MtDNA damage as a biomarker for environmental mitochondrial toxicity

证明或证实当前或过去暴露于环境线粒体毒素是 非常困难。对于任何给定的毒素，毒素的组织分布和药代动力学可 是未知的。低水平暴露可能不会产生临床表型， 选择，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，迫切需要开发一种生物标志物， 对于暴露于环境线粒体抑制剂，其是（i）敏感的，（i）至少半- 定量，（iii）在毒素暴露停止后持久，（iv）在样本采集后稳定，以及（v） 高度可重复。另一个问题是，在暴露于某些线粒体毒素后， 组织选择性地易受损伤，而其他组织具有抵抗力。例如，当老鼠 长期暴露于鱼藤酮，它们的黑质纹状体多巴胺发生选择性变性， 类似于帕金森病。这种异质性的分子和生理基础， 生物反应不明确，也不存在选择性脆弱性的生物标志物。我们的初步 数据表明，血液或骨骼肌中的mtDNA损伤可能提供了一种生物标志物， 过去或正在进行的毒素暴露，和核DNA（nDNA）损伤可能是一个 选择性易感性的临床前生物标志物。对于这些研究，我们将使用一个非常 灵敏的基于PCR的DNA损伤检测（mtDNA和nDNA），同时允许 评估多种形式的损伤，并进一步允许评估mtDNA和nDNA损伤 在相同的样品中，不需要线粒体分离。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中检测到mtDNA损伤， 肌肉. (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mtDNA所需的复合物I抑制的最低水平 损害 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损伤是否是全身活动性 复合物I抑制剂。(b)确定mtDNA损伤是否是其他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，我们的测定将提供一种相对简单、非常有效的生物标志物。 灵敏、定量、接触停止后持久、收集后稳定， 可复制的

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.