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

描述(申请人提供)：许多生物标记物对氧化应激和抗氧化剂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，这些标记物与疾病的相关性尚不确定。我们认为突变是与疾病相关的目标终点，因为它与衰老和癌症的因果关系是已知的关联。我们还认为线粒体DNA(MtDNA)突变导致的异质性很可能是氧化应激的一个超敏感的前哨生物标志物。(“异质性”是指出现新的线粒体DNA突变，导致细胞内线粒体DNA种群混合。)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.Cloway)建立了一种灵敏的线粒体DNA突变/异质性分析方法，该方法在法医学中得到了广泛的应用。在这里，她建议使用一种新颖的、超灵敏的测序方法来表征线粒体DNA突变的全谱。我们建议评估该检测方法对氧化应激的敏感性，不同原因引起的氧化应激突变谱的特异性，以及该检测方法对抗氧化剂调节作用的反应性。三种生物标记物将在F344大鼠身上进行评估：氧化剂诱导的线粒体DNA损伤(与B.Van Houten合作)；LCMS(J.Suh)的代谢组学检测氧化还原状态的变化；以及GCMS的丙二醛(MDA)检测氧化应激。这些检测的时间进程和灵敏度将在老年和缺铁引起的生理上相关的氧化应激后进行比较，我们预计这两种氧化应激在数量和质量上都不同。这些影响将与CCl4压力进行比较，CCl4压力是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分析，包括我们的GCMS-MDA分析。由于线粒体DNA突变/异质性分析的高度敏感性，氧化应激导致的线粒体DNA突变以及不同应激源引起的不同突变谱都应该被检测出来。异质性的逆转，由于功能失调的线粒体周转，将在清除氧化应激方面进行检查。与补充抗氧化剂对其他生物标志物(如线粒体DNA损伤、重建正常代谢氧化还原图谱)的影响相比，了解这些对突变/异质性的影响的时间进程和相对程度将是该项目的一个关键方面。如果线粒体DNA突变/异质性是氧化应激的敏感生物标记物，并且对饮食中补充抗氧化剂有反应，那么它将是与疾病相关的功能生物标记物，它将作为参考，其他更一般的氧化应激生物标记物可以被校准。它还将作为基因组DNA氧化突变的哨兵，预计数量要少得多，而且不能通过补充抗氧化剂来逆转。线粒体DNA突变/异质性检测适合于使用人头发、口腔拭子或少量血液进行临床试验。 公共卫生相关性：氧化应激导致线粒体功能障碍，线粒体功能障碍与许多退行性衰老疾病和衰老过程本身有关。线粒体DNA突变/异质性试验是检测氧化应激引起的细胞功能改变的一种新的、高度敏感的方法。这项测试将帮助科学家更好地了解氧化应激是如何导致疾病的，还将提供一种方法来衡量饮食中补充抗氧化剂的功能益处。

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.