Contamination of our environment with organic pollutants generates serious contemporary public health problems. A major mechanism contributing to human disease involves toxic activation of organic pollutants after they enter the body. This process features oxidation of the pollutants catalyzed by cytochrome P450 enzymes in the liver, followed by damage of genetic material (i. e. DNA) by activated pollutants. Chemicals activated in this way include styrene, benzo[a]pyrene, and many others. Organohalide pollutants such as chlorodioxins and polychlorinated biphenyls are not as easily activated, but they elevate cytochrome P450 levels in the body, promoting the chance of metabolic dysfunction. For example, excess cytochrome P450 can catalyze undesirable oxidation of membrane lipids or other functional biomolecules. These oxidation products may also damage DNA. Knowledge of molecular details and enzyme specificities of toxic activation and DNA damage can lead to new ways to treat pollutant-caused diseases and improve the future health of our citizens. The broad long-term goals of this project are to develop new, thin-film electrode coatings for in-vitro studies of DNA damage by activated pollutants, and as simple toxicity screens for new chemicals activated by enzymes.
Specific aims i nclude developing systems based on electrode-driven enzyme-catalyzed pollutant activation, using enzymes and DNA in thin films prepared by casting and layer-by-layer growth. Systems will be designed to mimic natural toxic activation of pollutants and DNA damage. Enzymes used will proceed from a model heme protein, myoglobin, to bacterial cytochrome P450, to human cytochromes P450. Natural redox partners of the cytochrome P450 enzymes may be included in the films if needed. Target devices will employ electron injection from electrodes to drive a series of events involving enzyme-catalyzed pollutant activation, and damage of DNA by the activated pollutants. Electrochemical and chromatographic analyses will be used to monitor DNA damage in the films. Validation of toxic screen assays will be done by correlating DNA damage rates for known toxic pollutants with mutagenicity and carcinogenicity data bases. In addition to new devices for screening toxic chemicals, this project will result in relatively simple probes for establishing detailed substrate specificities of human cytochrome P450s, the relative distribution of which may be important in individual susceptibility to pollutant-caused disease.
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