Cytoplasmic Damage and Genotoxicity

描述（申请人提供）：细胞质损伤的生物学后果在很大程度上是未知的。普遍的观点认为，多环芳烃和氡粒子等环境致癌物的遗传毒性作用主要是由于对细胞核的直接损害。申请人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，从而避免了细胞核的无意穿越，先前已经表明，细胞质辐照实际上在人-仓鼠杂交（AL）细胞的CD59位点具有诱变作用，同时造成最小的细胞毒性。此外，初步证据表明活性氧介导了这一过程。这就提出了以下问题：涉及到什么类型的氧自由基，它们的来源是什么？自由基生成过程是否涉及线粒体损伤？靶细胞质辐照诱导的突变是否也发生在人支气管上皮细胞（环境氡的靶组织）中？最后，细胞质辐照能否在哺乳动物细胞中诱导旁观者诱变效应，其方式与申请人最近通过AL细胞的核穿越所证明的类似。为了解决这些问题，提出了一系列5个具体目标来解决4个可测试的假设。将在AL细胞的CD59位点和人原代支气管上皮细胞的HPRT位点对突变进行评分。提出的研究将有助于解决细胞质辐照如何导致细胞核遗传事件的机制。与旁观者诱变效应一起，该研究将解决一些关于核外靶点和细胞质损伤如何在哺乳动物细胞中处理的基本问题。

DESCRIPTION (provided by applicant): The biological consequences of cytoplasmic damage are largely unknown. The prevailing dogma considered the genotoxic effects of environmental carcinogens such as polycyclic aromatic hydrocarbons and radon alpha particles as being due mostly to direct damage to the nucleus. 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. Furthermore, preliminary evidence suggests that reactive oxygen species mediate this process. This raised the following questions: What types of oxyradicals are involved and what are their origins? Does this radical generating process involve mitochondrial damage? Do the mutations induced by targeted cytoplasmic irradiation occur in human bronchial epithelial cells (target tissues of environmental radon) as well? And finally, can cytoplasmic irradiation induce bystander mutagenic effect in mammalian cells in a manner similar to what the applicant has recently demonstrated with nuclear traversal of the AL cells. To address these issues, a series of 5 specific aims are proposed to address the 4 testable hypotheses. Mutations will be scored at the CD59 locus of the AL cells and at the HPRT locus in primary human bronchial epithelial cells. 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.