Contamination of our environment with organic pollutants causes serious public health problems. A major pathway to human disease involves toxic bioactivation of organic pollutants after they enter the body. In this way, pollutants or drugs metabolized by cytochrome P450 (cyt P450) enzymes in the liver can damage genetic material (i. e., DNA). Chemicals in this class include styrene, benzo[a]pyrene, napthylamines and many others. Some chemicals elevate levels of cyt P450s and other metabolic enzymes, increasing the risk of damage to important biomolecules. In vitro biosensors that rapidly measure the ability of metabolites of pollutants or drugs to damage DNA would be valuable screening tools to predict the human toxicity of new organic chemicals. These devices can also be used to provide knowledge of enzyme specificities and pathways of toxic activation, possibly leading 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 enzyme/DNA sensors that bioactivate chemicals and measure the subsequent DNA damage as in-vitro toxicity screens for new chemicals, and as tools for elucidating mechanistic details of bioactivation. Prototype biosensors have been developed already in this project in which enzymes catalyze formation of metabolites, and subsequent electrochemical assays estimate DNA damage. The sensors employ peroxide or electron injection to drive enzyme-catalyzed bioactivation. Damage to DNA is then detected by catalytic or probe binding electrochemical assays.
Specific aims for the next grant period include: (1) validating the sensors as toxicity screens for a range of chemicals, (2) developing sensor arrays for voltammetry and electrode-driven light emission to profile the activity of human cyt P450s towards bioactivation, (3) developing films to explore sequential metabolic processes involving cyt P450s and glutathione-S-transferase (another important metabolic enzyme), and (4) applying the sensors to study inhibition of DNA damage by dietary antioxidants. Chromatography, mass spectrometry and electrophoresis will be used to measure specific DNA damage for molecular confirmation of sensor results. Validation will be assisted by correlating sensor results for toxic chemicals with databases of microbiological and animal mutagenicity and carcinogenicity. The project will also result in simple devices to establish substrate specificities of human cyt P450s, the inter-individual distribution of which may be important in individual susceptibility to pollutants, and for examining sequential metabolic processes. In summary, this work will provide novel in vitro tools for toxicity screening of new chemicals, and help expand the molecular knowledge base on pollutant-caused disease as a basis for future therapeutic protocols.
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