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（由申请人提供）：评估化学品引起染色体损伤的可能性是许多消费品、工业化学品和所有药剂的临床前遗传毒性安全测试的既定且重要的部分，目前哺乳动物红细胞微核测试是最常见的。采用体内评估染色体损伤的方法，但本报告特别报告了发生在骨髓中的遗传毒性测定，以便更全面地了解潜在的情况。遗传毒性，测试指南建议首选评估第二个组织，肝脏是代谢和在许多情况下激活基因毒性的场所，通常被视为第二个组织，但仍缺乏研究肝脏的高效工具。彗星和转基因啮齿动物突变模型可用于研究肝脏，但这些测定法受到方法学和成本问题的影响，限制了它们的实用性，另一个重要的考虑因素是这些测定法不太适合与正在进行的整合。毒理学研究，这意味着肝脏遗传毒性评估需要更多的动物，一种替代方法是检查肝细胞的微核形成，这是染色体损伤的既定指标。多步骤样品处理方案，然后通过显微镜进行手动评分，这种方法是主观的且劳动密集型的，并且导致被评分为可靠的细胞太少。微核肝细胞的计数，这种情况削弱了测试检测弱基因毒性物质的能力，我们将通过将简单、快速的组织处理和染色与高速流式细胞术分析相结合来克服这些缺陷，从而大大提高肝脏微核评分的执行。此外，我们将在肝脏微核测定中复用多种细胞毒性测量，从而提供我们预测对于解释遗传毒性结果非常重要的信息。该方法将在实践中得到简化。总体而言，该项目将满足遗传毒理学实践的关键需求。通过多种有意义的方式改进化学品安全评估。