Measuring DNA Damage and Repair Capacity in Human Populations

测量人群 DNA 损伤和修复能力

• 批准号: 8335872
• 负责人: michele k evans
• 金额: $ 48.88万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8335872
• 关键词: 1-Phosphatidylinositol 3-Kinase; Affect; African American; Age; Aging; Aging-Related Process; Alkaline Single-Cell Gel Electrophoresis Assay; Appearance; Autonomic nervous system; Behavior; Biological Assay; Biological Factors; Biological Markers; Biological Models; Blood Pressure; C-reactive protein; CCL2 gene; Cardiovascular system; Clinical; Complex; DNA; DNA Adducts; DNA Damage; DNA Repair; Defect; Demographic Factors; Detection; Development; Diagnosis; Diagnostic; Disease; Elderly; Environmental Risk Factor; Erythrocytes; Female; Gene Expression; Genetic; Glutathione; Health; Heme; Human; Individual; Inflammation; Inflammatory; Interleukin-17; Investigation; Laboratories; Lesion; Longevity; Longitudinal Studies; Lymphocyte; Malignant Neoplasms; Measures; MicroRNAs; Minority; Modification; Molecular Profiling; Monitor; Outcome; Oxidative Stress; Participant; Pathogenesis; Pathway interactions; Pattern; Peripheral Blood Mononuclear Cell; Phenotype; Plasma; Play; Population; Population Heterogeneity; Poverty; Predisposition; Premature aging syndrome; Prevalence; Process; Proteins; Proto-Oncogene Protein c-kit; Psychosocial Factor; Pulse Pressure; Race; Reverse Transcriptase Polymerase Chain Reaction; Role; Serum; Severities; Single Strand Break Repair; Small RNA; Socioeconomic Factors; Syndrome; TNF gene; Techniques; Time; Tissues; Uric Acid; Work; age related; aged; alpha Tocopherol; base; caucasian American; clinical application; cohort; cytokine; early onset; endonuclease III; frailty; genome-wide; health disparity; heart rate variability; high risk; inflammatory marker; insight; interleukin-23; male; novel; oxidant stress; oxidative DNA damage; oxidative damage; premature; protein expression; racism; sex

The alkaline comet assay is a sensitive and relatively inexpensive technique used for the detection of DNA damage. We have developed, and implemented modifications to the alkaline comet assay which may increase ability of the assay to detect subtle differences in DRC as well as baseline and induced levels of DNA damage measured as strand breaks and oxidative damage to DNA. Currently, there is no universally accepted panel of useful markers of oxidative stress in humans even though many lines of evidence suggest that oxidative DNA damage and other forms of oxidative stress influence the development of age-associated disease. The aim of this work is to determine whether demographic factors, such as age, sex and race affect the baseline level of oxidative DNA damage and to examine whether there were consistent relationships between frequently studied and novel measures of oxidative stress in diverse population. We are examining the level of single strand breaks and oxidative base lesions detected by endonuclease III and Fpg in cryopreserved PBMCs. In addition, we want to examine whether there is a correlation between the level of oxidative DNA damage in lymphocytes and other oxidative stress-related measures including: red blood cell glutathione (RBC GSH), heme degradation products, serum alpha-tocopherol, protein carbonyls in plasma and high-sensitivity C-reactive protein (hs-CRP). This study examines standard and unique markers of oxidative DNA damage and repair, oxidative stress and inflammation. We are studying four age-matched male and female whites and African-Americans aged 30-64 years. Our preliminary findings suggest that females have higher single strand break (SSB) levels than males (p=0.013). We observe a significant negative correlation between SSB repair capacity and SSB level (p=0.041). Thus far plasma carbonyl levels have shown no significant correlation with other markers. Our preliminary results suggest a complex relationship between measures of oxidative stress and clinical parameters used frequently and believed to reflect inflammation or oxidative stress. Our studies highlight the need for further examination to clarify the relationship between commonly used oxidative stress measures and DNA damage measures in diverse population cohorts so that we can understand how to accurately assess the potential clinical applications of these measures or to identify other more relevant and correlative markers. The lack of a strong overall relationship between hs-CRP, a marker of systemic inflammation, and markers of oxidative DNA damage may suggest that each of these markers represents a different pathway through which inflammation and oxidative stress occur and are monitored and/or modulated. Alternatively, it could also be difficult to correlate hs-CRP levels in serum with DNA damage in PMBCs. Therefore, we have begun to evaluate another marker of oxidant stress and oxidative DNA damage that can be detected in serum, the common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) and its correlation with inflammation, aging and other clinical parameters. We have assessed the relationship of 8-oxo-G levels with selected clinical and laboratory measures in a select part of our cohort. Our preliminary analyses revealed that the relationship between both systolic blood pressure and pulse pressure were significant. The relationship between 8-oxo-G and poverty status with race were not significant. 8-oxo-G levels increase with age and though we found no relationship between hs-CRP with SSB we have found that 8-oxo-G levels increase with increasing hs-CRP levels, suggesting a potential important relationship between a marker of inflammation and a marker of oxidative stress. We have also begun to pursue other biomarkers of age and disease. Human aging is a highly complex process that is characterized by an increase in age-associated diseases. Important in the study of aging is the discovery of new biomarkers that serve as indicators of tissue age and development and that also can aid in the diagnosis of age-related diseases. Studies in model systems suggest that longevity can be modulated by changes in gene and protein expression. However, little is known about how these small RNAs contribute to human aging. We have profiled the expression of over 800 miRNAs in peripheral blood mononuclear cells from young and old individuals by real-time RT-PCR analysis. This genome-wide assessment of miRNA expression revealed that the majority of miRNAs studied decreased in abundance with age. We identified nine miRNAs (miR-103, miR-107, miR-128, miR-130a, miR-155, miR-24, miR-221, miR-496, miR-1538) that were significantly lower in older individuals. Among them, five have been implicated in cancer pathogenesis. Predicted targets of several of these miRNAs, including PI3 kinase (PI3K), c-Kit and H2AX, were found to be elevated with advancing age, supporting a possible role for them in the aging process. Furthermore, we found that decreasing the levels of miR-221 was sufficient to cause a corresponding increase in the expression of the predicted target, PI3K. Taken together, these findings demonstrate that changes in miRNA expression occur with human aging and suggest that miRNAs and their predicted targets have the potential to be diagnostic indicators of age or age-related diseases.

