SEVEN BIOMARKERS OF ROS ACTIVITY IN DNA

DNA修饰，特别是8-羟基鸟嘌呤修饰， 被广泛用作活性氧（ROS）的指示剂， 活动 我们要解决的问题是大多数人的选择 合适的DNA修饰可用作ROS活性的指示剂。 在完全氧合的细胞中暴露于ROS的DNA寡聚体的分析 环境表明，最合适的修改是8- 羟基鸟嘌呤，嘧啶碱基的甲酰氨基残基， 两种修饰都存在于相邻基底上的基底病变。 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中评价作为ROS活性指标的修饰 60个牢房 这些细胞可以被佛波醇肉豆蔻酸酯醋酸酯刺激 (PMA)释放ROS。 DNA中七种修饰的数量 将从PMA刺激的细胞中提取的DNA与背景DNA进行比较 在未刺激的细胞中测量的水平。分析方法将是 通过32 P-后标记的调整，其中各种DNA 修饰以修饰的二核苷的形式测量 在提取的DNA的核酸酶P1酶中的单磷酸。 的 在二聚体水平上测量七种DNA修饰， 有几个优点并且是可行的，因为DNA修饰 兴趣增加磷酸酯键3'对磷酸酯键的抗性。 修饰的二核苷被核酸酶P1水解。 的特征 检测是使用二聚体载体明确定位病变 在检测的最后一步。 另一个特性是 引入内部对照以使测定定量。 我们 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 可能产生活性氧和特定的人类疾病。

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.