Epidemiological studies suggest that a significant fraction of human cancers in industrialized societies are induced by environmental factors such as genotoxic chemicals that are ubiquitous in urban environments. Rapid developments in the field of mass spectrometry promise to provide more and more extensive human DNA adductomics maps. However, for a deeper appreciation of the significance of the DNA adductomics results, it is important to establish which of these DNA adducts are a greater threat to human health than others. The first and critically important cellular defense against genotoxic DNA adducts is cellular DNA repair. Extensive experiments in human cell extracts in vitro have shown that there is a wide range of DNA adducts that persist in human cells and tissues for long periods of time, while others disappear more quickly. Research on the DNA repair capacities (DRC) of different isomeric and other forms of the same family of cancer-causing DNA adducts has shown that there are significant differences in DNA repair capacities in human cell extract model systems in vitro. There are classes of adducts that resist DNA repair entirely, and are therefore considered to be the most genotoxic ones. However, it is not known whether the results obtained in cell extract experiments in vitro carry over to the cellular environment. Therefore, In this project, the development of methods are proposed for measuring the repair of chemically defined DNA adducts in cellular environments in intact cells. Methods will be developed that will differentiate the repair of different DNA adducts by the three major human DNA repair systems: Nucleotide Excision Repair (NER), Transcription Coupled Repair (TCR) and Base Excision Repair (BER). Previous efforts to monitor these pathways in a unified manner using the same DNA substrates, were hindered by difficulties in recovering the products of the different DNA repair mechanisms from intact cells. Preliminary results show that this approach is now feasible using 32P-labeling techniques. In this project, plasmid vectors containing single chemically and structurally defined DNA lesions belonging to different classes of DNA adducts will be transfected into single cells, and the repair of these lesions will be monitored as a function of time. The results from the proposed DNA repair capacity experiments will ultimately provide new information for assessing the significance of DNA adductomic patterns. Another objective is to improve the present biomarker technologies that simply document the presence of DNA lesions, but do not provide critical information about an individual?s capacity to remove that damage from the genome. An accurate knowledge of individual DNA repair capacities will be useful for assessing the risk of such individuals for developing cancers associated with exposure to environmental carcinogens. In summary, the biological significance of DNA adductomics data for evaluating the risk to human health due to exposure to toxic chemicals in the environment or work place, would be markedly enhanced by a knowledge of the cellular DRC of the different DNA adducts.
This project is focused on developing a deeper understanding of the human DNA adductome by developing robust methods for measuring the efficiencies of human DNA repair mechanisms to clear different, well defined DNA adducts from intact human cells. By measuring the DNA repair capacities of human cells to remove genotoxic and cancer-causing DNA adducts, a better understanding of the significance of DNA adductomes to human health and disease will be achieved.
