SEVEN BIOMARKERS OF ROS ACTIVITY IN DNA

DNA修饰，特别是8-羟基鸟嘌呤修饰，具有 被广泛用作活性氧物种(ROS)的指示剂 活动。我们建议解决的问题是对大多数 适当的DNA修饰可用作ROS活性的指示剂。 全氧条件下接触ROS的DNA寡聚体分析 环境表明，最合适的修改是8- 羟基鸟嘌呤，嘧啶碱甲酰胺残基及其串联 两种修饰都出现在相邻碱基上的碱基病变。 随着氧气变得越来越少，适当的DNA修饰是 胸腺嘧啶二醇、5-羟甲基尿嘧啶、6-羟基-5，6-二氢胸腺嘧啶和 二氢胸腺嘧啶。这七种DNA的相对有用性 将在DMSO-HL中评估作为ROS活性指标的修饰物 60个细胞。这些细胞可以被佛波酯醋酸酯刺激。 (PMA)释放ROS。DNA中七种修饰的数量 从PMA刺激的细胞中提取的物质将与背景进行比较 在未受刺激的细胞中测量的水平。分析的方法将是 通过32P-后标记的改编，其中各种DNA 修饰以修饰二核苷的形式进行测量 提取的DNA的核酸酶P1酶切中的一磷酸。这个 在二聚体水平上测量七种DNA修饰 几个优点和可行性，因为DNA修饰 利息增加了磷酸酯键3‘对 用核酸酶P1将二核苷修饰为水解物。的一项功能 化验是使用二聚体载体来明确定位病变 对化验的最后一步很感兴趣。另一项功能是 引入内部对照，使检测定量化。我们的 观点认为，有意义和可靠的ROS活性生物标志物是关键 在环境因素之间建立可能的联系 这可能会产生ROS和特定的人类疾病。

DNA modifications, especially the 8-hydroxyguanine modification, have been widely used as indicators of reactive oxygen species (ROS) activity. The question we propose to address is the choice of most appropriate DNA modifications to be used as indicators of ROS activity. Analysis of DNA oligomers exposed to ROS in a fully oxygenated environment indicate that the most appropriate modifications are 8- hydroxyguanine, the formamido remnant of pyrimidine bases and tandem base lesions in which both modifications are present on adjacent bases. As oxygen becomes less available the appropriate DNA modifications are thymine glycol, 5-hydroxymethyluracil, 6-hydroxy-5,6-dihydrothymine and dihydrothymine. The comparative usefulness of these seven DNA modifications as indicators of ROS activity will be evaluated in DMSO-HL 60 cells. These cells can be stimulated by phorbol myristate acetate (PMA) to release ROS. The amounts of the seven modifications in DNA extracted from PMA stimulated cells will be compared with background levels measured in non-stimulated cells. The method of analysis will be by an adaptation of 32P-postlabeling wherein the various DNA modifications are measured in the form of modified dinucleoside monophosphates in nuclease P1 digests of the extracted DNA. The measurement of the seven DNA modifications at the dimer level has several advantages and is feasible because the DNA modifications of interest increase the resistance of the phosphoester bond 3' to the modified dinucleoside to hydrolysis by nuclease P1. A feature of the assay is the use of dimer carriers to unequivocally locate the lesion of interest in the final step of the assay. Another feature is the introduction of internal controls to make the assay quantitative. Our view is that meaningful and reliable biomarkers of ROS activity are key to establishing possible connections between factors in the environment that may generate ROS and specific human diseases.