Search

Anthocyanins are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. The phenolic structure of anthocyanins conveys marked antioxidant activity in model systems via donation of electrons or hydrogen atoms from hydroxyl moieties to free radicals. Dietary intakes of anthocyanins may exceed 200 mg/day, however, little is known about their antioxidant potency in vivo. Consequently, the aim of this study was to establish whether anthocyanins could act as putative antioxidant micronutrients. Rats were maintained on vitamin E-deficient diets for 12 weeks in order to enhance susceptibility to oxidative damage and then repleted with rations containing a highly purified anthocyanin-rich extract at a concentration of 1 g/kg diet. The extract consisted of the 3-glucopyranoside forms of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Consumption of the anthocyanin-repleted diet significantly improved (p <.01) plasma antioxidant capacity and decreased (p <.001) the vitamin E deficiency-enhanced hydroperoxides and 8-Oxo-deoxyguanosine concentrations in liver. These compounds are indices of lipid peroxidation and DNA damage, respectively. Dietary consumption of anthocyanin-rich foods may contribute to overall antioxidant status, particularly in areas of habitually low vitamin E intake.

Background: It is widely believed that antioxidant micronutrients obtained from fruit and vegetables afford significant protection against cancer and heart disease, as well as ageing. Flavonoids are potential antioxidants found in foods such as onions; information on their effectiveness in vivo is so far lacking., Aims: To determine uptake as well as in vivo antioxidant effects of flavonoids from foods., Methods: Six healthy non-obese normocholesterolaemic female volunteers in the age range 20-44 years participated in a randomised two-phase crossover supplementation trial to compare the antioxidant effects associated with (a) a meal of fried onions and (b) a meal of fried onions and fresh cherry tomatoes. Plasma flavonoids, lymphocyte DNA damage, plasma ascorbic acid, tocopherols and carotenoids, urinary malondialdehyde and 8-hydroxy-2'-deoxyguanosine were determined to assess flavonoid absorption and antioxidant efficacy., Results: Flavonoid glucosides (quercetin-3-glucoside and isorhamnetin-4-glucoside) were significantly elevated in plasma following ingestion of the onion meal and the increases were associated with an increased resistance of lymphocyte DNA to DNA strand breakage. A significant decrease in the level of urinary 8-hydroxy-2'-deoxyguanosine was evident at 4 h following ingestion of the onion meal. After the combined tomato and onion meal, only quercetin was detected in plasma. Endogenous base oxidation was decreased but resistance to strand breakage was unchanged. There was no significant change in the excretion of urinary malondialdehyde following either meal., Conclusion: Both meals--onions, and onions together with tomatoes--led to transient decreases in biomarkers of oxidative stress, although the particular biomarkers affected differ. It is possible that the differences in patterns of response reflect the different uptakes of flavonoids but the underlying mechanism is not understood.

Background: Epidemiological studies report an inverse relationship between intake of the B vitamin folic acid and colon cancer. Folate is important for DNA synthesis and repair. Moreover, the production of S-adenosylmethionine (SAM), essential for normal DNA methylation and gene expression, is dependent on folic acid. Folate deficiency may increase the risk of malignant transformation by perturbing these pathways., Aims of the Study: The principal aim of this study was to determine the effects of folate deficiency on DNA stability and DNA methylation in rat colonocytes in vivo. As the metabolic pathways of folate and other dietary methyl donors are closely linked, the effects of methionine and choline deficiency were also evaluated., Methods: Male Hooded-Lister rats were fed a diet deficient in folic acid, or in methionine and choline, or in folate, methionine and choline for 10 weeks. DNA strand breakage and misincorporated uracil were determined in isolated colonocytes using alkaline single cell gel electrophoresis. Global DNA methylation was measured in colonic scrapings. Folate was measured in plasma, erythrocyte and liver samples., Results: Methyl donor deficiency induced DNA strand breakage in colonocytes isolated from all experimental groups. Uracil levels in colonocyte DNA remained unchanged compared with controls. DNA methylation was unaffected either by folate and/or methionine and choline depletion. Rats fed a folate-deficient diet had less folate in plasma, red blood cells and liver than controls., Conclusions: Folate and methyl deficiency in vivo primarily affects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation.