碱性彗星试验是用于检测DNA损伤的敏感且相对便宜的技术。我们已经开发并实施了对碱性彗星试验的修改，这可能会增加该试验检测DRC的细微差异以及基线和诱导水平的DNA损伤（测量为DNA链断裂和氧化损伤）的能力。 目前，在人类中没有普遍接受的有用的氧化应激标志物，尽管许多证据表明氧化DNA损伤和其他形式的氧化应激影响年龄相关疾病的发展。 这项工作的目的是确定人口统计学因素，如年龄，性别和种族是否会影响DNA氧化损伤的基线水平，并检查在不同人群中经常研究和新的氧化应激指标之间是否存在一致的关系。我们正在研究单链断裂和氧化性碱基损伤的水平检测核酸内切酶III和Fpg在冷冻保存的PBMC。此外，我们想研究淋巴细胞中的氧化DNA损伤水平与其他氧化应激相关指标之间是否存在相关性，这些指标包括：红细胞谷胱甘肽（RBC GSH）、血红素降解产物、血清α-生育酚、血浆中的蛋白质羰基和高敏C反应蛋白（hs-CRP）。 这项研究检查了氧化DNA损伤和修复，氧化应激和炎症的标准和独特的标志物。 我们正在研究四个年龄匹配的男性和女性白人和非洲裔美国人，年龄在30-64岁之间。我们的初步研究结果表明，女性有较高的单链断裂（SSB）水平比男性（p=0.013）。我们观察到SSB修复能力和SSB水平之间的显着负相关性（p=0.041）。到目前为止，血浆羰基水平与其他标志物无显著相关性。我们的初步结果表明，氧化应激和临床参数的措施之间的复杂关系经常使用，并认为反映炎症或氧化应激。 我们的研究强调需要进一步研究，以澄清不同人群中常用的氧化应激指标和DNA损伤指标之间的关系，以便我们能够了解如何准确评估这些指标的潜在临床应用或识别其他更相关和相关的标志物。 hs-CRP（全身性炎症的标志物）和氧化性DNA损伤的标志物之间缺乏强的总体关系可能表明这些标志物中的每一个代表了炎症和氧化应激通过其发生并被监测和/或调节的不同途径。 或者，也可能难以将血清中的hs-CRP水平与PMBC中的DNA损伤相关联。 因此，我们已经开始评估另一种可以在血清中检测到的氧化应激和氧化DNA损伤的标志物，即常见的DNA碱基修饰8-氧代-7，8-二氢鸟嘌呤（8-oxo-G）及其与炎症、衰老和其他临床参数的相关性。 我们已经评估了8-oxo-G水平与选定的临床和实验室测量在我们的队列的一个选定的部分的关系。 我们的初步分析显示，收缩压和脉压之间的关系是显着的。 8-oxo-G与种族贫困状况的关系不显著。 8-氧代-G水平随着年龄的增长而增加，尽管我们发现hs-CRP与SSB之间没有关系，但我们发现8-氧代-G水平随着hs-CRP水平的增加而增加，这表明炎症标志物和氧化应激标志物之间存在潜在的重要关系。 我们还开始研究年龄和疾病的其他生物标志物。 人类衰老是一个高度复杂的过程，其特征是与年龄相关的疾病增加。在衰老研究中，重要的是发现新的生物标志物，作为组织年龄和发育的指标，也可以帮助诊断与年龄有关的疾病。对模型系统的研究表明，基因和蛋白质表达的变化可以调节寿命。然而，人们对这些小RNA如何促进人类衰老知之甚少。 我们通过实时RT-PCR分析了年轻人和老年人外周血单个核细胞中超过800种miRNA的表达。 对miRNA表达的全基因组评估显示，研究的大多数miRNA随着年龄的增长而减少。我们鉴定了9种在老年个体中显著较低的miRNAs（miR-103、miR-107、miR-128、miR-130 a、miR-155、miR-24、miR-221、miR-496、miR-1538）。其中，有五种与癌症发病机制有关。其中几种miRNAs的预测靶点，包括PI 3激酶（PI 3 K），c-Kit和H2 AX，被发现随着年龄的增长而升高，支持它们在衰老过程中的可能作用。此外，我们发现降低miR-221的水平足以导致预测靶点PI 3 K的表达相应增加。总之，这些发现表明，随着人类衰老，miRNA表达发生变化，并表明miRNA及其预测的靶点有可能成为年龄或年龄相关疾病的诊断指标。