DNA stability and genomic methylation status in colonocyte isolated from methyl-donor-deficient rats

Summary Background: Epidemiological studies report an inverse relationship between intake of the B vitamine 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, methionne 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 colonocytes 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 afects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation. Received: 16 February 2000, Accepted: 25 April 2000     

Effect of complex polyphenols and tannins from red wine on DNA oxidative damage of rat colon mucosa in vivo

Summary Background: Dietary polyphenols have been reported to have a variety of biological actions, including anti-carcinogenic, antioxidant and anti-inflammatory activities. Aim of the study: In the present study we have evaluated the effect of an oral treatment with complex polyphenols and tannins from red wine and tea on DNA oxidative dammage in the rat colon mucosa. Methods: Isolated colonocytes were prepared from the colon mucosa of rats treated for ten days with either wine complex polyphenols (57.2 mg/kg/d) or thearubigin (40 mg/kg/d) by oral gavage. Colonocyte oxidative DNA damage was analysed at the single cell level using a modification of the comet assay technique. Results: These results show that wine complex polyphenols and tannins induce a significant decrease (−62% for pyrimidine and −57% for purine oxidation) in basal DNA oxidative damage in colon mucosal cells without affecting the basal level of single-strand breaks. On the other hand, tea polyphenols, namely a crude extract of thearubigin, did not affect either strand breaks or pyrimidine oxidation in colon mucosal cells. Conclusions Our experiments are the first demonstration that dietary polyphenols can modulate in vivo oxidative damage in the gastrointestinal tract of rodents. These data support the hypothesis that dietary polyphenols might have both a protective and a therapeutic potential in oxidative damage-related pathologies. Received: 17 April 2000, Accepted: 27 July 2000     

Absorption and DNA protective effects of flavonoid glycosides from an onion meal

Summary 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 food 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. Received: 1 April 2000, Accepted: 1 August 2000     

Lack of effect of the flavonoids, myricetin, quercetin, and rutin, on repair of H2O2-induced DNA single-strand breaks in Caco-2, Hep G2, and V79 cells

In the present study the effects of three flavonoids on the repair of H2O2-induced DNA strand breaks were investigated in Caco-2, Hep G2, and V79 cells. At the concentrations used, myricetin, quercetin, rutin, and H2O2 did not significantly affect cell viability in all the cell lines. Catalase activity was measured in V79 cells and was found to be considerably lower than activities previously measured in Caco-2 and Hep G2 cells. Cells were exposed to 50 microM H2O2 for 0.5 hour at 37 degrees C. After treatment, DNA strand break repair in H2O2-treated cells was monitored at various time points over a 48-hour period using the alkaline single-cell gel electrophoresis assay. Caco-2 cells repaired faster than Hep G2 cells, which repaired considerably faster than V79 cells. Preincubation with 50 microM quercetin for 24 hours significantly decreased the extent of H2O2-induced DNA single-strand breaks throughout repair time points in Caco-2 cells (p < 0.05), but not in Hep G2 cells. Myricetin (50 microM) and rutin (50 microM) had no effect on repair in Caco-2 and Hep G2 cells. Preincubation for 10 hours with quercetin and rutin, but not myricetin, significantly decreased the initial extent of DNA damage induced by H2O2 in V79 cells (p < 0.05). However, from the results of this study, none of the three flavonoids increased the rate of repair of strand breaks in any of the cell types.     

Folate deficiency in vitro induces uracil misincorporation and DNA hypomethylation and inhibits DNA excision repair in immortalized normal human colon epithelial cells

