Automation of a Liver Genotoxicity Assay

 描述(由申请人提供)：评估化学品造成染色体损伤的可能性是对许多消费品、工业化学品和所有药剂进行临床前遗传毒性安全测试的重要组成部分。目前，哺乳动物红细胞微核试验是最常用的体内染色体损伤评估方法，但该方法专门报道了发生在骨髓中的遗传毒性。为了更全面地了解潜在的遗传毒性，检测指南建议对第二个组织进行评估。肝脏是新陈代谢的场所，在许多情况下是遗传毒物的激活，通常被认为是首选的第二组织。即便如此，目前仍缺乏研究肝脏遗传毒性的有效工具。彗星试验和转基因啮齿动物突变模型可以用来研究肝脏，但这些分析方法和成本问题限制了它们的实用。另一个重要的考虑是，这些分析不太容易与正在进行的毒理学研究相结合，这意味着需要更多的动物来进行肝脏遗传毒性评估。一种替代方法是检查肝细胞是否有微核形成，这是染色体损伤的既定指标。然而，现有的检测肝脏微核的方法仍然是新兴的，目前的方法是基于多步骤的样品处理方案，然后通过显微镜手动评分。这种方法是主观的和劳动密集型的，并且导致为可靠而评分的细胞太少 微核肝细胞计数，这种情况降低了检测弱遗传毒性物质的能力。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仪分析相结合来克服这些不足，极大地提高肝微核评分的执行力。此外，我们将在肝脏微核试验中多重测量几种细胞毒性，从而提供我们预测将对解释遗传毒性结果重要的信息。该方法将以商用试剂盒的形式付诸实践，并将有助于减少和完善动物测试，因为它将使将肝脏遗传毒性分析与正在进行的毒理学研究相结合成为可能。总体而言，该项目将通过以几种有意义的方式改进化学安全评估，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.