Assessing toxicity with an ex vivo erythropoiesis system

描述（由申请人提供）：人类通过各种来源暴露于有毒化学和物理制剂。由于遗传毒性与癌症、动脉粥样硬化和出生缺陷有关，因此非常值得关注的是可损伤DNA的药剂。本文提出的研究工作旨在开发能够检测通过致染色体断裂和非整倍体活性损伤染色体的试剂的鼠离体培养系统。目前，化学诱导的染色体损伤是通过对细胞系或实验室啮齿动物的染色体畸变或微核形成进行评分来评估的。 通过开发离体红细胞生成系统，可在培养物中生成网织红细胞，作为微核评分的源材料。与永生化细胞系相比，原代培养物应提供更多相关的毒性信息。此外，开发流式细胞术评分技术以计数离体网织红细胞中的微核的可行性较高，并克服了与标准体外试验自动评分相关的人为因素。相对于啮齿动物试验，拟定的原代培养系统将需要较少的动物。为了研究基于离体红细胞生成的遗传毒性筛选系统的可行性和实用性，我们将： 1.通过离体培养物纯化网织红细胞生产。 2.测量离体衍生网织红细胞中的微核频率。 3.确定系统对已知染色体断裂剂和模型非整倍体的反应性。

DESCRIPTION (provided by applicant): Humans are exposed to toxic chemical and physical agents through a variety of sources. Of extreme concern are agents which can damage DNA, as genotoxicity is associated with cancer, atherosclerosis, and birth defects. The research effort proposed herein is aimed at developing murine ex vivo culture system that is capable of detecting agents which damage chromosomes through clastogenic and aneugenic activity. Currently, chemical-induced chromosome damage is evaluated by scoring chromosome abberations or micronucleus formation in cell lines or in laboratory rodents. By developing an ex vivo erythropoiesis system, reticulocytes can be generated in culture which serve as source material for micronucleus scoring. The primary cultures should provide more relevant toxicity information compared to immortilized cell lines. Furthermore, the feasibility of developing a flow cytometric scoring technique to enumerate micronuclei in ex vivo-derived reticulocytes is high, and overcomes artifacts associated with automated scoring of the standard in vitro assay. Relative to rodent tests, the proposed primary culture system will require few animals. To investigate the feasibility and utility of a genetic toxicity screening system based on ex vivo erythropoiesis, we will: 1. EVALUATE reticulocyte cell production by ex vivo culture(s). 2. MEASURE the frequency of micronuclei in ex vivo derived reticulocytes. 3. DETERMINE the responsiveness of the system to known clastogens and a model aneugen.