Quantitative analysis of damage to the nucleotide pool

核苷酸库损伤的定量分析

• 批准号: 8638724
• 负责人: Peter C Dedon
• 金额: $ 19.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-11-20 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638724
• 关键词: 8-hydroxyguanosine; Address; Adenine; Affect; Anabolism; Anions; Antibiotics; Applications Grants; Area; Attention; Biochemical; Biomedical Research; Cell Death; Cell model; Cells; Cellular Stress; Chromatography; Coupled; DNA; DNA Damage; DNA Repair Enzymes; Data; Deamination; Defect; Deoxyribonucleotides; Development; Ensure; Enzymes; Escherichia coli; Exploratory/Developmental Grant; Future; Genetic; Genetic Variation; Goals; Guanine; Health; Hereditary Disease; Human; Human Genetics; Hydrogen Peroxide; Hygiene; Hypoxanthines; Inflammation; Knockout Mice; Lesion; Lipid Peroxidation; Liquid Chromatography; Mammalian Cell; Mass Spectrum Analysis; Metabolic Pathway; Methods; Modeling; Mono-S; Morphologic artifacts; Mutagenesis; Nucleic Acids; Nucleotides; Oxidative Stress; Pathology; Polymerase; Population; Protein Dephosphorylation; Purine Nucleotides; RNA; Reaction; Role; Solvents; Source; System; Technology; Testing; Toxic effect; Toxicogenetics; Walkers; Xanthines; adduct; analytical method; anticancer research; cell killing; chemical carcinogenesis; cytotoxic; experience; human disease; human tissue; inorganic phosphate; metabolomics; mutant; new technology; nucleotide metabolism; oxidation; phosphatase inhibitor; public health relevance; purine metabolism; pyrophosphatase; toxicant

Project Summary/Abstract The goal of these R21 Exploratory/Developmental studies is to develop a sensitive metabolomic platform to quantify damaged components of the nucleotide pool as a source of DNA and RNA damage. While toxic and mutagenic lesions arise in nucleic acids by direct reaction with environmental and endogenous toxicants, incorporation of damaged ribo- and 2-deoxyribonucleotides into DNA and RNA represents a potentially important source of genetic and cellular toxicity. The impact of the nucleotide pool has long been suspected from studies of highly conserved pool sanitizing enzymes, such as the pyrophosphatases that target damaged and non-canonical (d)NTP. The loss of these enzymes leads to increased levels of DNA damage. In spite of this evidence, there have been few quantitative studies of nucleotide pool damage due to a lack of analytical methods. To address this problem, we will develop a specific, sensitive and precise analytical method to quantify damaged nucleotide mono-, di- and tri-phosphates in the nucleotide pool. Following development with standards, the method will applied to two cellular models of genetic pathology and chemical carcinogenesis in humans: defects in purine nucleotide metabolism and oxidative stress. We recently discovered that defects in purine nucleotide metabolic pathways cause up to 600-fold increases in hypoxanthine, but not xanthine, into both DNA and RNA, presumably due to imbalances in guanine (G) and adenine (A) precursors in the nucleotide pool. The second application, which poses a greater challenge in terms of sensitivity, addresses oxidation of purine nucleotides in cells subjected to oxidative stress. These applications allow us to test and optimize the analytical platform for future studies in mammalian cells and human tissues, in which we address the full range of genotoxic and cytotoxic nucleotide pool damage from endogenous and environmental sources. Furthermore, both the results obtained and the novel technologies developed will find broad application in a variety of areas of biomedical research, including antibiotic development, genetic toxicology, inflammation and oxidative stress, and, at a systems level, any of the dozens of hereditary disorders of purine nucleotide metabolism.

项目总结/摘要 这些R21探索性/开发性研究的目标是开发一个敏感的代谢组学平台， 量化作为DNA和RNA损伤来源的核苷酸库的受损组分。虽然有毒， 诱变损伤通过与环境和内源性毒物的直接反应在核酸中产生， 将受损的核糖核苷酸和2-脱氧核糖核苷酸掺入DNA和RNA代表了一种潜在的 遗传和细胞毒性的重要来源。核苷酸库的影响一直受到怀疑 从高度保守的游泳池消毒酶的研究，如焦磷酸酶，目标受损 和非正则（d）NTP。这些酶的损失导致DNA损伤水平增加。尽管 这一证据表明，由于缺乏分析，很少有定量研究核苷酸库损伤。 方法.为了解决这个问题，我们将开发一种特异、灵敏和精确的分析方法， 定量核苷酸库中受损的核苷酸单磷酸、二磷酸和三磷酸。随着发展， 标准，该方法将应用于遗传病理学和化学致癌作用的两种细胞模型， 人类：嘌呤核苷酸代谢缺陷和氧化应激。我们最近发现， 嘌呤核苷酸代谢途径导致次黄嘌呤增加高达600倍，但不是黄嘌呤， DNA和RNA，可能是由于鸟嘌呤（G）和腺嘌呤（A）前体的不平衡， 核苷酸库。第二项申请在敏感性方面提出了更大的挑战， 在受到氧化应激的细胞中嘌呤核苷酸的氧化。这些应用程序允许我们测试和 优化分析平台，用于未来在哺乳动物细胞和人体组织中的研究， 来自内源性和环境来源的全方位遗传毒性和细胞毒性核苷酸库损害。 此外，所获得的结果和开发的新技术都将在一个 生物医学研究的各个领域，包括抗生素开发，遗传毒理学，炎症和 氧化应激，以及在系统水平上，嘌呤核苷酸的数十种遗传性疾病中的任何一种 新陈代谢.