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

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 todayandapos 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 we will leverage this technology to screen up to 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 PUBLIC HEALTH RELEVANCE Knowing that a chemical has the potential to induce cancer is critical to public health DNA damage is known to promote toxicity and mutations that drive cancer but there is a major gap in our current assays for DNA damage due to the fact that lesions that bind DNA rather than breaking DNA are not detected We already know of examples where DNA binding chemicals cause cancer therefore it is critical to develop a screening method that ensure that agents that bind to DNA are identified before they cause cancer in people who are exposed