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.

碱性彗星测定是一种灵敏且相对便宜的技术，用于检测 DNA 损伤。我们开发并实施了对碱性彗星测定的修改，这可能会提高测定的能力，以检测 DRC 的细微差异以及以链断裂和 DNA 氧化损伤测量的基线和诱导的 DNA 损伤水平。 目前，尽管许多证据表明氧化 DNA 损伤和其他形式的氧化应激会影响与年龄相关的疾病的发展，但目前还没有普遍接受的人类氧化应激有用标记物。 这项工作的目的是确定年龄、性别和种族等人口因素是否影响氧化 DNA 损伤的基线水平，并检查不同人群中经常研究的氧化应激指标和新的氧化应激指标之间是否存在一致的关系。我们正在检查冷冻保存的 PBMC 中核酸内切酶 III 和 Fpg 检测到的单链断裂和氧化碱基损伤的水平。此外，我们想要检查淋巴细胞中氧化DNA损伤的水平与其他氧化应激相关指标之间是否存在相关性，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标记。 我们正在研究四名年龄匹配的男性和女性白人以及非裔美国人，年龄在 30-64 岁之间。我们的初步研究结果表明，女性的单链断裂 (SSB) 水平高于男性 (p=0.013)。我们观察到 SSB 修复能力和 SSB 水平之间存在显着的负相关性 (p=0.041)。迄今为止，血浆羰基水平与其他标志物没有显着相关性。我们的初步结果表明，氧化应激测量值与经常使用的临床参数之间存在复杂的关系，并被认为反映了炎症或氧化应激。 我们的研究强调需要进一步检查，以澄清不同人群中常用的氧化应激测量和 DNA 损伤测量之间的关系，以便我们能够了解如何准确评估这些测量的潜在临床应用或识别其他更相关和相关的标记。 hs-CRP（全身炎症标记物）和氧化性 DNA 损伤标记物之间缺乏牢固的整体关系，这可能表明这些标记物中的每一个都代表了炎症和氧化应激发生、监测和/或调节的不同途径。 或者，将血清中的 hs-CRP 水平与 PMBC 中的 DNA 损伤关联起来也可能很困难。 因此，我们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。 我们评估了我们队列中选定部分的 8-oxo-G 水平与选定的临床和实验室测量的关系。 我们的初步分析表明，收缩压和脉压之间存在显着的关系。 8-oxo-G 与贫困状况与种族的关系不显着。 8-oxo-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-oxo-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。 我们还开始寻找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，其特点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，这些标志物可以作为组织年龄和发育的指标，也可以帮助诊断与年龄相关的疾病。模型系统的研究表明，寿命可以通过基因和蛋白质表达的变化来调节。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，分析了年轻人和老年人外周血单核细胞中 800 多种 miRNA 的表达情况。 这种对 miRNA 表达的全基因组评估表明，大多数研究的 miRNA 丰度随着年龄的增长而减少。我们发现了九种 miRNA（miR-103、miR-107、miR-128、miR-130a、miR-155、miR-24、miR-221、miR-496、miR-1538）在老年人中显着降低。其中，有五种与癌症发病机制有关。研究发现，其中一些 miRNA 的预测靶标，包括 PI3 激酶 (PI3K)、c-Kit 和 H2AX，随着年龄的增长而升高，这支持了它们在衰老过程中可能发挥的作用。此外，我们发现降低 miR-221 的水平足以导致预测靶标 PI3K 的表达相应增加。总而言之，这些发现表明 miRNA 表达的变化随着人类衰老而发生，并表明 miRNA 及其预测靶点有可能成为年龄或与年龄相关的疾病的诊断指标。