Automation of a Liver Genotoxicity Assay

 描述（由申请方提供）：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临床前遗传毒性安全性试验的既定和重要部分。目前，哺乳动物红细胞微核试验是体内评估染色体损伤最常用的试验，但该试验专门报告了骨髓中发生的遗传毒性。为了更全面地了解潜在遗传毒性，试验指南建议评价第二种组织。肝脏是遗传毒物的代谢部位，在许多情况下是遗传毒物的活化部位，通常被视为首选的第二组织。即便如此，仍然缺乏有效的工具来研究肝脏遗传毒性。彗星试验和转基因啮齿动物突变模型可用于研究肝脏，但这些试验存在方法和成本问题，限制了其实用性。另一个重要的考虑因素是，这些试验不太适合与正在进行的毒理学研究整合，这意味着需要额外的动物进行肝脏遗传毒性评估。一种替代方法是检查肝细胞的微核形成，这是一种确定的染色体损伤指标。然而，用于检查肝脏微核的现有方法仍然是新兴的，并且目前基于多步样品处理方案，然后通过显微镜进行手动评分。这种方法是主观的和劳动密集型的，并且导致太少的细胞被评分以用于可靠的评估。 微核肝细胞计数，这种情况会降低检测弱遗传毒性试剂的能力。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，我们将在肝微核试验中进行多项细胞毒性测量，从而提供我们预测对解释遗传毒性结果至关重要的信息。该方法将以市售试剂盒的形式简化为实践，并将有助于减少和完善动物试验，因为它将使肝脏遗传毒性试验与正在进行的毒理学研究相结合成为可能。总的来说，该项目将通过以几种有意义的方式改进化学品安全评估来满足遗传毒理学实践中的关键需求。

 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.