Phase II year
2015
(last award dollars: 2020)
Phase II Amount
$3,750,517
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.
Public Health Relevance Statement: 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.
Project Terms: Address; adduct; Aflatoxins; Alkalies; Aromatic Polycyclic Hydrocarbons; base; Beds; Benign; Benzo(a)pyrene; Binding (Molecular Function); Biological Assay; Blinded; Cancer Etiology; cancer risk; carcinogenicity; Carcinogens; Cell Line; cell type; Cells; Chemical Industry; Chemicals; Comet Assay; Detection; Development; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA lesion; Dose; drug development; Engineering; Ensure; Environment; Environmental Pollution; Enzymes; Experimental Designs; Family; Food; genotoxicity; Glucuronosyltransferase; hazard; Health; Hepatocyte; high throughput screening; high throughput technology; Human; improved; innovation; Knowledge; Lead; Lesion; Libraries; Malignant neoplasm of liver; Malignant Neoplasms; Mammalian Cell; man; Measures; Metabolic; Metabolic Activation; Metabolic Biotransformation; Methods; Microfabrication; Mutagenicity Tests; Mutation; National Institute of Environmental Health Sciences; National Toxicology Program; novel; Nucleotide Excision Repair; oxidative damage; Pharmaceutical Preparations; Phase; Phosphorylation; Play; Procedures; Process; public health medicine (field); Publications; repaired; research study; response; Role; Safety; screening; Sensitivity and Specificity; Sepharose; Site; Small Business Innovation Research Grant; Specificity; sulfotransferase; System; Technology; Testing; Time; tool; Toxic effect; Transferase; UV induced; Variant; Xenobiotics
