Validation of a Rodent Mutagenicity Assay

描述(申请人提供)：DNA突变是癌症发生的主要机制。这些事件也与动脉粥样硬化等疾病和衰老等过程有关。因此，重要的是需要灵敏的分析方法来促进对突变的研究，以及识别可以使DNA突变的化学或物理因素。目前存在测量体内突变的方法，每种方法都有自己的优点和局限性。有些是基于群体形成，需要组织培养工作，而另一些则依赖昂贵的、专有的转基因啮齿动物。这个实验室已经开发出一种基于Pig-a基因座的体内突变检测方法。Pig-a基因产物是糖基磷脂酰肌醇(GPI)锚的生物合成所必需的。导致非功能性GPI锚定的突变阻止了某些蛋白质在细胞表面的表达，这代表了一种可以通过流式细胞术测量的表型。本文提出的工作通过广泛的实验室间验证工作扩展了这一调查路线，通过使用短期和28天重复给药计划，用已知的诱变剂和非诱变剂治疗大鼠。这项测试验证工作将集中在基于红细胞的测试上，这是迄今为止研究最深入的目标细胞群。与此同时，其他工作将针对开发方法来测量猪--其他组织中的突变。社会将从拟议的工作中受益，因为制药和化学公司获得了进行安全评估工作的改进方法。 与公共卫生相关：众所周知，DNA损伤是癌症和其他重大疾病发展的先兆。因此，减少环境、药物和工作场所中的致突变化学物质的发生符合公众健康的利益。这项研究项目将验证一种强大的基于血液的方法，该方法可以检测诱变剂，从而增强国家有效减少接触这些有毒化合物的能力。其他工作将针对开发与其他组织兼容的突变细胞评分方法。

DESCRIPTION (provided by applicant): Mutation to DNA is a primary mechanism by which cancers arise. These events have also been implicated in diseases such as atherosclerosis, and processes such as aging. Therefore, there is an important need for sensitive analytical methods that facilitate the study of mutagenesis, as well as the identification of chemical or physical agents that can mutate DNA. Methods for measuring in vivo mutation currently exist, each with their own advantages and limitations. While some are based on colony formation and require tissue culture work, others rely on expensive, proprietary trangenic rodents. This laboratory has developed an in vivo mutation assay that is based on the Pig-a locus. The Pig-a gene product is essential for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Mutations giving rise to nonfunctional GPI anchors prevent certain proteins from being expressed on the cell surface, and this represents a phenotype that can be measured by flow cytometry. The work proposed herein extends this line of investigation through an extensive inter-laboratory validation effort whereby rats will be treated with known mutagens and non-mutagens using both a short-term as well as a 28-day repeat dosing schedule. This assay validation effort will focus on an erythrocyte-based assay, a target cell population that has been studied most intensely to date. Concurrently, other work will be directed at developing means to measure Pig-a mutation in other tissues. Society will benefit from the proposed work as pharmaceutical and chemical companies are provided improved methods for conducting safety assessment work. PUBLIC HEALTH RELEVANCE: It is well known that DNA damage is a precursor to the development of cancer and other significant diseases. Therefore, it is in the interest of public health to reduce the occurrence of mutagenic chemicals in the environment, in our drugs, and from our workplaces. This research project will validate a powerful blood-based method that detects mutagenic agents, thereby enhancing the nation's ability to effectively reduce exposure to these toxic compounds. Additional work will be directed at developing mutant cell scoring methods that are compatible with other tissues.