RESPONSE TO DNA DAMAGE--COLON VS SMALL INTESTINE

结肠癌是癌症死亡的第二大原因 在今天的美国，小肠癌相对来说是一种 罕见事件。肠道肿瘤由一系列体细胞突变发展而来 DNA 烷基化或氧化等起始事件之后。我们的 主要假设是小肠 (SI) 和大肠 (LI) 对这两种重要形式的 DNA 损伤（烷基化和 氧化），这是肿瘤发生率差异的一个关键原因 这两个网站。我们的第二个假设是 SI 细胞受到保护 肿瘤诱导，因为它们产生较少的活性氧（ROS）和 对 DNA 和 LI 细胞的氧化损伤较小。在具体目标#1中，我们将注入 大鼠与 DNA 烷化剂氧化偶氮甲烷并测量体内 DNA 损伤， 注射后 48 小时内 SI 和 LI 的修复和凋亡。脱氧核糖核酸 通过定量免疫组织化学来测量损伤和修复 O6-甲基鸟嘌呤加合物及其修复酶；并通过以下方式检测细胞凋亡 TUNEL 测定。在具体目标#2中，我们将确定对 DNA 氧化剂右旋糖酐硫酸钠在大鼠 SI 和 LI 中的第 48 期内 除去氧化剂后h。 DNA损伤通过定量测量 8oxodG 加合物的免疫组织化学；修复酶特异性活性 通过核酸内切酶测定检测 8oxodG；和通过TUNEL测定进行细胞凋亡。在 具体目标#3 我们将确定氧化 DNA 损伤的稳态水平 年轻和年老大鼠 SI 和 LI 中 ROS 的生成以及是否有 ROS SI 和 LI 的产生是由于线粒体电子的差异 运输。 DNA 损伤将通过 FLARE 测定进行评估，ROS 产生则通过 细胞中重要染料 2',7'-二氯荧光素的氧化 并且不含电子传输抑制剂。

Colon cancer is the second leading cause of death from cancer in the United States today whereas cancer of the small intestine is a relatively rare event. Intestinal tumors develop from a series of somatic mutations subsequent to an initiating event such as DNA-alkylation or oxidation. Our primary hypothesis is that the small intestine (SI) and large intestine (LI) respond differently to these two important forms of DNA damage (alkylation and oxidation), which is a key reason for the difference in tumor incidence at these two sites. Our secondary hypothesis is that SI cells are protected from tumor induction because they produce less reactive oxygen species (ROS) and less oxidative damage to DNA and LI cells. In specific aim # 1 we will inject rats with the DNA alkylating agent azoxymethane and measure in vivo DNA damage, repair and apoptosis in SI and LI over the 48 h period post injection. DNA damage and repair are measured by quantitative immunohistochemistry of O6-methylguanine adducts and its repair enzyme; and apoptosis is detected by the TUNEL assay. In specific aim #2 we will determine in vivo response to the DNA oxidizing agent dextran sodium sulfate in rat SI and LI within the first 48 h after removal of the oxidizing agent. DNA damage is measured by quantitative immunohistochemistry of 8oxodG adducts; activity of the repair enzyme specific for 8oxodG by an endonuclease assay; and apoptosis by the TUNEL assay. In specific aim #3 we will determine steady state levels of oxidative DNA damage and ROS generation in SI and LI in young and old rats and whether or not ROS generation in SI and LI is due to differences in mitochondrial electron transport. DNA damage will be assessed by the FLARE assay and ROS production by oxidation of the vital dye 2',7'-dichlorofluorescein in cells incubated with and without electron transport inhibitors.