MtDNA damage as a biomarker for environmental mitochondrial toxicity

描述（由申请人提供）：证明或验证当前或过去暴露于环境线粒体毒素是非常困难的。对于任何给定的毒素，毒素的组织分布和药代动力学可能是未知的。低水平暴露可能不会产生临床表型，并且根据所选择的测定，功能性线粒体损伤或酶抑制可能不会持续超过急性暴露（或毒素的代谢/排泄）。因此，迫切需要开发一种用于暴露于环境线粒体抑制剂的生物标志物，其是（i）敏感的，（i）至少半定量的，（iii）在毒素暴露停止后持久的，（iv）在样本收集后稳定的，和（v）高度可再现的。另一个问题是，在暴露于某些线粒体毒素后，一些组织选择性地容易受到损伤，而另一些组织则具有抵抗力。例如，当大鼠长期暴露于鱼藤酮时，它们的黑质纹状体多巴胺系统发生类似于帕金森病的选择性变性。生物反应的这种异质性的分子和生理基础是模糊的，不存在选择性脆弱性的生物标志物。初步数据表明，血液或骨骼肌中的mtDNA损伤可能提供了过去或正在进行的毒素暴露的生物标志物，而核DNA（nDNA）损伤可能是选择性脆弱性的临床前生物标志物。对于这些研究，我们将使用一种非常灵敏的基于PCR的DNA损伤（mtDNA和nDNA）检测方法，可以同时评估多种形式的损伤，并进一步评估相同样本中的mtDNA和nDNA损伤，而无需分离线粒体。该提案的具体目标是：1。(a)确定鱼藤酮暴露后多久可以在血液和肌肉中检测到mtDNA损伤。(b)确定单次暴露后可以检测到mtDNA损伤的持续时间; 2.确定引起可检测的mtDNA损伤所需的复合物I抑制的最低水平; 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变性的组织的标志物;和4. (a)确定外周mtDNA损伤是否是系统活性复合物I抑制剂的共同特征。(b)确定mtDNA损伤是否是其他ETC抑制剂的共同特征，包括作用于复合物II-IV的抑制剂。初步结果表明，该测定法将提供一种相对简单、极其灵敏、定量、暴露停止后持久、收集后稳定且高度可重复的生物标志物。 公共卫生相关性：暴露于环境线粒体毒素是非常难以证明或验证。我们已经采用了一种非常敏感的线粒体DNA损伤检测方法，并表明它可以检测亚临床暴露，甚至可以在毒素的主要作用结束后检测暴露。我们现在建议进一步表征这种生物标志物的敏感性和实用性。

DESCRIPTION (provided by applicant): 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. 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; and 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 this assay will provide a biomarker that is relatively simple, extremely sensitive, quantitative, enduring after exposure has ceased, stable after collection, and highly reproducible. PUBLIC HEALTH RELEVANCE: Exposures to environmental mitochondrial toxins are extremely difficult to demonstrate or verify. We have employed an extremely sensitive assay for mitochondrial DNA damage and have shown that it can detect subclinical exposures, and can detect an exposure even after the primary effect of the toxin has ended. We now propose to further characterize the sensitivity and utility of this biomarker.