Automation of a Liver Genotoxicity Assay

肝脏基因毒性测定的自动化

基本信息

批准号：
9038484
负责人：
Jeffrey C Bemis
金额：
$ 17.26万
依托单位：
LITRON LABORATORIES, LTD.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9038484
关键词：
Affect Age Animal Testing Animals Antibodies Apoptotic Automation Benchmarking Biological Assay Bone Marrow Cell Nucleus Cells Chemicals Comet Assay DNA DNA Damage DNA Double Strand Break Data Detergents Development Diethylnitrosamine Dose Dyes Erythrocytes Evaluation Experimental Designs Exposure to Family suidae Female Flow Cytometry Fluorescence Microscopy Frequencies Genetic Goals Harvest Hepatocyte Histopathology Japan Kinetics Label Laboratories Liver Manuals Measurement Measures Metabolism Methodology Methods Micronucleus Tests Microscopy Mitotic Modeling Mutagenicity Tests Mutagens Mutation Organ Performance Pharmaceutical Preparations Pharmacologic Substance Play Procedures Process Rattus Reagent Reporting Research Design Rodent Safety Sampling Schedule Scheme Scoring Method Site Speed Temperature Testing Time Tissue Harvesting Tissue Stains Tissues Toxic effect Toxicogenetics Toxicology Transgenic Organisms Validation age group base consumer product cost cytotoxicity design genotoxicity high throughput screening improved in vivo indexing male meetings method development micronucleus pre-clinical prototype public health relevance quinoline safety testing sex tissue processing tool treatment duration treatment group

项目摘要

DESCRIPTION (provided by applicant): Assessment of chemicals' potential to cause chromosomal damage is an established and important part of preclinical genotoxicity safety testing for many consumer products, industrial chemicals, and all pharmaceutical agents. Currently the mammalian erythrocyte micronucleus test is the most commonly employed assay for in vivo assessment of chromosomal damage, but this assay reports specifically on genotoxicity that occurs in the bone marrow. In order to obtain a more comprehensive understanding of potential genotoxicity, testing guidance's recommend evaluation of a second tissue. The liver, the site of metabolism and in many cases activation of genotoxicants, is usually regarded as the preferred second tissue. Even so, there is a lack of efficient and effective tools for studying liver genotoxicity. The Comet assay and transgenic rodent mutation models can be employed to study the liver, but these assays suffer from methodological and cost issues that limit their utility. Another important consideration is that these assays are not highly amenable to integration with on-going toxicology studies, meaning additional animals are required for the liver genotoxicity assessment. One alternative approach is to examine liver hepatocytes for the formation of micronuclei, an established indicator of chromosomal damage. However existing methods for examining liver micronuclei are still emerging and currently based on a multi-step sample processing scheme followed by manual scoring by microscopy. This approach is subjective and labor- intensive, and results in too few cells being scored for reliable enumeration of micronucleated hepatocytes, a situation that diminishes the ability of the test to detect weakly genotoxic agents. We will overcome these deficiencies by combining simple, rapid tissue processing and staining with high-speed flow cytometric analysis to greatly improve the execution of liver micronucleus scoring. Furthermore, we will multiplex several cytotoxicity measurements into the liver micronucleus assay, thereby providing information that we predict will be important for interpreting the genotoxicity results. The methodology will be reduced to practice in the form of commercially available kits, and will contribute to the reduction and refinement of animal testing, as it will make it feasible to integrate a liver genotoxicity assay ito ongoing toxicology studies. Overall, this project will meet a critical need in the practice of genetic toxicology by improving chemical safety assessments in several meaningful ways.

描述（通过应用提供）：化学物质引起染色体损伤的潜力的评估是许多消费产品，工业化学品和所有药物的临床前遗传毒性安全测试的确定且重要的部分。目前，哺乳动物红细胞微核测试是对染色体损伤的体内评估最常见的评估，但该测定特别报告了骨髓中发生的遗传毒性。为了获得对潜在遗传毒性的更全面的了解，测试指南对第二组织的建议评估。肝脏，代谢部位和在许多情况下激活遗传毒性，通常被认为是首选的第二组织。即便如此，缺乏研究肝遗传毒性的有效工具。可以聘请彗星测定法和转基因啮齿动物突变模型来研究肝脏，但是这些测定法患有限制其效用的方法论和成本问题。另一个重要的考虑因素是，这些评估与正在进行的毒理学研究相结合并不高度适应，这意味着肝遗传毒性评估需要其他动物。一种替代方法是检查肝脏肝细胞的形成微核（已建立的染色体损伤指标）。但是，现有的检查肝微核的方法仍在出现，目前基于多步样本处理方案，然后是通过显微镜进行手动评分。这种方法是主观和劳动力密集的，导致对可靠的细胞得分太少微核肝细胞的枚举，这种情况降低了检测弱遗传毒性剂的能力。我们将通过将简单的，快速的组织加工和染色与高速流式细胞仪分析相结合，从而极大地改善肝脏小核评分的执行，从而克服这些缺陷。此外，我们将对肝脏微核测定法进行多次多次细胞毒性测量，从而提供我们预测的信息对于解释遗传毒性结果至关重要。该方法将以市售套件的形式减少为实践，并将有助于减少和完善动物测试，因为这将使整合肝遗传毒性测定ITO ITO持续的毒理学研究，这将使其可行。总体而言，通过以几种有意义的方式改善化学安全评估，该项目将满足遗传毒理学实践的关键需求。