Prostate cancer is one of the most common male cancers in Western countries, yet the incidence of this fatal disease remains low in Asian populations. Environmental factors such as diet play an important role in hormone-dependent cancer etiology, and a high phytoestrogen intake may be one factor contributing to the low prostate cancer mortality in Eastern populations. In this study, we investigated the effects of the phytoestrogens genistein, daidzein, coumestrol, and equol on cell growth and DNA damage (strand breakage) in two human prostate tumor cell lines: androgen receptor-positive LNCaP and androgen receptor-negative PC-3. Each compound caused growth inhibition at physiologically relevant concentrations (<10 microM). Genistein induced DNA damage in both cell lines at <10 microM. Daidzein inhibited cell growth at 10-100 microM yet had no effect on DNA damage at up to 500 microM. Thus, despite their structural similarities, different phytoestrogens inhibit prostate tumor cell growth by independent mechanisms.

The effect of increasing dietary intakes of polyunsaturated fatty acids (PUFAs) and vitamin E on indices of oxidative DNA damage was investigated. Twenty-one healthy male, nonsmokers aged 28.9 +/- 1.3 years participated in a free-living, split plot/change over trial in which half the volunteers consumed diets containing 5% PUFA as food energy for 4 wk and, after a 10 wk washout period, consumed a 15% PUFA diet for another 4 wk. The other volunteers followed an identical protocol, except that they consumed the 15% PUFA diet first. The diets were provided to volunteers either with or without an additional 80 mg dalpha-tocopherol acetate/day; otherwise total fat, carbohydrates, protein, and basal vitamin E contents remained unchanged. DNA damage induced by 200 microM H(2)O(2) in lymphocytes from volunteers as well as endogenous DNA damage in the form of oxidized pyrimidines, measured by alkaline single-cell gel electrophoresis (the comet assay), significantly decreased after consumption of the 5% PUFA diet (P<0.001 and P=0.01, respectively), but significantly increased after consumption of the 15% PUFA diet when alpha-tocopherol levels were in the range of 5-7 mg/day (P=0. 008 and P=0.03, respectively). These changes were abolished by an additional 80 mg dalpha-tocopherol/day. This study indicates that increasing dietary levels of PUFA to 15% may adversely affect some indices of DNA stability. However, increasing the dietary intake of vitamin E by 80 mg/day ameliorates the damaging effects of PUFA. -Jenkinson, A. McE., Collins, A. R., Duthie, S. J., Wahle, K. W. J., Duthie, G. G. The effect of increased intakes of polyunsaturated fatty acids and vitamin E on DNA damage in human lymphocytes.

Background & Aims: Epidemiological studies suggest that antioxidant polyphenols in the human diet may protect against diseases such as cancer. In this study we investigated the cytoprotective potential of the flavonoids, quercetin, myricetin, kaempferol and rutin against oxidative DNA damage in human colonocytes in vitro., Methods: Caco-2 cells, which display specialised enterocyte/colonocyte cell functions, were used as an in vitro model for human colonocytes. Hydrogen peroxide was employed as the oxidant. DNA damage (strand breakage, oxidised purines and oxidised pyrimidines) was determined using the alkaline single cell gel electrophoresis or comet assay. Cell growth and viability were measured., Results: Hydrogen peroxide caused a dose-dependent increase in DNA strand breakage in human colonocytes, presumably via oxygen free radical generation. Quercetin and myricetin protected Caco-2 cells against oxidative attack. In addition, quercetin decreased hydrogen peroxide-mediated inhibition of growth. Neither rutin nor kaempferol was effective. However, quercetin, while inhibiting DNA strand breakage, did not alter the levels of oxidised bases following peroxide treatment. The antifungal agent ketoconazole, prevented quercetin cytoprotection in Caco-2 cells, indicating that P450-mediated metabolism may alter the efficacy of the flavonoids against oxidative DNA damage., Conclusion: Flavonoids, particularly quercetin, the most abundant flavonoid in the human diet, are likely to be important in defending human colonocytes from oxidative attack.

Objective: To investigate their effects on lymphocyte damage with supplementation of high-dose vitamins E, C and beta-carotene., Methods: Healthy men aged 50 - 59 years were selected and randomized into the trial and control groups with 50 in each one. In the trial group, 25 mg of beta-carotene, 100 mg vitamin C and 280 mg vitamin E were given to each subject every day for 20 weeks. Whole blood was collected in both the trial and control groups and its lymphocytes separated. Damage to DNA was analyzed with a"comet" electrophoresis technique, and serum levels of vitamin C, beta-carotene and alpha-tocopherol were determined by high performance liquid chromatography., Results: There was no significant difference in the proportions of spontaneous damage to DNA in lymphocytes between the trial and control groups (6.1% vs. 6.8%). But, proportions of damage to DNA in peripheral lymphocytes increased to 36.14%, 59.45, and 69.62%, respectively, after treatment with 30 micromol/L, 100 micromol/L and 300 micromol/L of H(2)O(2)., Conclusion: Supplementation of vitamin E, C and beta-carotene could effectively reduce the damage to DNA caused by H(2)O(2).

