Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure

Szeto, Yim Tong Savio; Collins, Andrew; Benzie, Iff

doi:10.1016/s0027-5107(01)00298-6

Cited by 57 publications

(49 citation statements)

References 25 publications

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“…First, the cells were permeabilized on ice (for 1 h) before incubation with A2E-epoxides (for 3 h) or A2E-bisepoxide (for 5 h). As a second approach, a modified comet assay was performed in which the agaroseembedded cells were incubated with A2E-epoxides or A2E-bisepoxide (at 1 h) before lysis (29). The results were similar with these two approaches, and the data were pooled.…”

Section: Methodsmentioning

confidence: 99%

A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells

Sparrow

Vollmer-Snarr

Zhou

et al. 2003

Journal of Biological Chemistry

247

106

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The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.The di-retinal conjugate A2E forms as a consequence of light related vitamin A cycling in the retina. This orange-emitting fluorophore is formed synthetically as the condensation product of all-trans-retinal and ethanolamine (1-3). NMR and corroborative total chemical synthesis revealed A2E to be a pyridinium bisretinoid consisting of an unprecedented pyridinium polar head group and two hydrophobic retinoid tails (4, 5). A2E, its slightly less polar photoisomer, iso-A2E, and other minor cis-isomers together constitute the most prominent age-related hydrophobic pigments (lipofuscin) in retinal pigment epithelial (RPE) 1 cell extracts assayed by reverse phase HPLC (3, 6). In vivo, A2E is generated by phosphate hydrolysis of the fluorophore phosphatidylpyridinium bisretinoid, the latter precursor forming from reactions between all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane (3,7,8).Although certain levels of A2E are clearly tolerated by RPE cells, adverse effects of its accumulation have also been reported. Thus, not only can A2E mediate detergent-like effects on cell membranes (9), its accumulation can also lead to the alkalinization of lysosomes (10) and to the detachment of proapoptotic proteins from mitochondria (11). A2E also bestows a sensitivity to blue light damage (11-13) that is proportional to t...

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Section: Methodsmentioning

confidence: 99%

A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells

Sparrow

Vollmer-Snarr

Zhou

et al. 2003

Journal of Biological Chemistry

247

106

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“…The comet assay has been used to determine the effects of these cellular processes on the amount of DNA damage induced (Kasamatsu et al, 1996;Szeto et al, 2002;Tice et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Determination of the effects of some artificial sweeteners on human peripheral lymphocytes using the comet assay

Findikli

Türkoğlu²

2014

J. Toxicol. Environ. Health Sci.

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In this study, the genotoxicity of the artificial sweeteners acesulfame potassium, aspartame, saccharin, and sorbitol, which are used in food industry and by patients with diabetes, was investigated in human peripheral lymphocyte cells using the single-cell gel electrophoresis (comet) technique. Human lymphocyte cells were treated with the substances for three hour at each of the three dosages (1.25, 2.5, and 5 ppm). The chemical additives were studied, and the related DNA damages in the study group were compared to the control group for each of the treatment dosages. The DNA breakages observed in the comet assay were assessed in terms of tail moment and tail DNA percent using the comet parameters. The statistical and photographic analyses were performed using SPSS 15 and BAB BS 200 Pro software, respectively. Based on the results for the short-term in vitro treatments, the 4 different food flavorings were found to have genotoxic effects.

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“…Previous studies demonstrated increased resistance to oxidant DNA damage in lymphocytes with the antioxidant pretreatment by using vitamin E and plant-derived compound such as quercetin, epicatechin and ginseng (Szeto, 2007b;Szeto et al, 2002Szeto et al, , 2011a.…”

Section: Introductionmentioning

confidence: 99%

“…Under normal intracellular conditions with continuous oxidant challenge of varying intensity, there are four important intracellular mechanisms of DNA protection for the maintenance of low baseline level of DNA damage including scavenging of damaging ROS, enzymatic inactivation of ROS, binding of iron and activation of DNA repair enzymes (Szeto et al, 2002). In this study, both SAC assay and LCC assay were used to elucidate the effect of Yunzhi on human lymphocytes.…”

Section: Introductionmentioning

confidence: 99%

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Direct human DNA protection byCoriolus versicolor(Yunzhi) extract

Szeto

Lau

Kalle

et al. 2013

Pharmaceutical Biology

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Context and objective: Scientific evidence has shown Coriolus versicolor (L. ex Fr.) Quel (also known as Yunzhi) has the role of immunomodulator in therapeutic effect. The aim of this in vitro study was to investigate the antioxidative effect of Yunzhi and to explore the mechanisms behind its DNA protection. Materials and methods: Commercial Yunzhi extract was dissolved in water and diluted in five concentrations (105 mg/L) with appropriate buffers. Lymphocytes harvested from three healthy subjects were incubated with Yunzhi extract for 30 min. Cells were then subjected to 5 min oxidant challenge by 45 mM hydrogen peroxide. The standard alkaline comet (SAC) assay and lysed cell comet (LCC) assay were performed in parallel. DNA damage of each treatment was scored under a fluorescence microscope and compared with the cells without Yunzhi pretreatment. Results: U-shaped dose-response was seen in both versions of the comet assay. Yunzhi at 10 4 mg/L demonstrated a genoprotective effect against oxidative damage in the SAC assay (25% decrease in comet score). In the LCC assay, a trend of protection in lymphocytes was observed but it did not reach statistical significance. Conclusion: A direct antioxidant effect of Yunzhi against oxidant challenge on the DNA of lymphocytes was evidenced. The active component in Yunzhi was likely to be membrane permeable.

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Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure

Cited by 57 publications

References 25 publications

A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells

A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells

Determination of the effects of some artificial sweeteners on human peripheral lymphocytes using the comet assay

Direct human DNA protection byCoriolus versicolor(Yunzhi) extract

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