Cytoplasmic Damage and Genotoxicity

细胞质损伤和遗传毒性

• 批准号: 7842077
• 负责人: Tom K. Hei
• 金额: $ 39.34万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842077
• 关键词: Address; Alpha Particles; Biological; Biological Assay; Bystander Effect; Cell Nucleus; Cells; Charge; Chromatids; Cytoplasm; Cytosol; DNA; Dose; Dyes; Educational process of instructing; Event; Fibroblasts; Fluorochrome; Funding; Gap Junctions; Generations; Genetic; Glutathione; Hamsters; Human; Hybrids; Incidence; Ionizing radiation; Link; Lipid Peroxidation; Low Dose Radiation; Mammalian Cell; Mediating; Membrane; Membrane Potentials; Mitochondria; Mitochondrial DNA; Mutagenesis; Mutation; Nuclear; Oxidants; Parents; Point Mutation; Process; Proteins; Radiation; Radiobiology; Reactive Oxygen Species; Research Personnel; Respiratory Chain; Risk Assessment; Roentgen Rays; Role; Science; Signal Transduction; Skin; Students; Succinate Dehydrogenase; Universities; cytochrome c oxidase; cytotoxicity; genotoxicity; inhibitor/antagonist; irradiation; mitochondrial DNA mutation; mitochondrial membrane; mutant; oxidative damage; particle; public health relevance; radiation effect; research study; response

DESCRIPTION (provided by applicant): For over a century since the discovery of X-rays by Rventgen, students in radiological sciences have been taught that the quintessential target for radiation induced genetic damages resides in the DNA of the nucleus. The biological consequences of cytoplasmic damage are largely unknown. Using a precision charged particle microbeam and dual fluorochrome dyes to locate nucleus and cellular cytoplasm respectively, thereby avoiding inadvertent traversal of nuclei, the applicant has shown previously that cytoplasmic irradiation is, in fact, mutagenic at the CD59 locus of human-hamster hybrid (AL) cells while inflicting minimal cytotoxicity. With the funding support of the parent R01, we have shown recently that 1) reactive oxygen species mediate this process; 2) targeted cytoplasmic irradiation results in lipid peroxidation of membranes as shown by the induction of 4-hdyroxy-2-nonenal, a major lipid peroxidation byproduct; 3) generated mitochondrial DNA depleted (Ao) AL cells and human fibroblasts; and finally, targeted cytoplasmic irradiation induces bystander mutations and chromatid breaks among neighboring, non-irradiate cells through a gap junction-mediated process. This raised the following questions: What are the effects of targeted cytoplasmic irradiation on mitochondrial DNA mutations and does mitochondrial DNA depleted human fibroblasts demonstrated reduced induction of mutagenesis upon cytoplasmic irradiation? The central hypotheses for this one year supplemental application is targeted cytoplasmic irradiation induces a dose dependent mutagenesis of mitochondrial DNA in human fibroblasts and that mitochondrial DNA depleted cells respond poorly to genotoxic signaling. To address these hypotheses, 2 specific aims are proposed. Mutations will be scored at the HPRT locus in human fibroblasts and mitochondrial DNA mutations (depletion, heteroplasmic deletions and point mutations will be determined. The proposed studies will help to address the mechanisms of how cytoplasmic irradiation results in a genetic event in the nucleus. Together with the bystander mutagenic effect, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells. PUBLIC HEALTH RELEVANCE: Generations of students in radiation biology have been taught that heritable biological effects induced by ionizing radiation are the consequence of a direct radiation-nuclear interaction. Using the Columbia University charged particle microbeam and the human hamster hybrid (AL) cell mutagenic assay, there is evidence that targeted cytoplasmic irradiation is mutagenic in mammalian cells. This first, unequivocal demonstration of an extranuclear effect of ionizing radiation provides strong support of the subsequent, broad reaching bystander/ non-targeted effects of radiation. There is evidence that targeted cytoplasmic irradiation can also induce a bystander effect. As such, a better understanding of the genotoxic mechanism of cytoplasmic irradiation is critical in low dose radiation risk assessment. Mitochondria are widely distributed in the cytosol and mitochondrial DNA is highly susceptible to oxidative damage. The objective of the proposed study is to ascertain the possible effects of cytoplasmic irradiation on mitochondrial DNA mutations and the subsequent modulation on mitochondrial function. The study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells.

描述（由申请人提供）：自rventgen发现X射线以来，放射科学的学生被教导，辐射诱导的遗传损害的典型目标位于原子核的DNA中。细胞质损伤的生物学后果在很大程度上未知。使用精确的电荷颗粒微膜和双荧光组染料分别定位核和细胞胞质，从而避免了核的无意间遍历，先前申请人表明，在人类汉斯特杂交（Al）Cytit（Al）cytit的CD59基因座上，实际上是细胞辐射，实际上是诱变的。在母体R01的资金支持下，我们最近表明1）活性氧介导了这一过程； 2）靶向细胞质辐照导致膜的脂质过氧化，如4-Hdyroxy-2-nonenal诱导，这是一种主要的脂质过氧化副产品； 3）产生的线粒体DNA耗竭（AO）Al细胞和人成纤维细胞；最后，靶向的细胞质辐照通过间隙连接介导的过程引起旁观者突变和相邻非辐射细胞之间的染色单体断裂。这提出了以下问题：靶向细胞质辐照对线粒体DNA突变有什么影响，线粒体DNA耗竭的人成纤维细胞会显示诱变对细胞质辐照的诱导降低？这一年补充施用的中心假设是靶向细胞质辐照，在人成纤维细胞中诱导线粒体DNA的剂量依赖性诱变，并且线粒体DNA耗尽的细胞对遗传毒性信号的反应不佳。为了解决这些假设，提出了2个具体目标。突变将在人类成纤维细胞和线粒体DNA突变中的HPRT基因座上进行评分（耗尽，异质质缺失和点突变。拟议的研究将有助于解决细胞质辐射在遗传学中的细胞质辐射结果的机制。靶标以及如何在哺乳动物细胞中处理细胞质损伤。 公共卫生相关性：已经教导了几代辐射生物学中的学生，即电离辐射引起的可遗传生物学作用是直接辐射核互动的结果。使用哥伦比亚大学带电的粒子微束和人仓鼠杂种（AL）细胞诱变测定法，有证据表明，靶向细胞质辐照在哺乳动物细胞中是诱变的。电离辐射的核外效应的第一个明确的证明为随后的，广泛的旁观者/非靶向辐射效应提供了强有力的支持。有证据表明，靶向细胞质辐照也可以诱导旁观者效应。因此，更好地理解细胞质照射的遗传毒性机制对于低剂量辐射风险评估至关重要。线粒体广泛分布在细胞质中，线粒体DNA高度易受氧化损伤。拟议的研究的目的是确定细胞质辐照对线粒体DNA突变的可能影响以及随后对线粒体功能的调节。这项研究将解决有关核外靶标以及哺乳动物细胞中如何处理细胞质损害的一些基本问题。