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 射线以来的一个多世纪以来，放射科学专业的学生一直被教导说，辐射引起的遗传损伤的典型目标存在于细胞核的 DNA 中。细胞质损伤的生物学后果很大程度上未知。使用精密带电粒子微束和双荧光染料分别定位细胞核和细胞质，从而避免无意中穿过细胞核，申请人之前已经证明，细胞质照射实际上对人仓鼠杂种（AL）的CD59位点具有诱变作用。 ）细胞，同时造成最小的细胞毒性。在母公司 R01 的资助下，我们最近证明：1) 活性氧介导这一过程； 2) 靶向细胞质照射导致膜的脂质过氧化，如诱导 4-羟基-2-壬烯醛（一种主要的脂质过氧化副产物）所示； 3) 生成线粒体 DNA 耗尽 (Ao) 的 AL 细胞和人成纤维细胞；最后，靶向细胞质照射通过间隙连接介导的过程诱导邻近未照射细胞之间的旁观者突变和染色单体断裂。这就提出了以下问题：靶向细胞质照射对线粒体 DNA 突变有何影响？线粒体 DNA 耗尽的人成纤维细胞是否表现出细胞质照射后诱变诱导减少？这项为期一年的补充应用的中心假设是，靶向细胞质照射会诱导人类成纤维细胞中线粒体 DNA 的剂量依赖性诱变，并且线粒体 DNA 耗尽的细胞对基因毒性信号反应较差。为了解决这些假设，提出了 2 个具体目标。将在人类成纤维细胞的 HPRT 位点进行突变评分，并确定线粒体 DNA 突变（将确定耗尽、异质性缺失和点突变）。拟议的研究将有助于解决细胞质照射如何导致细胞核中遗传事件的机制。利用旁观者诱变效应，该研究将解决有关核外靶点以及哺乳动物细胞中细胞质损伤如何处理的一些基本问题。 公共健康相关性：一代又一代的放射生物学学生被教导，电离辐射引起的遗传性生物效应是直接辐射与核相互作用的结果。使用哥伦比亚大学带电粒子微束和人仓鼠杂交（AL）细胞诱变试验，有证据表明靶向细胞质照射对哺乳动物细胞具有诱变作用。这首次明确地证明了电离辐射的核外效应，为后续广泛影响旁观者/非目标辐射效应提供了强有力的支持。有证据表明，靶向细胞质照射也可以引起旁观者效应。因此，更好地了解细胞质辐射的基因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 极易受到氧化损伤。本研究的目的是确定细胞质照射对线粒体 DNA 突变以及随后对线粒体功能的调节的可能影响。该研究将解决有关核外靶标以及哺乳动物细胞如何处理细胞质损伤的一些基本问题。