Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes

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Abstract

The effects of the flavonoids quercetin and myricetin, and the antihepatotoxic agent silymarin, on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide increased the levels of DNA strand breaks and oxidised pyrimidine bases in these cells. Quercetin was protective at concentrations above 10 μM and myricetin decreased oxidant-induced DNA strand breakage at concentrations of 100 μM. Cellular metabolism may alter the antioxidant efficacy of the flavonoids. Silymarin had no protective effect at any of the concentrations tested. None of these flavonoids was itself genotoxic. Neither α-tocopherol nor β-carotene decreased hydrogen peroxide-induced DNA breakage. The differences in effectiveness of these dietary compounds against oxidative DNA damage may be explained by differences in their chemical structure or location within the cell.

Introduction

Reactive oxygen species, from both endogenous and exogenous sources, may be involved in the aetiology of diverse human diseases, such as coronary heart disease, stroke, rheumatoid arthritis and cancer 1, 2. Diets rich in fruit and vegetables are associated with a reduced risk for these pathologies 3, 4, 5and protection has been attributed to antioxidant vitamins such as vitamin C, vitamin E and β-carotene 6, 7, 8, 9.

However, other dietary components may be important protective agents. Flavonoids are polyphenolic compounds, ubiquitous in plants, and found in significant quantities in vegetables, fruits, seeds, nuts and beverages such as tea and wine. Quercetin is the major flavonoid found in the average Western diet (16 mg/day). It is found mainly in onions, apples and tea [10]. Myricetin occurs in many plant-derived foodstuffs, notably tea, grapes and cranberries 10, 11. Silymarin, isolated from the milk thistle, Silybum marianum, is a hepatoprotective agent, used principally in the treatment of alcohol-induced liver toxicity [12]. The dietary intake for flavonoids in humans has been estimated to lie between 16 and 1000 mg/day 13, 14, but little is known of their absorption, distribution and metabolism.

Flavonoids have a number of biological effects in vivo and in vitro. They are anti-inflammatory, antiviral and antiallergic agents and can modulate a wide range of mammalian enzyme activities, such as cytochrome P450, epoxide hydrase, glutathione transferase, DNA and RNA polymerases and topoisomerase 15, 16. They are best known, however, for their antioxidant properties, and can act in vitro as reducing agents, hydrogen donors, free radical quenchers and metal ion chelators (17, 18, 19, 20). While this may account for the antimutagenic activities of flavonoids in experimental systems, relatively little is known about the potential cytoprotection afforded by these dietary components in humans.

This study compares the ability of the flavonoids, quercetin, myricetin and silymarin to modulate the DNA damage induced in human lymphocytes by oxygen free radicals.

Section snippets

Materials

α-Tocopherol, β-carotene, l-glutamine, histopaque 1077, myricetin, penicillin G, pyruvic acid, quercetin and streptomycin sulphate were from Sigma (Poole, UK). Silymarin was obtained from Aldrich Chemical Co. (Dorset, UK). Dutch modified RPMI 1640 medium containing 20 mM HEPES buffer was from ICN Flow (Irvine, UK). Fetal calf serum (FCS; batch 150) was obtained from Globepharm Ltd. (Surrey, UK). Nunc sterile tissue culture plastics were supplied by Gibco Life Technologies Inc. (Paisley, UK).

Results

The genotoxic effects of hydrogen peroxide and the protective ability of quercetin, myricetin and silymarin were assessed in normal human lymphocytes using the comet assay. The concentrations of flavonoids used were non-toxic [29]and DMSO, the vehicle used throughout, had no effect on either DNA strand breakage or base damage (data not shown). Neither hydrogen peroxide (200 μM) nor quercetin (50 μM), incubated either singly or in combination, affected human lymphocyte viability or cell number (

Discussion

Epidemiological studies suggest that dietary flavonoids protect against coronary heart disease and stroke 5, 34. The beneficial effects of flavonoids have been attributed to their antioxidant properties, including their ability to inhibit xanthine oxidase activity, to scavenge superoxide anions and hydroxyl radicals, and to inhibit lipid peroxidation in a variety of experimental systems 17, 20, 35, 36. Free radical scavenging efficiency is associated with the presence and number of hydroxy

Acknowledgements

The authors are grateful to Professor Ian Bremner for critically reviewing the manuscript.This work was funded by the Scottish Office Agriculture Environment and Fisheries Department and the Ministry of Agriculture Fisheries and Food.

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