Carotenoids are thought to act as antioxidants in vivo, decreasing oxidative damage to biomolecules and thus protecting against coronary heart disease and cancer. However, human intervention studies with beta-carotene have given equivocal results in terms of cancer incidence. In an alternative molecular epidemiological approach, we have employed the 'comet assay' (single cell alkaline gel electrophoresis) to measure strand breaks, oxidized pyrimidines and altered purines in the DNA of lymphocytes from volunteers supplemented with alpha/beta-carotene, lutein, lycopene or placebo. In addition, we measured concentrations of the main serum carotenoids, and vitamins E and C, by HPLC. We report a significant negative correlation between basal concentrations of total serum carotenoids and oxidized pyrimidines. A similar correlation was seen between individual carotenoids (notably lutein and beta-carotene) and oxidized pyrimidines. However, carotenoid supplementation did not have a significant effect on endogenous oxidative damage. This suggests that there are some factors in the basal diet, probably found in fruit and vegetables, that decrease oxidative damage to DNA. In this case, basal serum carotenoids may simply be markers of consumption of fruit and vegetables, they themselves having little or no protective value.

Folic acid is essential for the synthesis and repair of DNA. We report the effects of folate depletion on DNA stability in normal human lymphocytes in vitro. DNA strand breakage, uracil misincorporation, oxidative DNA base damage, and DNA repair capability were determined using variants of the comet assay (single cell gel electrophoresis). Lymphocyte proliferation was measured as an indicator of normal replication. Lymphocytes isolated from human venous blood were stimulated to grow in either complete medium containing folic acid (1 ng/ml-2 microgram/ml) or medium deficient in folic acid for up to 10 days. Cells prepared for comet analysis were treated either with the bacterial DNA repair enzyme endonuclease III to determine the level of oxidized pyrimidines in lymphocyte DNA or with uracil DNA glycosylase, which detects misincorporated uracil. Cell number and viability were measured. Normal human lymphocyte DNA contained detectable amounts of misincorporated uracil (estimated as approximately 1000 per cell). DNA strand breakage and uracil misincorporation increased in a time- and concentration-dependent manner after lymphocytes were cultured with decreasing amounts of folic acid. DNA damage was induced at folic acid concentrations routinely observed in plasma from the human population (1-10 ng/ml). Lymphocytes cultured under folate-deficient conditions failed to grow normally compared with control cells. However, all lymphocytes remained viable as measured by Trypan blue exclusion. Cells deprived of folate were unable to efficiently repair oxidative DNA damage induced by hydrogen peroxide. Inhibition of repair was maximal after 8 days in culture. Folate supply had no effect on the level of oxidized pyrimidines in lymphocyte DNA, even after 10 days in culture, suggesting that folate deficiency increases uracil misincorporation relatively specifically. These in vitro results help to determine the mechanism(s) through which folic acid maintains DNA stability.

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 microM and myricetin decreased oxidant-induced DNA strand breakage at concentrations of 100 microM. 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 alpha-tocopherol nor beta-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.

The effects of the flavonoids quercetin, myricetin and silymarin on DNA damage and cytotoxicity in human cells were investigated. DNA strand breaks and oxidised pyrimidines were determined using alkaline single cell gel electrophoresis (the comet assay). Inhibition of cell growth was also measured. Caco-2 (colon), HepG2 (liver), HeLa (epithelial) cells and normal human lymphocytes showed different, dose-dependent susceptibilities (in terms of strand breakage) to the various flavonoids, quercetin being the most damaging. This agreed well with the ability of the flavonoids to inhibit cell growth. None of the flavonoids induced DNA base oxidation above background levels. All of the flavonoids under investigation caused depletion of reduced glutathione, which, in the case of quercetin, occurred prior to cell death. Neither cytotoxicity nor genotoxicity was associated with the antioxidant enzyme capacity (glutathione, glutathione reductase, glutathione peroxidase and catalase) of the cells.

