The goal of these R21 Exploratory/Developmental studies is to develop a sensitive metabolomic platform to quantify damaged components of the nucleotide pool as a source of DNA and RNA damage. While toxic and mutagenic lesions arise in nucleic acids by direct reaction with environmental and endogenous toxicants, incorporation of damaged ribo- and 2-deoxyribonucleotides into DNA and RNA represents a potentially important source of genetic and cellular toxicity. The impact of the nucleotide pool has long been suspected from studies of highly conserved pool sanitizing enzymes, such as the pyrophosphatases that target damaged and non-canonical (d)NTP. The loss of these enzymes leads to increased levels of DNA damage. In spite of this evidence, there have been few quantitative studies of nucleotide pool damage due to a lack of analytical methods. To address this problem, we will develop a specific, sensitive and precise analytical method to quantify damaged nucleotide mono-, di- and tri-phosphates in the nucleotide pool. Following development with standards, the method will applied to two cellular models of genetic pathology and chemical carcinogenesis in humans: defects in purine nucleotide metabolism and oxidative stress. We recently discovered that defects in purine nucleotide metabolic pathways cause up to 600-fold increases in hypoxanthine, but not xanthine, into both DNA and RNA, presumably due to imbalances in guanine (G) and adenine (A) precursors in the nucleotide pool. The second application, which poses a greater challenge in terms of sensitivity, addresses oxidation of purine nucleotides in cells subjected to oxidative stress. These applications allow us to test and optimize the analytical platform for future studies in mammalian cells and human tissues, in which we address the full range of genotoxic and cytotoxic nucleotide pool damage from endogenous and environmental sources. Furthermore, both the results obtained and the novel technologies developed will find broad application in a variety of areas of biomedical research, including antibiotic development, genetic toxicology, inflammation and oxidative stress, and, at a systems level, any of the dozens of hereditary disorders of purine nucleotide metabolism.
The goal of these exploratory studies is to develop a new bioanalytical technology to study imbalances in the cellular pool of DNA and RNA building blocks in cells. To demonstrate the power of this method, we will tackle two major human health problems caused by imbalances in the nucleotide pool in cells: how human genetic variation affects the presence of mutagenic RNA and DNA nucleotides in the pool, and how oxidative stress kills cells - does it cause damage to the nucleotide pool? Both the results obtained from our studies on mutagenic nucleotide damage and the novel technologies that we develop will find broad application in many areas of biomedical research, including cancer research, mutagenesis, inflammation, and any of the dozens of human diseases caused by disruption of nucleotide metabolism.
