RESPONSE TO DNA DAMAGE--COLON VS SMALL INTESTINE

结肠癌是#年癌症死亡的第二大原因。 今天的美国，而小肠癌是一种相对 罕见的事件。肠道肿瘤由一系列体细胞突变发展而来 在诸如DNA烷基化或氧化之类的启动事件之后。我们的 基本假设是小肠(SI)和大肠(LI) 对这两种重要的DNA损伤形式(烷基化和 氧化)，这是肿瘤发病率不同的关键原因 这两个地点。我们的第二个假设是SI细胞受到保护 诱导肿瘤，因为它们产生较少的活性氧簇(ROS)和 减少对DNA和LI细胞的氧化损伤。在具体目标#1中，我们将注入 用DNA烷化剂偶氮甲烷和测量体内DNA损伤的大鼠， 注射后48h内SI和LI的修复和细胞凋亡。脱氧核糖核酸 损伤和修复通过定量免疫组织化学方法测量 O6-甲基鸟嘌呤加合物及其修复酶； 末端标记法。在特定的目标#2中，我们将确定体内对 DNA氧化剂葡聚糖硫酸钠在大鼠SI和LI中的前48 H后去除氧化剂。DNA损伤是通过定量的 8oxodG加合物的免疫组织化学；特异性修复酶的活性 用核酸内切酶法检测8oxodG的表达，用TUNEL法检测细胞凋亡率。在……里面 具体目标#3我们将确定DNA氧化损伤的稳定水平 幼年和老年大鼠SI和LI中ROS的产生以及是否存在ROS SI和LI的产生是由于线粒体电子的差异 运输。DNA损伤将通过耀斑试验和ROS产生进行评估 活性染料2‘，7’-二氯荧光素在细胞中的氧化 而且没有电子传递抑制剂。

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.