Single-cell gel electrophoresis (the comet assay) is a sensitive method for detecting strand breaks at the level of individual cells. Cells embedded in agarose are lysed, electrophoresed, and fluorescently stained. Breaks in the DNA release its supercoiling and allow DNA to extend toward the anode, resembling a comet. We have used the comet assay to investigate the influence of growth state, xenobiotic detoxifying enzymes, and DNA repair processes on the response of cultured human cells to oxidative damage. HepG2 and Caco-2 cells are differentiated liver and colon cell lines, respectively. HeLa and GM1899A cells are relatively unspecialized epithelial and lymphoblastoid cells. Substrate-dependent cells showed a cyclical fluctuation of glutathione (GSH) with respect to growth. Enzyme activities (glutathione reductase, glutathione peroxidase, and catalase) varied considerably between cell types and changed with cell growth state. Hydrogen peroxide induced more DNA damage in actively dividing cells than in confluent cultures. Sensitivity to oxidative injury did not correlate with detoxifying enzyme activity. Rather, differences in susceptibility between cells could be correlated with differences in DNA repair capacity. This study highlights the need to standardize experimental conditions if the comet assay is to be employed in the study of genotoxicity.

The association between high intake of fruit and vegetables and low incidence of certain cancers is well established. Dietary antioxidants present in these foods are thought to decrease free radical attack on DNA and hence to protect against mutations that cause cancer, but this causal mechanism remains conjectural. We have adopted a molecular epidemiological approach to this question, based on a modified alkaline single-cell gel electrophoresis assay ("comet assay") which specifically detects oxidation of pyrimidines in the DNA of human lymphocytes. In a survey of men 50-59 years of age living in the northeast of Scotland, smokers initially showed significantly more base damage than nonsmokers. Correlations between oxidative base damage and plasma concentrations of various antioxidants were generally negative but not statistically significant. Supplementation of the diet for 20 weeks with vitamin C (100 mg/day), vitamin E (280 mg/day), and beta-carotene (25 mg/day) resulted in a highly significant (P < 0.002) decrease in endogenous oxidative base damage in the lymphocyte DNA of both smokers and nonsmokers. In addition, lymphocytes of antioxidant-supplemented subjects showed an increased resistance to oxidative damage when challenged in vitro with H2O2. These findings strongly support the hypothesis that fruit and vegetables exert a cancer-protective effect via a decrease in oxidative damage to DNA.

The endogenous production of oxidative damage in DNA by free radicals released as a by-product of respiration is a likely cause of mutations which, if they occur in appropriate genes, may lead to cancer. Using an endonuclease specific for oxidized pyrimidines, in conjunction with the highly sensitive method of single cell gel electrophoresis, we have detected significant oxidative damage in untreated, freshly isolated lymphocytes from normal, healthy individuals.

We have previously reported that supplementation with folic acid (1.2 mg day(-1) for 12 week) elicited a significant improvement in the folate status of 61 healthy volunteers. We have examined effects of this supplement on markers of genomic stability. Little is known about the effect of folate supplementation on DNA stability in a cohort, which is not folate deficient. Preintervention, there was a significant inverse association between uracil misincorporation in lymphocyte DNA and red cell folate (P < 0.05). In contrast, there were no associations between folate status and DNA strand breakage, global DNA methylation or DNA base excision repair (measured as the capacity of the lymphocyte extract to repair 8-oxoGua ex vivo). Folate supplementation elicited a significant reduction in uracil misincorporation (P < 0.05), while DNA strand breakage and global DNA methylation remained unchanged. Increasing folate status significantly decreased the base excision repair capacity in those volunteers with the lowest preintervention folate status (P < 0.05). Uracil misincorporation was more sensitive to changes in folate status than other measures of DNA stability and therefore could be considered a specific and functional marker of folate status, which may also be relevant to cancer risk in healthy people. [ABSTRACT FROM AUTHOR]

DNA damage in lymphocytes, as measured by alkaline single cell gel electrophoresis (pH 12.7), is greatly increased by the concurrent lysis of whole blood in both freshly isolated samples and in PHA-stimulated cultures over a period of 7 days. Further, there is a marked progressive increase in DNA damage with time in PHA-stimulated lymphocytes cultured in whole blood even when the lymphocytes are separated before analysis; no such increase is seen in lymphocytes cultured alone. This indicates that there are components in whole blood that can cause DNA damage in lymphocytes, with granulocytes and lysis of red blood cells likely candidates. The DNA damage is greatly reduced in granulocyte-depleted whole blood cultures, but even in these significant increases are seen at later sampling times. Consequently, careful sample preparation is of paramount importance if the Comet assay is to be successfully used to assess DNA damage in human peripheral blood lymphocytes. Further, the progressive increase in DNA damage in whole blood cultures may influence other methods using lymphocytes for population biomonitoring and may be significant for in vitro genotoxicity testing. [ABSTRACT FROM PUBLISHER]