45083-GB4
The Role of Oxygen in Photoinduced DNA Damage
Overview:
This past year, my research program with undergraduates has focused on examining the products that result from the photoexcitation of the anthracycline antibiotic daunomycin in the presence of DNA and the role of oxygen in this process. Our results are consistent with a charge transfer mechanism whereby an electron is transferred from the DNA base guanine to daunomycin. The resulting daunomycin radical can then reduce molecular oxygen forming a superoxide radical. This year, our work was presented at the ACS meeting and published in J. Photochem. Photobiol A: Chem with an undergraduate co-author.
Research Outcomes:
The role of molecular oxygen on oxidative DNA damage induced by photoactivated daunomycin-DNA complexes.
Our goals were to characterize the DNA damage profile that is induced by photoactivated daunomycin, a DNA intercalator, and identify the oxygen species involved in the mechanism. The plasmid relaxation assay, which monitors the conversion of supercoiled to open-circular forms of DNA, showed that photoactivated daunomycin induces DNA strand scission. When this assay is coupled with various DNA repair endonucleases, enzyme-sensitive modifications such as oxidative base damage and abasic sites could also be examined. Last year, we established the protocols in my laboratory and determined that photoexcitation of daunomycin induces oxidative DNA damage- with a prevalence of oxidized guanine bases- that is dose and irradiation time dependent. This year, we finalized these results so we could report the quantification of open-circular DNA, and thereby determine the relative levels of purine and pyrimidine base modifications, abasic sites, and strand breaks induced by photoactivated daunomycin.
Our next goal was to evaluate the role of oxygen in the DNA damage mechanism. Oxidized guanine formation decreased when the solution was depleted with oxygen while the level of DNA strand breaks increased under anaerobic conditions. These results suggest that DNA strand scission can arise from an oxygen independent mechanism while guanine specific oxidation is indeed oxygen dependent.
Identification of the oxygen species involved in the DNA damage mechanism. In summary, we have shown that photoactivated daunomycin damages DNA leading to mostly guanine specific oxidation that is oxygen dependent. Furthermore, the cytochrome c assay demonstrated that superoxide is produced under these conditions. Together, these results are consistent with a charge transfer mechanism where activated daunomycin abstracts an electron from guanine producing a guanine cation radical and a daunomycin anion radical which can subsequently reduce molecular oxygen generating superoxide. In the absence of oxygen, electron back transfer occurs forming the initial reactants. Our results identified the reaction products formed when daunomycin is photoactivated in the presence of DNA and we conclude that a charge transfer is a main driving force of the mechanism. Consequences of superoxide formation on DNA damage. This upcoming year, we will further evaluate the consequence of superoxide production on DNA damage. We will also investigate the role of metal ions in this process and examine if metal association with DNA or daunomycin is required. Finally, we will extend our studies to examine other types of DNA intercalators. Student Outcomes:
By using spectroscopic methods, we were able to indirectly detect the presence of superoxide radicals formed from irradiation of daunomycin-DNA complexes using a cytochrome c assay. The formation of reduced cyctochrome c can be monitored spectrally by measuring the increase in absorbance at 550 nm. In the presence of DNA, the spectra showed cytochrome c reduction, which was suppressed in the presence of superoxide dismutase. These results confirm that photoirradiation of DM-DNA complexes generates superoxide radicals. In addition, we were able to rule out singlet oxygen as a major oxygen species because reactions carried out in D2O, which increases the lifetime of singlet oxygen, did not increase the level of damage.
Although superoxide itself cannot damage DNA, it can generate DNA-damaging hydroxyl radicals through Fenton-type reactions catalyzed by transition metal ions. Hence, oxidative DNA damage could arise from two different mechanisms—from the direct formation of guanine cation radicals produced by charge transfer and from reactive oxygen species (ROS) derived from superoxide. The level of oxidative DNA damage was measured in the presence of specific ROS scavengers and metal ion chelators. Our initial results suggest the involvement of hydroxyl radicals and transition metal ions in the induction of damage.
One goal of my work with undergraduates is to fully engage them in exciting research. This project has proven to be highly effective in advancing their education and in instilling a passion for research. For example, the undergraduate student who initiated this project with me and continued to work on it for the past two years finished her undergraduate career with a successful honors capstone and as co-author of our publication. Another student in my laboratory, who is interested in attending graduate school in part because of her research experience, is currently evaluating the role of ROS and transition metals on DNA damage.
