Contamination of our environment with organic pollutants causes serious public health problems. This proposal addresses major toxicity pathways involving bioactivation of pollutants after they enter the body. Reactive metabolites formed by cytochrome P450s and other metabolic enzymes damage genetic material and proteins. Examples include a range of common chemicals in food, air and water. We are developing novel high throughput devices to rapidly identify metabolites that damage DNA to reveal chemical pathways in genotoxicity. In the next project period, we will extend our devices to organ specific genotoxicity and DNA oxidation, and detect tumor suppressor gene damage to help predict cancer target organs. The project will generate valuable new tools to help predict genotoxicity of new organic chemicals at early development stages, revealing chemical pathways of genotoxicity not obviated by bioassays. Genotoxicity pathways discovered in this way should moderate pollutant-caused disease, and ultimately improve public health. We developed new bioanalytical approaches featuring ultrathin, layered films of metabolic enzymes and DNA in the last funding period. These assays address the chemistry and dynamics of metabolite-related genotoxicity in cell-free solutions, and thus complement toxicity bioassays. First, metabolic bioactivation is done in DNA/enzyme films, then resulting DNA damage is measured. Novel arrays assess chemical pathways and rates of metabolite-DNA reactions, enzyme specificities, inhibition, and interspecies toxicity differences for organic pollutants and drugs. Establishing these parameters for new chemicals is critical for individual safety. Our most advanced devices are high throughput microfluidic arrays for reactive metabolite screening of test chemicals, and biocolloid reactors in 96-well formats to generate samples for LC-MS/MS that provide DNA adduct structures and formation rates correlated with genotoxicity. Plans for the next funding period are aimed at greatly increasing specificity and selectivity of genotoxicity prediction of our approaches by introducing representative organ specific enzymes, and incorporating measurements of metabolite-driven DNA oxidation. In addition, we will combine the bioreactor approach with LC-MS/MS sequencing to detect metabolite codon damage patterns to p53 tumor suppressor gene to predict possible cancer target organs. Summary of Specific Aims: (1) Develop microfluidic arrays to measure DNA oxidation and general DNA damage, test with known toxic chemicals, and validate with LC-MS/MS. (2) Evaluate microfluidic arrays and LC-MS/MS approaches using enzymes from liver, lung, intestine, and kidney to screen test compounds for organ specific genotoxicity. (3) Couple DNA/enzyme biocolloid reactors with LC-MS/MS sequencing to identify specific codons on p53 tumor suppressor gene where metabolites react, and analyze results using the p53 database to predict possible cancer target organs. (4) Develop a global microfluidic array to monitor organ specific DNA adduct formation and oxidation, and validate with LC-MS/MS studies.

Public Health Relevance

Toxicity is often caused by conversion of chemicals (bio-activation) by enzymes in the body to genotoxic species that damage key biological molecules. This project aims at developing mechanism-based devices to screen new chemicals that produce genotoxic species, and to facilitate designing out genotoxicity. These advanced methods hold great promise for improving public health by screening out potential toxicants from many sources.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
2R01ES003154-31A1
Application #
8756198
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Balshaw, David M
Project Start
1983-03-01
Project End
2019-03-31
Budget Start
2014-06-01
Budget End
2015-03-31
Support Year
31
Fiscal Year
2014
Total Cost
$448,694
Indirect Cost
$125,936
Name
University of Connecticut
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
614209054
City
Storrs-Mansfield
State
CT
Country
United States
Zip Code
06269
Kanyong, Prosper; Rawlinson, Sean; Davis, James (2016) Immunochemical Assays and Nucleic-Acid Detection Techniques for Clinical Diagnosis of Prostate Cancer. J Cancer 7:523-31
Song, Boya; Shen, Min; Jiang, Di et al. (2016) Microfluidic array for simultaneous detection of DNA oxidation and DNA-adduct damage. Analyst 141:5722-5729
Liu, Ben; Yao, Huiqin; Song, Wenqiao et al. (2016) Ligand-Free Noble Metal Nanocluster Catalysts on Carbon Supports via ""Soft"" Nitriding. J Am Chem Soc 138:4718-21
Hvastkovs, Eli G; Rusling, James F (2016) State-of-the-Art Metabolic Toxicity Screening and Pathway Evaluation. Anal Chem 88:4584-99
Bist, Itti; Song, Boya; Mosa, Islam M et al. (2016) Electrochemiluminescent Array to Detect Oxidative Damage in ds-DNA Using [Os(bpy)2(phen-benz-COOH)](2+)/Nafion/Graphene Films. ACS Sens 1:272-278
Malla, Spundana; Kadimisetty, Karteek; Fu, You-Jun et al. (2015) CHEMICAL SELECTIVITY OF NUCLEOBASE ADDUCTION RELATIVE TO IN VIVO MUTATION SITES ON EXON 7 FRAGMENT OF P53 TUMOR SUPPRESSOR GENE. Chem Sci 6:5554-5563
Li, Dandan; Fu, You-Jun; Rusling, James F (2015) Characterizing protein modifications by reactive metabolites using magnetic bead bioreactors and LC-MS/MS. Chem Commun (Camb) 51:4701-3
Rusling, James F; Wasalathanthri, Dhanuka P; Schenkman, John B (2014) Thin multicomponent films for functional enzyme devices and bioreactor particles. Soft Matter 10:8145-56
Song, Boya; Pan, Shenmin; Tang, Chi et al. (2013) Voltammetric microwell array for oxidized guanosine in intact ds-DNA. Anal Chem 85:11061-7
Mani, Vigneshwaran; Kadimisetty, Karteek; Malla, Spundana et al. (2013) Paper-based electrochemiluminescent screening for genotoxic activity in the environment. Environ Sci Technol 47:1937-44

Showing the most recent 10 out of 92 publications