IN VIVO OXIDATIVE DNA DAMAGE IN CULTURED MAMMALIAN CELLS

氧自由基被认为是主要的可能因素之一 一系列病理疾病的致病因素，如癌症， 衰老、关节炎、肺气肿和婴儿视网膜病变等。一 氧衍生物种的作用方式是导致DNA损伤， 导致细胞突变或死亡。这项研究的目标是 定量地确定氧自由基的程度 对培养的哺乳动物细胞中的体内DNA损伤负责。一位模特 产生单抗的小鼠杂交瘤系统已经被 被选为我们的研究对象。为了实现我们的目标，以下是 具体问题将按顺序回答： 一)氧化DNA损伤是否可以定量测量 染色质？DNA化学修饰的当代测量 都是在分离的DNA上制作的，这并不能描绘出真实的画面 在染色质水平发生的实际损伤。这是因为DNA 存在于染色质中的染色质在结构上是隔离的并与组蛋白结合。 此外，目前最先进的DNA损伤测量方法 哺乳动物细胞主要由识别链断裂组成。那里 需要一种更强大、更定量的分析方法。一位上级 可通过使用气相色谱/质谱法获得方法 精确定量鉴定DNA的光谱技术 损伤。通过该项目确定了几种基础产品 通过将分离的DNA暴露在自由基攻击下的技术。它是 建议将分离的完整染色质暴露在自由基攻击下 控制条件，并随后确定损害的特征。 Ii)在活体细胞中发现的损伤是否相同？这个问题 在哺乳动物细胞中还没有达到任何显著的程度。我们 建议使用体外生物反应器培养技术，这允许 对所有环境和生理因素的精确控制。活着 受控环境中的单元格将被暴露(如 分离的染色质)到已知的自由基生成系统。基于 为回答第一个问题而发展的方法论，这种方法 将允许对培养的哺乳动物体内DNA损伤的准确研究 细胞。此外，研究DNA的动力学也将成为可能 通过监测受损碱基在时间上的消失进行修复 细胞暴露于自由基后的细胞染色质。 除了对当代研究产生重大影响之外 在氧毒性领域，这项工作将有助于满足 RFA的目标不仅是通过发展杂交瘤作为高 哺乳动物系统的连通性也促进了体外的使用 组织培养实验作为动物用途的替代。在 从长远来看，这里提出的研究将为以下方面奠定基础 了解氧化DNA损伤和氧化损伤的条件和机制 随后的修复发生在哺乳动物细胞中。

Oxygen free radicals are believed to be among the major possible causative agents of a number of pathological conditions such as cancer, aging, arthritis, emphysema and infant retinopathy among others. One mode of action of oxygen-derived species is to cause DNA lesions, resulting in cell mutation or death. The goal of this study is to quantitatively establish the extent to which oxygen free radicals are responsible for in vivo DNA damage in cultured mammalian cells. A model murine hybridoma system producing monoclonal IgG antibodies has been chosen for our studies. In order to attain our objective, the following specific questions will be answered sequentially: i) Can oxidative DNA lesions be quantitatively measured in chromatin? Contemporary measurements of chemical modifications to DNA have been made on isolated DNA, which do not portray a realistic picture of actual damage occurring at the chromatin level. This is because DNA present in chromatin is structurally sequestered and bound to histones. Furthermore, present state-of-the-art measurements of DNA damage in mammalian cells consist primarily of identifying strand breakage. There is a need for a more powerful and quantitative assay. A superior approach is available through the use of Gas Chromatography/Mass Spectrometry techniques to precisely and quantitatively identify DNA lesions. Several base products have been identified through this technique by exposing isolated DNA to free radical attack. It is proposed to expose isolated intact chromatin to free radical attack under controlled conditions and subsequently characterize the damage. ii) Are the same lesions found in cells in vivo? This question has not been addressed to any significant degree in mammalian cells. We propose to use in vitro bioreactor culture techniques, which allow for the precise control of all environmental and physiological factors. Live cells in controlled environments will be exposed (as in the case of isolated chromatin) to known free radical generating systems. Based on the methodology developed to answer the first question, this approach will permit accurate studies of in vivo DNA damage in cultured mammalian cells. Additionally, it will be possible to study the kinetics of DNA repair by monitoring the temporal disappearance of damaged bases from cellular chromatin after exposing cells to free radicals. In addition to having a major impact on contemporary research in the field of oxygen toxicity, this work will assist in meeting the objectives of the RFA by not only developing hybridomas as a high connectivity mammalian system but also in promoting the use of in vitro tissue culture experiments as an alternative to animal use. In the longer term, the studies proposed here will lay the groundwork for understanding conditions and mechanisms by which oxidative DNA damage and subsequent repair occurs in mammalian cells.