Quantitative analysis of damage to the nucleotide pool

项目摘要/摘要 这些R21探索性/发育性研究的目标是开发一个敏感的代谢组平台 量化核苷酸池中受损成分作为DNA和RNA损伤的来源。虽然有毒，但 通过与环境和内源性毒物的直接反应，在核酸中产生突变损伤， 将受损的核糖核酸和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。长期以来，人们一直怀疑核苷酸池的影响 来自高度保守的泳池消毒酶的研究，例如针对受损的焦磷酸酶 和非规范(D)NTP。这些酶的丧失会导致DNA损伤水平的增加。尽管 这一证据表明，由于缺乏分析，对核苷酸池损害的定量研究很少。 方法：研究方法。为了解决这个问题，我们将开发一种具体、灵敏和精确的分析方法来 量化核苷酸池中受损的单磷酸、二磷酸和三磷酸。遵循以下发展： 标准，该方法将应用于遗传病理和化学致癌的两个细胞模型。 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的增加高达600倍，但不是黄嘌呤。 DNA和RNA，推测是由于体内鸟嘌呤(G)和腺嘌呤(A)前体的失衡所致 核苷酸池。第二个应用程序在敏感度方面提出了更大的挑战，解决了 氧化应激下细胞中嘌呤核苷酸的氧化。这些应用程序允许我们测试和 优化未来哺乳动物细胞和人类组织研究的分析平台，我们在其中解决 来自内源和环境来源的全方位的遗传毒性和细胞毒性核苷酸池损害。 此外，所获得的结果和开发的新技术都将在 生物医学研究的各种领域，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，嘌呤核苷酸的几十种遗传性疾病中的任何一种 新陈代谢。

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.