Epidemiological studies have indicated that folic acid protects against a variety of cancers, particularly cancer of the colorectum. Folate is essential for efficient DNA synthesis and repair. Moreover, folate can affect cellular S-adenosylmethionine levels, which regulate DNA methylation and control gene expression. We have investigated the mechanisms through which folate affects DNA stability in immortalized normal human colonocytes (HCEC). DNA strand breakage, uracil misincorporation, and DNA repair, in response to oxidative and alkylation damage, were determined in folate-sufficient and folate-deficient colonocytes by single cell gel electrophoresis. In addition, methyl incorporation into genomic DNA was measured using the bacterial enzyme Sss1 methylase. Cultured human colonocyte DNA contained endogenous strand breaks and uracil. Folate deficiency significantly increased strand breakage and uracil misincorporation in these cells. This negative effect on DNA stability was concentration dependent at levels usually found in human plasma (1-10 ng/ml). DNA methylation was decreased in HCEC grown in the absence of folate. Conversely, hypomethylation was not concentration dependent. Folate deficiency impaired the ability of HCEC to repair oxidative and alkylation damage. These results demonstrate that folic acid modulates DNA repair, DNA strand breakage, and uracil misincorporation in immortalized human colonocytes and that folate deficiency substantially decreases DNA stability in these cells.     

Protection by the flavonoids myricetin, quercetin, and rutin against hydrogen peroxide-induced DNA damage in Caco-2 and Hep G2 cells

Flavonoids are reported to exhibit a wide variety of biological effects, including antioxidant and free radical-scavenging activities. Reactive oxygen species have been implicated in a range of human pathological diseases such as atherosclerosis and certain cancers. The aims of this present study were 1) to investigate the effect of the flavonoids myricetin, quercetin, and rutin on cell viability, endogenous antioxidant enzyme activities, and DNA integrity in Caco-2 and Hep G2 cells and 2) to determine whether these flavonoids could protect against H2O2-induced DNA damage. Both cell lines were supplemented with various concentrations (0-200 microM) of myricetin, quercetin, and rutin for 24 hours or H2O2 (50 microM) for 30 minutes, and cell viability was assessed. Over the concentration range tested, neither the flavonoids nor H2O2 significantly affected cell viability. The effect of the flavonoids on the activities of the antioxidant enzymes catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) and on DNA integrity was assessed. The flavonoids did not significantly affect catalase or superoxide dismutase activity and did not induce DNA damage in either cell line. Exposure to 50 microM H2O2 for 30 minutes at 37 degrees C resulted in significant DNA damage, and preincubation with the flavonoids before H2O2 exposure significantly (p < 0.05) protected Caco-2 and Hep G2 cells against H2O2-induced DNA damage.     

DNA stability and lipid peroxidation in vitamin E–deficient rats in vivo and colon cells in vitro

Summary Background Fruit and vegetable consumption protects against cancer. This is attributed in part to antioxidants such as vitamin E combating oxidative DNA damage. Anthocyanins are found in significant concentrations in the human diet. However, it remains to be established whether they are bioactive in vivo. Aim To investigate the consequence both of vitamin E deficiency on oxidative damage to DNA and lipids and the cytoprotective effect of nutritionally relevant levels of cyanidin–3–glycoside both in vivo in rats and in vitro in human colonocytes. Methods Male Rowett Hooded Lister rats were fed a diet containing less than 0.5 mg/kg vitamin E or a vitamin E supplemented control diet containing 100 mg d–tocopherol acetate/kg. Half of the controls and vitamin E–deficient rats received cyanidin–3–glycoside (100 mg/kg). After 12 weeks endogenous DNA stability in rat lymphocytes (strand breaks and oxidised bases) and response to oxidative stress ex vivo (H₂O₂; 200 µM) was measured by single cell gel electrophoresis (SCGE). Tissue levels of 8–oxo–7,8–dihydro–2–deoxyguanosine (8–Oxo–dG) were measured by HPLC with EC detection. D–tocopherol and lipid peroxidation products (thiobarbituric acid reactive substances; TBARS) were measured by HPLC. Rat plasma pyruvate kinase and the production of reactive oxygen by phagocytes were detected spectrophotometrically and by flow cytometry respectively. Immortalised human colon epithelial cells (HCEC) were preincubated in vitro with the anthocyanins cyanidin and cyanidin–3–glycoside and the flavonol quercetin (all 50 µM) before exposure to H₂O₂ (200 µM). DNA damage was measured by SCGE as above. Results Plasma and liver d–tocopherol declined progressively over 12 weeks in rats made vitamin E deficient. Lipid peroxidation was increased significantly in plasma, liver and red cells. Reactive oxygen levels in phagocytes and plasma pyruvate kinase were increased. Vitamin E deficiency did not affect DNA stability in rat lymphocytes, liver or colon. Cyanidin–3–glycoside did not alter lipid peroxidation or DNA damage in rats. However, it was chemoprotective against DNA damage in human colonocytes.DNA strand breakage was decreased 38.8 ± 2.2 % after pretreatment with anthocyanin. Conclusion while it is accepted that vitamin E alters lipid oxidation in vivo, its role in maintaining DNA stability remains unclear. Moreover, whereas cyanidin–3–glycoside protects against oxidative DNA damage in vitro, at nutritionally relevant concentrations it is ineffective against oxidative stress in vivo.     

