mtDNA mutation/heteroplasmy: a sensitive functional biomarker of oxidative stress

mtDNA 突变/异质性：氧化应激的敏感功能生物标志物

• 批准号: 7820696
• 负责人: Bruce N Ames
• 金额: $ 41.26万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7820696
• 关键词: Age; Aging; Aging-Related Process; Antioxidants; Biological Assay; Biological Markers; Blood; Carbon Tetrachloride; Cells; Clinical Trials; Collaborations; DNA; DNA Damage; Degenerative Disorder; Dietary Iron; Disease; Enzymes; Excision; Forensic Medicine; Future; Generic Drugs; Genomics; Hair; Half-Life; Human; Inbred F344 Rats; Intake; Iron; Isoprostanes; Laboratories; Link; Malignant Neoplasms; Malondialdehyde; Mass Fragmentography; Measures; Metabolic; Methods; Mitochondria; Mitochondrial DNA; Modeling; Mus; Mutation; Mutation Spectra; Nuclear; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Induction; Pathology; Performance; Phase; Physiological; Plasma; Population; Process; Production; Rattus; Relative (related person); Scientist; Sensitivity and Specificity; Sentinel; Somatic Mutation; Source; Specificity; Staging; Stress; Supplementation; Swab; System; Testing; Thioctic Acid; Time; Vitamin E; age related; alcohol exposure; dietary antioxidant; dietary supplements; disorder risk; functional outcomes; in vivo; metabolomics; mitochondrial DNA mutation; mitochondrial dysfunction; novel; oxidant stress; oxidative damage; public health relevance; response; stressor

DESCRIPTION (provided by applicant): Many biomarkers are responsive to oxidative stress and modulating effects of antioxidant dietary supplements, but the disease relevance of these markers is uncertain because of the lack of known functional or mechanistic linkages. We propose that mutation is a disease-relevant target endpoint because of its known association with aging and causal relationship to cancer. We also propose that mitochondrial DNA (mtDNA) mutation leading to heteroplasmy is likely to be an ultra sensitive sentinel biomarker of oxidative stress. ("Heteroplasmy" is when a new mtDNA mutation arises leading to a mixed intracellular mtDNA population.) MtDNA is unusually sensitive to oxidative damage because of proximity to the main source of cellular oxidants. We (C. Calloway) developed a sensitive assay of mtDNA mutation/heteroplasmy which is widely used in forensics. Here, she proposes to use a novel, ultra sensitive sequencing method to characterize the full spectrum of mutations in mtDNA. We propose to evaluate the sensitivity of this assay to oxidative stress, the specificity of mutational spectra resulting from different causes of oxidative stress, and the responsiveness of the assay to modulatory effects of antioxidants. Three biomarker assays will be evaluated in F344 rats: oxidant-induced mtDNA damage (with B. Van Houten); metabolomics by LCMS (J. Suh) for changes to redox status; and malondialdehyde (MDA) by GCMS for oxidative stress developed in our lab and widely used. The time course and sensitivity of these assays will be compared following physiologically relevant oxidative stresses induced by old age and by iron deficiency, which we expect to be different quantitatively and qualitatively. These effects will be compared to CCl4 stress, which was the oxidative stress utilized in the NIHinitiated multi-center BOSS study using the common oxidative stress assays, including our GCMS-MDA assay. MtDNA mutations resulting from oxidative stress should be detected because of the exquisite sensitivity of the mtDNA mutation/heteroplasmy assay, as will different mutational spectra resulting from different stressors. Reversal of heteroplasmy, due to turnover of dysfunctional mitochondria, will be examined on removal of oxidative stress. Understanding the time course and relative magnitude of these effects on mutation/heteroplasmy as compared to the effects of antioxidant supplementation on other biomarkers (e.g., mtDNA damage, re-establishment of a normal metabolomic redox profile) will be a key aspect of the project. If mtDNA mutation/heteroplasmy is a sensitive biomarker of oxidative stress and is responsive to dietary antioxidant supplementation, it will be a functional biomarker linked to disease and it will serve as a reference against which other more generic oxidative stress biomarkers can be calibrated. It will also be useful as a sentinel for oxidative mutations of genomic DNA, which are expected to be in much smaller numbers and would not be reversible by antioxidant supplementation. The mtDNA mutation/heteroplasmy assay is suitable for use in clinical trials using human hair, buccal swabs, or small amounts of blood. PUBLIC HEALTH RELEVANCE: Oxidative stress causes mitochondrial dysfunction which is associated with many degenerative diseases of aging and with the aging process itself. The mitochondrial DNA mutation/heteroplasmy assay being proposed is a novel, highly sensitive method to detect functional cellular changes caused by oxidative stress. The assay will help scientists better understand how oxidative stress leads to disease and will also provide a way to measure functional benefits of dietary antioxidant supplementation.

