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45083-GB4
The Role of Oxygen in Photoinduced DNA Damage
Overview: This past year, my research program with undergraduates has been focused on examining the DNA damage products produced in the presence of irradiated daunomycin. My students and I have successfully characterized the DNA damage profile. We presented this work at the ACS National Meeting in Chicago this past spring and are in the process of finalizing these findings as a scientific publication.
Research Outcomes (2006-2007): Photoexcitation of daunomycin leads to oxidative DNA base damage. Daunomycin, an anthracycline antibiotic, intercalates DNA with GC base pair preference. The cytotoxicity of daunomycin is enhanced in the presence of visible light. Interestingly, femtosecond dynamic experiments suggest that photoactivation leads to charge separation between deoxyguanine and daunomycin.1 Under aerobic conditions, an electron can then be transferred from the daunomycin radical anion to oxygen, generating a superoxide radical anion. Although superoxide itself cannot damage DNA, it can generate DNA-damaging hydroxyl radicals through Fenton-type reactions. In order to obtain a more detailed picture of the mechanism of DNA damage, we quantitatively assessed the types of DNA damage, such as strand breaks, oxidized bases, and abasic sites, that are induced by photoactivated daunomycin. Ultimately, we wish to monitor the damage products under anaerobic conditions and with scavengers for reactive oxygen species (ROS) in order to establish if oxygen is involved in generating DNA damage. Thus far, we have demonstrated the following:
1) Photoexcitation of daunomycin induces oxidative DNA damage that is dose and irradiation time dependent. We first examined if irradiated daunomycin-DNA complexes can induce DNA strand breaks. The plasmid relaxation assay was used to assess the level of single strand breaks by monitoring the conversion of nicked circular DNA (Form II) from supercoiled plasmid DNA (Form I). Upon irradiation, UV light had a slight increase in single strand break formation. However, in the presence of daunomycin, there is a distinct effect in which spontaneous DNA cleavage is generated in a dose- and irradiation time dependent manner.
To further characterize the DNA damage profile, DNA repair enzymes were used to convert oxidized purines or pyrimidines, and abasic sites into DNA strand breaks. The DNA glycosylase hOGG1 has specificity for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (fapy) guanine while endo III and APE1 have specificity for oxidatively damaged pyrimidines and abasic sites, respectively. Our results show that the largest increase in strand breaks occurs when the reaction mixture is treated with hOGG1, indicating that oxidized purines are substantially produced under these conditions. Indeed, our preliminary data using HPLC with electrochemical detection confirms that 8-oxoguanine is being generated in a daunomycin dose dependent and irradiation time dependent manner. On the other hand, oxidized pyrimidine bases and abasic sites, which can result from the oxidation of deoxyribose or nucleobases, did not occur to a significant extent.
2) Irradiated daunomycin-DNA complexes generate superoxide. 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. However, the addition of superoxide dismustase (SOD) did not completely abolish the absorbance peak at 550 nm, even at high SOD concentrations. These results suggest that the superoxide anion is being generated, which could be produced either from irradiated daunomycin alone or from a multi-step electron transfer from the DNA base guanine to daunomycin and then to superoxide. The fact that addition of SOD did not completely remove the absorbance peak at 550 nm suggests that superoxide is not the only species capable of transferring an electron to cytochrome c. It is possible that the daunomycin radical anion is being formed from the oxidation of deoxyguanine.
In summary, we have shown that photoactivated daunomycin oxidatively damages DNA leading to DNA strand scission and purine base modification. Furthermore, the cytochrome c assay demonstrated that superoxide is produced under these conditions. We hypothesize that DNA damage can arise from two different mechanisms: from direct formation of guanine radical cation caused by charge transfer or from ROS production derived from superoxide anion. The femtosecond dynamic experiments, although they did not isolate the product, provided the timescale for charge separation, which is much shorter than any diffusion process. This mechanism is consistent with our results. We are currently evaluating the role of ROS in inducing damage.
Student Outcomes (2006-2007): The goals of my work with undergraduates are to fully engage them in exciting research and actively encourage them to present and disseminate their work to the scientific community. During this past year, this grant supported the research of two undergraduate biochemistry majors, one of whom presented this work at the ACS National Meeting in Chicago and was selected by the department to use her research experience to fulfill an honors capstone. We are currently finalizing our results as a scientific publication.
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1Qu, X., et. al (2001) Proc. Natl. Acad. Sci. USA 98, 14212-14217
