Bioactivation of Dietary Phenols by Hemoproteins

一个成熟的小鼠肺两阶段致癌实验模型涉及单剂量 的引发剂，随后是多剂量的酚类抗氧化剂BHT。戏剧性的增强 该系统中的肿瘤发生是由于肺细胞色素P450形成醌甲基化物。 这些代谢物是亲电的，并与细胞蛋白质结合，产生许多效应， 最终导致肿瘤的形成。本项目下一阶段的目标是确定 BHT衍生的醌甲基化物的重要目标，并确定这种加合物在促进作用。的 所得数据将解决我们的工作假设，即亲电启动子通过形成 与参与细胞信号传导的蛋白质形成共价加合物。我们将利用一些有效的工具， 检测和鉴定重要的加合物，包括具有不同促进效力的BHT结构类似物， 来自促进敏感（B+）和促进抗性（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，多克隆 识别酚基的抗体和各种质谱技术。结果 从这项工作将使更好地了解肿瘤促进机制的亲电 代谢物。具体目标如下。目的1：鉴定醌甲基化物的选定蛋白质靶点 在来自小鼠的促进敏感（B+）和促进抗性（B-）品系的肺细胞中以及在致瘤性 和非致瘤性上皮细胞系，并评估它们在促进选择性克隆扩增中的作用。 启动的细胞。将标记比较细胞组中以不同量形成的蛋白加合物 以确认身份目的2：研究与促进相关的生物化学效应， 在上皮细胞系和从小鼠分离的细胞中产生的代谢产生的醌甲基化物 肺。氧化应激和对抗氧化酶的影响将在表达 细胞色素P450以及Clara和分离自B+/B-小鼠的2型细胞中的细胞毒性。醌甲基化物的影响 将确定对间隙连接细胞间通讯和凋亡的影响;蛋白质表达和 将使用同位素编码的亲和标签在细胞系和分离的细胞中检查蛋白质磷酸化 和质谱分析。

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.