Presentation Title

Inhibition of Oxidative DNA Damage by an Aqueous Extract of Spinach

Faculty Mentor

Eric Stemp

Start Date

17-11-2018 8:30 AM

End Date

17-11-2018 8:45 AM

Location

C327

Session

Oral 1

Type of Presentation

Oral Talk

Subject Area

physical_mathematical_sciences

Abstract

Oxidative damage can induce DNA protein cross-linking. Here, we explored whether aqueous extracts of spinach can inhibit oxidative cross-linking between DNA and protein. In the flash-quench method, the intercalator Ru(phen)2dppz2+ (phen = phenanthroline, dppz = dipyridophenazine) is excited with blue light, releasing an electron to the quencher, Co(NH3)5Cl2+. The oxidized intercalator then extracts an electron from guanine and the guanine radical reacts to produce DNA-protein cross-links. Samples containing Co(NH3)5Cl2+, histone, Ru(phen)2dppz2+, and DNA were subjected to flash quench treatment in the absence or presence of a spinach extract. Cross-linking was then detected in two ways. With calf thymus DNA, the chloroform extraction assay was used, and this uv spectroscopy method showed that the spinach extract significantly decreased the loss of the free DNA absorbance due to cross-linking. In gel shift assays, pUC19 DNA subjected to the flash quench technique decreased greatly in mobility, with a considerable amount of cross-linked material visible within the wells. In the presence of an aqueous extract of spinach, cross-linking is strongly inhibited and the DNA runs as a single band. This inhibition is maintained out to significant dilutions of the green extract. More recently, we have examined kaempferol, one antioxidant found in spinach, and have observed protection of DNA via a nicking assay. Future studies will be conducted to ascertain whether any of this inhibition is due to trivial effects such as interference with the flash quench oxidation process or absorption of the laser light used to initiate oxidation. Since one likely explanation for these results would be the reduction of the guanine radical by antioxidants present in spinach (such as kaempferol), we will employ nicking and gel shift assays to assess the DNA damage and attempt to monitor the formation of antioxidant radicals by transient absorption spectroscopy.

Summary of research results to be presented

Cross-linking was detected in two ways to show the effect of aqueous spinach extract on DNA oxidation. With calf thymus DNA, the chloroform extraction assay was used, and the uv spectroscopy method showed that the spinach extract significantly decreased the loss of the free DNA absorbance due to cross-linking; we will show raw data (spectra) and a plot of % cross-linked vs time. In gel shift assays, pUC19 DNA subjected to the flash quench technique decreased greatly in mobility, with a considerable amount of cross-linked material visible within the wells. In the presence of an aqueous extract of spinach, cross-linking is strongly inhibited and the DNA runs as a single band. Similar gels are shown for kaempferol as an inhibitor of oxidative damage.

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Nov 17th, 8:30 AM Nov 17th, 8:45 AM

Inhibition of Oxidative DNA Damage by an Aqueous Extract of Spinach

C327

Oxidative damage can induce DNA protein cross-linking. Here, we explored whether aqueous extracts of spinach can inhibit oxidative cross-linking between DNA and protein. In the flash-quench method, the intercalator Ru(phen)2dppz2+ (phen = phenanthroline, dppz = dipyridophenazine) is excited with blue light, releasing an electron to the quencher, Co(NH3)5Cl2+. The oxidized intercalator then extracts an electron from guanine and the guanine radical reacts to produce DNA-protein cross-links. Samples containing Co(NH3)5Cl2+, histone, Ru(phen)2dppz2+, and DNA were subjected to flash quench treatment in the absence or presence of a spinach extract. Cross-linking was then detected in two ways. With calf thymus DNA, the chloroform extraction assay was used, and this uv spectroscopy method showed that the spinach extract significantly decreased the loss of the free DNA absorbance due to cross-linking. In gel shift assays, pUC19 DNA subjected to the flash quench technique decreased greatly in mobility, with a considerable amount of cross-linked material visible within the wells. In the presence of an aqueous extract of spinach, cross-linking is strongly inhibited and the DNA runs as a single band. This inhibition is maintained out to significant dilutions of the green extract. More recently, we have examined kaempferol, one antioxidant found in spinach, and have observed protection of DNA via a nicking assay. Future studies will be conducted to ascertain whether any of this inhibition is due to trivial effects such as interference with the flash quench oxidation process or absorption of the laser light used to initiate oxidation. Since one likely explanation for these results would be the reduction of the guanine radical by antioxidants present in spinach (such as kaempferol), we will employ nicking and gel shift assays to assess the DNA damage and attempt to monitor the formation of antioxidant radicals by transient absorption spectroscopy.