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: 7942870
• 负责人: Bruce N Ames
• 金额: $ 40.97万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942870
• 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。我们认为突变是一个与疾病相关的目标终点，因为它与衰老和癌症有因果关系。我们还提出，导致异质性的线粒体DNA （mtDNA）突变可能是氧化应激的超敏感前哨生物标志物。（“异质性”是指新的mtDNA突变产生，导致细胞内mtDNA群体混合。）由于接近细胞氧化剂的主要来源，MtDNA对氧化损伤异常敏感。我们（C. Calloway）开发了一种广泛应用于法医学的mtDNA突变/异质性的灵敏测定方法。在这里，她建议使用一种新颖的，超灵敏的测序方法来表征mtDNA突变的全谱。我们建议评估该方法对氧化应激的敏感性，不同氧化应激原因引起的突变谱的特异性，以及该方法对抗氧化剂调节作用的响应性。将在F344大鼠中评估三种生物标志物：氧化诱导的mtDNA损伤（与B. Van Houten合作）；通过LCMS代谢组学（J. Suh）检测氧化还原状态的变化；和丙二醛（MDA）在氧化应激中的作用是我们实验室开发并广泛应用的。这些试验的时间过程和敏感性将在老年和缺铁引起的生理相关氧化应激后进行比较，我们预计这在定量和定性上是不同的。这些影响将与CCl4应激进行比较，CCl4应激是nih发起的多中心BOSS研究中使用的氧化应激，包括我们的GCMS-MDA测定。由于MtDNA突变/异质性分析的敏感性，氧化应激引起的MtDNA突变应该被检测出来，不同应激源引起的不同突变谱也应该被检测出来。异质性的逆转，由于功能失调的线粒体的周转，将检查氧化应激的去除。与补充抗氧化剂对其他生物标志物（如mtDNA损伤、正常代谢组氧化还原谱的重建）的影响相比，了解这些对突变/异质性影响的时间过程和相对程度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有反应，那么它将是与疾病相关的功能性生物标志物，并将作为校准其他更通用的氧化应激生物标志物的参考。它还可以作为基因组DNA氧化突变的哨兵，预计这种突变的数量要少得多，并且不能通过补充抗氧化剂来逆转。mtDNA突变/异质性测定适用于使用人类头发、口腔拭子或少量血液的临床试验。