描述（由申请人提供）：许多生物标志物对氧化应激和抗氧化膳食补充剂的调节作用有反应，但由于缺乏已知的功能或机制联系，这些标志物的疾病相关性尚不确定。我们认为突变是疾病相关的目标终点，因为它与衰老和癌症的因果关系有关。我们还提出，线粒体DNA（mtDNA）突变导致异质性可能是一个超敏感的氧化应激的哨兵生物标志物。（“异质性”是指当一个新的mtDNA突变出现时，导致细胞内mtDNA群体的混合。线粒体DNA对氧化损伤异常敏感，因为它接近细胞氧化剂的主要来源。我们（C. Calloway）开发了一种广泛用于法医学的mtDNA突变/异质性的灵敏测定方法。在这里，她建议使用一种新的，超灵敏的测序方法来表征mtDNA突变的全谱。我们建议评估该测定对氧化应激的敏感性，由不同原因引起的氧化应激的突变谱的特异性，以及该测定对抗氧化剂的调节作用的响应性。将在F344大鼠中评价三种生物标志物测定：氧化剂诱导的mtDNA损伤（用B.货车豪滕）; LCMS（J. Suh）代谢组学用于氧化还原状态的变化;以及我们实验室开发并广泛使用的GCMS检测氧化应激的丙二醛（MDA）。这些检测的时间过程和灵敏度将进行比较后，生理相关的氧化应激诱导的老年和缺铁，我们预计是不同的定量和定性。这些影响将与CCl 4应激进行比较，CCl 4应激是NIH发起的多中心BOSS研究中使用的氧化应激，使用常见的氧化应激试验，包括我们的GCMS-MDA试验。由于mtDNA突变/异质性检测的灵敏度极高，因此应检测氧化应激导致的mtDNA突变，以及不同应激源导致的不同突变谱。将在去除氧化应激后检查由于功能障碍的线粒体的周转而引起的异质性恢复。了解与抗氧化剂补充对其他生物标志物（例如，线粒体DNA损伤，重建正常的代谢组学氧化还原谱）将是该项目的一个关键方面。如果mtDNA突变/异质性是氧化应激的敏感生物标志物，并且对膳食抗氧化剂补充剂有反应，那么它将是与疾病相关的功能性生物标志物，并且它将作为其他更通用的氧化应激生物标志物可以校准的参考。它也将作为基因组DNA氧化突变的哨兵，预计其数量要小得多，并且不会通过抗氧化剂补充而逆转。mtDNA突变/异质性检测适用于使用人头发、口腔拭子或少量血液的临床试验。 公共卫生相关性：氧化应激导致线粒体功能障碍，这与许多衰老的退行性疾病和衰老过程本身有关。提出的线粒体DNA突变/异质性分析是一种新的，高度敏感的方法来检测氧化应激引起的功能细胞变化。该试验将帮助科学家更好地了解氧化应激如何导致疾病，也将提供一种方法来衡量膳食抗氧化剂补充剂的功能益处。