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.
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.
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