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调节抗氧化剂补充剂的作用有反应，但是由于缺乏已知的功能或机械联系，这些标记的疾病相关性尚不确定。我们建议突变是一个与疾病相关的靶向终点，因为它已知与衰老和因果关系与癌症的关系。我们还建议导致异质的线粒体DNA（mtDNA）突变可能是氧化应激的超灵敏的前哨生物标志物。 （“杂质”是当新的mtDNA突变导致混合细胞内mtDNA种群的时候。）mtDNA由于靠近主要的细胞氧化剂来源而对氧化损伤异常敏感。我们（C。Calloway）开发了一种敏感的mtDNA突变/异质分析，该测定法广泛用于取证。在这里，她建议使用一种新型的超灵敏测序方法来表征mtDNA中的突变。我们建议评估该测定法对氧化应激的敏感性，由于氧化应激的不同原因引起的突变光谱的特异性以及测定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测定：氧化剂诱导的mtDNA损伤（带有B. van Houten）； LCMS（J。SUH）的代谢组学改变了氧化还原状态； GCMS和丙二醛（MDA）在我们的实验室中开发并广泛使用的氧化应激。在由老年和铁缺乏症引起的生理相关的氧化应激之后，将比较这些测定的时间过程和敏感性，我们预计这在定量和定性上会有所不同。这些作用将与CCL4应激进行比较，CCL4应激是使用常见的氧化应激测定法（包括我们的GCMS-MDA测定法）中NIHINIAD的多中心研究中使用的氧化应激。由于mtDNA突变/异质分析的灵敏度，应检测到由氧化应激产生的mtDNA突变，以及由不同的应激源引起的不同突变光谱也是如此。由于线粒体功能障碍的流失，杂质的逆转将在去除氧化应激时检查。与补充抗氧化剂对其他生物标志物的影响相比，了解这些对突变/异质的影响的时间过程和相对大小将是该项目的关键方面。如果mtDNA突变/异质性是氧化应激的敏感生物标志物，并且对饮食中的抗氧化剂补充有反应，它将是与疾病相关的功能性生物标志物，它将作为其他更通用的氧化应激生物标志物的参考。它也将是用于基因组DNA的氧化突变的前哨，预计该突变的数量将少得多，并且补充抗氧化剂不会可逆。 mtDNA突变/异质分析适用于使用人头发，颊拭子或少量血液的临床试验。 公共卫生相关性：氧化应激会导致线粒体功能障碍，这与许多退化性疾病以及衰老过程本身有关。提出的线粒体DNA突变/异质分析是一种新型，高度敏感的方法，可检测由氧化应激引起的功能性细胞变化。该测定法将帮助科学家更好地了解氧化应激如何导致疾病，还将提供一种衡量饮食抗氧化剂补充功能益处的方法。