CometChip: Development of a high throughput DNA damage assay in hepatocytes

描述(申请人提供)：每年生产数千种新的工业化学品。因此，了解这些化合物的潜在致癌性对公众健康至关重要。众所周知，DNA损伤会导致导致癌症的突变。因此，能够筛查化学品是否有可能损害DNA为改善公共健康提供了一种有效的战略。已经开发了几种遗传毒性筛查，并通过高通量筛查技术有效地使用了这些筛查技术。然而，在我们目前可用的工具中仍然存在一个严重的缺口。具体地说，虽然使用现有技术可以检测到人类类型的DNA损伤，但巨大的DNA加合物仍然无法检测到。众所周知，巨大的病变可能致癌。例如，众所周知，黄曲霉毒素在导致世界各地的肝癌方面发挥着重要作用。此外，众所周知，环境中存在的多环芳烃会增加患癌症的风险。使用当今的技术，一种导致类似DNA损伤的新型化学物质不会被确定为潜在危险。关键挑战之一在于，许多人 DNA损伤化合物的类型是良性的，除非它们被代谢激活，这通常发生在肝细胞中。因此，使用肝细胞以外的细胞类型限制了检测能力。为了克服这些限制，我们提出了一种科学和工程创新的结合，以创建一种高通量的基于彗星分析的技术，该技术可以检测巨大的DNA损伤。彗星试验的原理是，在电泳法中，受损的DNA可以通过其比未受损的DNA更容易迁移的能力来检测。虽然对检测链断裂、碱性位点和碱敏感部位有效，但在标准条件下使用彗星分析无法检测到巨大的病变。在这里，我们建议改进现有的方法，以开发一种检测DNA碱基加合物的方法。此外，我们建议改进这些方法，用于能够代谢激活潜在致癌物的肝细胞。最后，我们建议利用我们最近开发的CometChip平台，这是一种极大地提高了灵敏度和吞吐量的微制造系统。在这项建议的第一阶段，我们建议建立有效的方法来检测肝细胞中的大块病变，并通过分析一个小的精选化合物库来证明高通量筛选的有效性。在第二阶段，我们将利用这项技术筛选NIEHS国家毒理学计划提供的多达400种化合物，并开发一个通用筛选平台来检测来自各种化学类别的制剂造成的DNA损伤。拟议研究的结果将填补一个关键的空白 在国内和国际上进行遗传毒性测试。由此产生的技术将提供一种 NTP的有效平台，以及提高工业环境中遗传毒性测试的效率。因此，预计拟议的技术将对公众健康产生重大影响。

DESCRIPTION (provided by applicant): Thousands of new industrial chemicals are produced each year. Knowledge about the potential carcinogenicity of these compounds is therefore critical to public health. It is well-established that DNA damage can lead to the mutations that cause cancer. Therefore, being able to screen chemicals for their potential to damage DNA offers an effective strategy for improving public health. Several genotoxicity screens have been developed and are used today effectively with high throughput screening technology. However, there remains a critical gap in the tools that we currently have available. Specifically, while man types of DNA damage can be detected using existing technologies, bulky DNA adducts remain undetectable. It is well-established that bulky lesions can be carcinogenic. For example, aflatoxin is known to play a major role in causing liver cancer around the world. Furthermore, polycyclic aromatic hydrocarbons present in our environment are known to increase the risk of cancer. Using today's technologies, a novel chemical that causes analogous DNA lesions would not be identified as being a potential hazard. One of the key challenges lies in the fact that many types of DNA damaging compounds are benign unless they are metabolically activated, which usually happens in hepatocytes. Therefore, use of cell types other than hepatocytes limits detection ability. To overcome these limitations, we propose a combination of scientific and engineering innovation to create a high throughput comet assay-based technology that detects bulky DNA lesions. The principle of the comet assay is that damaged DNA can be detected by its ability to migrate more readily than undamaged DNA when electrophoresed. While effective for detecting strand breaks, abasic sites and alkali sensitive sites, bulky lesions are not detectable using the comet assay under standard conditions. Here we propose to refine established approaches to develop a method for detecting DNA base adducts. Furthermore, we propose refining these approaches for use with hepatocytes that are able to metabolically activate potential carcinogens. Finally, we propose to harness our recently developed, CometChip platform, a microfabricated system that dramatically increases both sensitivity and throughput. In Phase I of this proposal we propose to establish approaches that are effective for detecting bulky lesions in the hepatocytes and to demonstrate efficacy of high throughput screening by analyzing a small library of select compounds. In Phase 2, we will leverage this technology to screen up to 400 compounds available through the National Toxicology Program of the NIEHS and develop a general screening platform to detect DNA damage caused by agents from a variety of chemical classes. Results of the proposed studies will fill a critical gap in genotoxicity testing both nationally and internationally. Resulting technologies will provide an effective platform for the NTP, as well as enhancing the efficacy of genotoxicity testing in industrial settings. As such, it is anticipated that the proposed technology will have a significan impact on public health.