MtDNA damage as a biomarker for environmental mitochondrial toxicity

证明或验证当前或过去接触环境线粒体毒素是 非常困难。对于任何给定的毒素，该毒素的组织分布和药代动力学 默默无闻。低水平暴露可能不会产生临床表型，这取决于检测方法。 选定的功能性线粒体损伤或酶抑制可能不会持续到急性 暴露(或毒素的代谢/排泄)。因此，迫切需要开发一种生物标志物 对于暴露于(I)敏感的、(I)至少半- 数量，(Iii)在毒素暴露停止后持续，(Iv)在样本采集后稳定，和(V) 重复性很强。另一个问题是，在接触到某些线粒体毒素后，一些 组织选择性地容易受到损害，而其他组织则具有抵抗力。例如，当老鼠 长期接触鱼藤酮，他们会出现黑质纹状体多巴胺的选择性退化 类似帕金森氏症的系统。这种异质性的分子和生理基础 生物反应模糊不清，不存在选择性脆弱性的生物标志物。我们的预赛 数据表明，血液或骨骼肌中的mtdna损伤可能提供了一个生物标记物。 过去或正在进行的毒素暴露，以及核DNA(NDNA)损伤可能是 选择性易损性的临床前生物标记物。对于这些研究，我们将使用一个极端的 基于PCR的DNA损伤(包括mtDNA和nDNA)的灵敏分析，同时允许 评估多种形式的损伤，并进一步评估mtDNA和nDNA损伤 在相同的样本中，不需要分离线粒体。这项建议的具体目标是： 1.(A)确定接触鱼藤酮后多久可在血液中检测到线粒体DNA损伤 肌肉。(B)确定在一次暴露后可检测到线粒体DNA损伤的持续时间。 2.确定产生可检测到的mtDNA所需的复合体I抑制的最低水平 损坏。 3.确定核DNA(NDNA)损伤是否是选择性地 易受线粒体毒素诱导的退化。！！ 4.(A)确定外周mtDNA损伤是否是系统性活动性疾病的共同特征 复合I类抑制剂。(B)确定线粒体DNA损伤是否为其他ETC的共同特征 抑制剂，包括作用于络合物II-IV的那些。 初步结果表明，我们的检测将提供一种相对简单、极其简单的生物标志物 敏感的、定量的、停止暴露后的耐受性、收集后的稳定性和高度 可重现的。

Demonstrating or verifying a current or past exposure to an environmental mitochondrial toxin is extraordinarily difficult. For any given toxin, tissue distribution and pharmacokinetics of the toxin may be unknown. Low-level exposure may not produce a clinical phenotype, and depending on the assay chosen, functional mitochondrial impairment or enzyme inhibition may not persist beyond the acute exposure (or metabolism/excretion of the toxin). Thus, there is a pressing need to develop a biomarker for exposure to environmental mitochondrial inhibitors that is (i) sensitive, (i) at least semi- quantitative, (iii) enduring after toxin exposure has ceased, (iv) stable after specimen collection, and (v) highly reproducible. Another problem is that after exposure to certain mitochondrial toxins, some tissues are selectively vulnerable to damage while others are resistant. For example, when rats are exposed to rotenone chronically, they develop selective degeneration of the nigrostriatal dopamine system similar to Parkinson disease. The molecular and physiological basis for such heterogeneity in biological response is obscure and no biomarker of selective vulnerability exists. Our preliminary data suggest that mtDNA damage in blood or skeletal muscle may provide a biomarker of past or ongoing toxin exposure, and nuclear DNA (nDNA) damage may be a preclinical biomarker of selective vulnerability. For these studies, we will use an extremely sensitive PCR-based assay of DNA damage (both mtDNA & nDNA) that simultaneously allows assessment of multiple forms of damage, and further allows assessment of mtDNA and nDNA damage in the same samples, without a need for mitochondrial isolation. The Specific Aims of this proposal are: 1. (a) Determine how soon after rotenone exposure mtDNA damage can be detected in blood and muscle. (b) Determine the duration, after a single exposure, that mtDNA damage can be detected. 2. Determine the minimal level of complex I inhibition that is required to cause detectable mtDNA damage. 3. Determine whether nuclear DNA (nDNA) damage is a marker of tissues that are selectively vulnerable to mitochondrial toxin-induced degeneration.!! 4. (a) Determine whether peripheral mtDNA damage is a common feature of systemically active complex I inhibitors. (b) Determine whether mtDNA damage is a common feature of other ETC inhibitors, including those acting at complexes II-IV. Preliminary results suggest our assay will provide a biomarker that is relatively simple, extremely sensitive, quantitative, enduring after exposure has ceased, stable after collection, and highly reproducible.