Sierra Evelyn Dabell and Dr. Heidi R. Vollmer-Snarr, Chemistry and Biochemistry
Introduction
A2E, a common fluorophore found in the retinal pigment epithelial cells of the eye has been shown to have phototoxic properties. For this reason A2E has been implicated in the progressive etiology of Age-related Macular Degeneration (AMD). If the mechanism of cell death can be understood, a possible intervention can be produced. Moreover, the benefits of researching A2E are two-fold. An understanding of bisoxirane-DNA interactions holds the possibility of revealing information about the pathology of AMD and presents itself as a possible chemotherapy through photo-dynamic therapy.
The intent of the research was to clarify the interaction between the epoxide form of A2E (bisoxirane) and DNA, specifically deoxyguanosine. The decision to research the A2E-DNA interaction as a pathological trigger is rooted in two facts. First, researchers in the past found 8-oxo-deoxyguanosine using antibodies after cells treated with the bisoxirane. 8-oxo-deoxyguanosine is a characteristic marker of oxidative DNA damage. The second fact spurring this research is the precedent established through previous research showing the highly reactive nature between nucleophilic deoxyguanosine and unstable epoxides.
Methods
The bisoxirane was obtained by irradiating A2E with 430nm (blue) light to induce epoxidation. The presence of the epoxide was confirmed through High Performance Liquid Chromatography (HPLC). HPLC standards of A2E, irradiated A2E, deoxyguanosine, irradiated deoxyguanosine and 8-oxo-deoxyguanosine were also made for comparison. Once initial controls were established, reaction conditions were experimented with. Conditions tried included basic, neutral and acidic pH in an aprotic solvent; neutral and acidic conditions using a protic solvent and finally a phosphate buffer. All reaction trials were analyzed by HPLC before irradiation, at various times after irradiation and after a reaction period.
Discussion and Future Work
Throughout my research, I have failed to find sufficient evidence that an interaction is occurring.
No 8-oxo-deoxyguanosine has been detected. The only unexplainable peak formed over the course of the HPLC injections does not fit the hypothesized adduct profile. This lack of evidence does not mean a reaction between the two can not occur or does not occur in vivo. More research will be done experimenting with Lewis acids as a catalyst and with gel electrophoresis as an easier method to assay strand breakage.
Resources
- Sparrow Janet R; Vollmer-Snarr Heidi R; Zhou Jilin; Jang Young P; Jockusch Steffen; Itagaki Yasuhiro; Nakanishi Koji A2E-epoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation. JOURNAL OF BIOLOGICAL CHEMISTRY (2003 May 16), 278(20), 18207-13.
- Richter, Sara; Gatto, Barbara; Fabris, Daniele; Takao, Ken-Ichi; Kobayashi, Susumu; Palumbo, Manlio. Clerocidin alkylates DNA through its epoxide function: evidence for a fine tuned mechanism of action. Nucleic Acids Research (2003), 31(17), 5149-5156.
- Sparrow Janet R; Zhou Jilin; Cai Bolin DNA is a target of the photodynamic effects elicited in A2E-laden RPE by blue-light illumination. INVESTIGATIVE OPHTHALMOLOGY AND VISUAL SCIENCE (2003 May), 44(5), 2245-51.
- Ben-Shabat, S.; Parish, C. A.; Hashimoto, M.; Liu, J.; Nakanishi, K.; Sparrow, J. R. Fluorescent pigments of the retinal pigment epithelium and age-related macular degeneration. Bioorganic & Medicinal Chemistry Letters (2001), 11(12), 1533-1540.