Contamination of our environment with organic pollutants causes serious public health problems. A major toxicity pathway involves bioactivation of pollutants after they have entered the body. Thus, metabolism mediated by cytochrome P450s and other enzymes causes the toxicity of many foreign chemicals. Resulting reactive metabolites can damage genetic material, proteins and other biomolecules. Examples of bioactivated pollutants include styrene, benzo[a]pyrene, arylamines, and N-nitrosamines. We are developing in vitro biosensor arrays to rapidly measure the ability of metabolites to damage DNA and serve as valuable screening tools to predict toxicity of new organic chemicals. Similar films on nanoparticles provide samples for LC-MS analysis of metabolite-DNA adducts. These approaches will also provide enzyme specificities, site specificities of DNA damage, and pathways of DNA damage. Our broad long-term goals are to develop structure-based methods for toxicity assessment based on enzyme/DNA films that mimic natural bioactivation. Prototype electro-optical arrays have been developed in which enzymes catalyze formation of metabolites, and subsequent electrocatalytic-optical detection estimates DNA damage. Similar films on nanoparticles generate nucleobase adducts for measurement by LC-MS, providing structural identification and formation rates that correlate with array responses and rodent mutagenicity. Capillary electrophoresis arrays will be used to identify specific sites of damage on key genes implicated in carcinogenesis. This structural information is valuable for """"""""designing out toxicity"""""""" of synthetic target molecules while retaining desired bioactivity. Microsomes or pure enzymes can be used.
Specific aims summarized for the next grant period include: (1) increase throughput of electro-optical arrays using ink jet spotting, and test them against a wide range of toxic chemicals;(2) develop films of bioconjugation enzymes in sequential multi-enzyme pathways leading to reactive metabolites for incorporation into arrays and nanoreactors;(3) pursue selected applications of DNA/enzyme nanoreactors for detailed LC- MS studies of chemicals with reactive metabolites;(4) develop array methods to measure inhibition aimed at uncovering multi-substrate metabolic interactions with toxic impact;(5) couple DNA/enzyme nanoreactors and capillary electrophoresis arrays to identify codons where metabolites react on genes implicated in cancer. Validation will be done by correlating array and LC-MS-derived DNA damage rates for toxic chemicals with animal mutagenicity databases. These approaches will also be useful to establish effects of substrate specificities, multi-chemical interactions, and enzyme polymorphism. Resulting bioanalytical advances will facilitate screening new toxic chemicals early in their development and promote new therapeutic approaches for diseases caused by pollutant exposure.
Major pathways of chemical toxicity involve bioactivation by enzymes in the body to make toxic species that damage key biomolecules. This project aims at developing novel mechanism-based methods to screen new chemicals that produce toxic species, and to facilitate designing out the toxicity. These advanced methods hold great promise for improving public health by screening out potential toxicants from many sources.
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