will be determined in cell lines expressing cytochrome P450 and in Clara and type 2 cells isolated from B+/B- mice. The effects of quinone methides on gap junctional intercellular communication and apoptosis will be determined; protein expression and protein phosphorylation will be examined in the cell lines and isolated cells using isotope-coded affinity tags and mass spectrometry.