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损伤的一种灵敏且相对便宜的技术。我们已经开发并实施了对碱性彗星试验的修改，这可能会提高该分析的能力，以检测DRC以及基线和诱导的DNA损伤水平的细微差异，以链断裂和DNA氧化损伤的形式测量。 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影响年龄相关疾病的发展，但还没有普遍接受的人类氧化应激有用标记物的小组。这项工作的目的是确定人口统计因素，如年龄、性别和种族是否影响氧化DNA损伤的基线水平，并检查在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。我们正在用核酸内切酶III和FPG检测冷冻保存的PBMC中单链断裂和氧化碱基损伤的水平。此外，我们还想研究淋巴细胞DNA氧化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唯一标记物。我们正在研究四名年龄匹配的白人和非裔美国人，年龄在30-之间。我们的初步发现表明，女性的单链断裂水平高于男性(p=0.013)。我们观察到SSB修复能力与SSB水平呈显著负相关(p=0.041)。到目前为止，血浆羰基水平与其他标志物没有显示出明显的相关性。我们的初步结果表明，氧化应激的测量方法和临床参数之间存在复杂的关系，这些参数被认为是反映炎症或氧化应激的常用参数。我们的研究强调了进一步研究的必要性，以澄清不同人群队列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确定其他更相关和相关的标记物。全身炎症标记物hs-CRP与DNA氧化损伤标记物之间缺乏很强的整体相关性，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的不同途径，并受到监测和/或调节。或者，也可能很难将血清中hs-CRP水平与PMBC中的DNA损伤联系起来。因此，我们已经开始评估在血清中可以检测到的另一种氧化应激和氧化DNA损伤的标志物--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、衰老等临床参数的相关性。我们评估了8-oxo-G水平与我们队列中选定的部分临床和实验室指标的关系。我们的初步分析表明，收缩压和脉压之间的关系是显著的。8-oxo-G与贫困状况和种族的关系不显著。8-oxo-G水平随着年龄的增长而升高，虽然我们没有发现hs-CRP与SSB之间的关系，但我们发现8-oxo-G水平随着hs-CRP水平的升高而升高，提示炎症标志和氧化应激标志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记物。人类老龄化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记物，作为组织年龄和发育的指标，并有助于诊断与年龄有关的疾病。在模型系统中的研究表明，长寿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。我们已经通过实时RT-PCR分析了超过800个miRNAs在年轻人和老年人的外周血单核细胞中的表达。这种对miRNA表达的全基因组评估表明，所研究的大多数miRNAs的丰度随着年龄的增长而减少。我们发现9个miRNAs(miR-103、miR-107、miR-128、miR-130a、miR-155、miR-24、miR-221、miR-496、miR-1538)在老年人中显著降低。其中，有5个与癌症发病机制有关。其中几个miRNAs的预测靶标，包括PI3激酶(PI3K)、c-Kit和H_2AX，被发现随着年龄的增长而升高，支持它们在衰老过程中可能的作用。此外，我们发现，降低miR-221的水平足以导致预测目标PI3K的表达相应增加。综上所述，这些发现表明miRNA的表达随着人类的衰老而发生变化，并表明miRNAs及其预测的靶点有可能成为年龄或与年龄相关的疾病的诊断指标。