Bioactivation of Dietary Phenols by Hemoproteins

描述（由申请方提供）：一种成熟的小鼠肺两阶段致癌作用实验模型涉及单次给药引发剂，随后多次给药酚类抗氧化剂BHT。该系统中肿瘤发生的显著增强是由于肺细胞色素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，产生许多最终导致肿瘤形成增强的效应。我们下一阶段的目标是确定BHT衍生的醌甲基化物的重要靶标，并确定这种加合物在促进中的作用。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导的蛋白质形成共价加合物来发挥作用。我们将利用许多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-）小鼠品系肺的Clara和2型细胞、来自小鼠肺上皮和致瘤性同胞的非致瘤性细胞系、识别苯酚基团的多克隆抗体，和各种质谱技术。这项工作的结果将使亲电代谢物促进肿瘤的机制得到更好的了解。具体目标如下。目标1：在来自小鼠的促进敏感（B+）和促进抗性（B-）品系的肺细胞以及致瘤性和非致瘤性上皮细胞系中鉴定醌甲基化物的选定蛋白质靶标，并评估其在促进起始细胞的选择性克隆扩增中的作用。将标记比较细胞组中以不同量形成的蛋白加合物以进行鉴别。目标二：研究从小鼠肺分离的上皮细胞系和细胞中产生的代谢产生的醌甲基化物与促进相关的生化效应。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型细胞中测定氧化应激和对抗氧化酶的影响。将确定醌甲基化物对间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法检查细胞系和分离细胞中的蛋白表达和蛋白磷酸化。

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.