Presentation Title

Investigation of the Molecular Mechanism of Inhibition of DNA Oxidation via Polyphenols from Green Tea

Faculty Mentor

Dr. Eric Stemp

Start Date

18-11-2017 9:15 AM

End Date

18-11-2017 9:30 AM

Location

9-279

Session

Physical Sciences 1

Type of Presentation

Oral Talk

Subject Area

physical_mathematical_sciences

Abstract

Oxidative stress causes many chronic diseases, such as cancer. Damage occurs particularly at the guanine base in DNA since it is the easiest to oxidize. In previous research, green tea has been shown to inhibit DNA-protein crosslinking. Green tea is composed of many polyphenols, such as Epigallocatechin-3-gallate (EGCG) and Quercetin. Here, we investigated whether EGCG and Quercetin can minimize the oxidative damage by reductive repair of guanine radical. The flash-quench method was utilized to selectively oxidize guanine and induce DNA-protein crosslinks. DNA-protein crosslinks produced by this method were detected two ways. In the chloroform extraction assay, uv spectroscopy showed that more DNA remained in the aqueous phase in samples containing the phenols, consistent with inhibition of oxidative damage. In the gel shift assay, the band of free DNA persisted in irradiated samples containing the polyphenols, in contrast to samples lacking the EGCG or quercetin, where the free DNA band decreased in intensity and lower mobility material was seen. In the transient absorption experiment, excitation gave rise to long-lived signals for both EGCG (l=405 nm) and quercetin (l=590 nm), consistent with formation of a phenoxy radical. During the transient absorption spectroscopy experiment, samples with EGCG turned pink. Thus, we examined the spectrum of flash quench samples containing EGCG as a function of irradiation time with a green diode laser at 532 nm. Upon irradiation, there were spectral changes in the visible region, with a new band showing up at ~490 nm, probably showing that ECGC is unstable under these conditions.

Summary of research results to be presented

In the chloroform extraction assay, uv spectroscopy showed more DNA remained in the aqueous phase in samples containing the phenols, consistent with a diminution of crosslinking. In the gel shift assay, the band of free DNA persisted in irradiated samples containing the polyphenols, in contrast to samples lacking the EGCG or quercetin, where the free DNA band decreased in intensity and lower mobility material was seen. In the transient absorption experiment, excitation of samples containing the flash quench components gave rise to long-lived signals for both EGCG( l=405nm) and quercetin (l=590nm), consistent with formation of a phenoxy radical. During the transient absorption spectroscopy experiment, we noted that samples with EGCG underwent a pink color change. Thus, we examined the spectrum of samples containing calf thymus DNA, Ruhex, Ru(phen)2dppz2+, and EGCG as a function of irradiation time with a green Nd-YAG diode laser at 532 nm to observe auto-oxidation. In addition, for the transient absorption spectroscopy experiment there was spectral changes in the EGCG sample seen in the visible light region of the spectrum. Hence, ECGC becomes unstable and auto-oxidizes.

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Nov 18th, 9:15 AM Nov 18th, 9:30 AM

Investigation of the Molecular Mechanism of Inhibition of DNA Oxidation via Polyphenols from Green Tea

9-279

Oxidative stress causes many chronic diseases, such as cancer. Damage occurs particularly at the guanine base in DNA since it is the easiest to oxidize. In previous research, green tea has been shown to inhibit DNA-protein crosslinking. Green tea is composed of many polyphenols, such as Epigallocatechin-3-gallate (EGCG) and Quercetin. Here, we investigated whether EGCG and Quercetin can minimize the oxidative damage by reductive repair of guanine radical. The flash-quench method was utilized to selectively oxidize guanine and induce DNA-protein crosslinks. DNA-protein crosslinks produced by this method were detected two ways. In the chloroform extraction assay, uv spectroscopy showed that more DNA remained in the aqueous phase in samples containing the phenols, consistent with inhibition of oxidative damage. In the gel shift assay, the band of free DNA persisted in irradiated samples containing the polyphenols, in contrast to samples lacking the EGCG or quercetin, where the free DNA band decreased in intensity and lower mobility material was seen. In the transient absorption experiment, excitation gave rise to long-lived signals for both EGCG (l=405 nm) and quercetin (l=590 nm), consistent with formation of a phenoxy radical. During the transient absorption spectroscopy experiment, samples with EGCG turned pink. Thus, we examined the spectrum of flash quench samples containing EGCG as a function of irradiation time with a green diode laser at 532 nm. Upon irradiation, there were spectral changes in the visible region, with a new band showing up at ~490 nm, probably showing that ECGC is unstable under these conditions.