Recovery of human lymphocytes from oxidative DNA damage; the apparent enhancement of DNA repair by carotenoids is probably simply an antioxidant effect

Summary Background: Many epidemiological studies have identified a protection against cancer associated with consumption of fruit and vegetables. One factor is this protection may be the enhancement of cellular DNA repair activity by micronutrients, such as carotenoids, found in these foods. Aims of the study: To measure the capacity of lymphocytes isolated from volunteers supplemented with β-carotene, lutein or lycopene to recover from DNA damage induced in vivo by treatment with H₂O₂. Methods: Healthy volunteers were given supplements of lutein (15 mg/day), lycopene (15 mg/day) and βcarotene (15 mg/day), each for 1 week, the supplementation periods being separated by 3-week wash-out periods. Blood samples were taken at the beginning and end of each supplementation, and at 1 week and 3 weeks during the wash-out period. Carotenoid levels were measured in plasma. Lymphocytes were isolated and frozen. Subsequently, they were treated with 100 μM H₂O₂ and incubated for up to 24 h; DNA damage was measured with the comet assay (single cell gel electrophoresis) after 0, 2, 4, 8, and 24 h. Results: Increases of 2- to 3-fold in mean plasma lutein and β-carotene concentrations were seen at the end of the respective supplementation periods; they returned virtually to basal levels after wash-out. Lycopene concentrations were less affected by supplementation, and were more variable. H₂O₂-induced DNA strand breaks were apparently only slowly rejoined by the lymphocytes. The rejoining of breaks in the first few hours appeared substantially faster in lymphocytes following supplementation with β-carotene, but no such effect was seen with lutein. In those individuals who showed increases in lycopene concentrations, the recovery was significantly faster. Lymphocytes that were not treated with H₂O₂ showed a transient increase in DNA breakage to about double the background level in 2 h, presumably as a result of exposure to atmospheric oxygen; this effect, too, was relieved by supplementation with lycopene or β-carotene. Conclusions: While certain carotenoids appear to enhance recovery from oxidative damage, this is probably in fact an antioxidant protective effect against additional damage induced by atmospheric oxygen, rather than a stimulation of DNA repair. Received: 6 December 1999, Accepted: 22 March 2000     

Effects of a soy milk supplement on plasma cholesterol levels and oxidative DNA damage in men – a pilot study

Summary Background: Phytoestrogens are a major component of Asian diets and may be protective against certain hormone-dependent cancers (breast and prostate) and coronary heart disease. They may also have antioxidant function in scavenging potentially harmful free radicals and thus decreasing oxidative attack on DNA. Aims of the study: A pilot study to determine the effects of a phytoestrogen supplement, in the form of soy milk, on plasma LDL and HDL cholesterol levels and DNA damage in men. Methods: Ten healthy men participated in the study and were assigned to one of three groups consuming 1 litre of either soy milk, rice dream (vegetable protein control) or semi-skimmed cow's milk (animal protein control) each day for 4 weeks. Results: The soy supplement caused significant increases in plasma genistein and daidzein concentrations despite considerable interindividual variation (P<0.001). Supplementation with soy resulted in a decrease in oxidative damage to DNA bases detected using the comet assay compared with controls (P<0.05). However, there was no significant effect of the soy supplement on plasma cholesterol or triglyceride levels in comparison with control groups. Conclusions: A 4 week soy milk supplementation in healthy volunteers does not alter serum cholesterol levels but can have a protective effect against oxidative DNA damage in lymphocytes. Received: 29 December 1998, Accepted: 24 March 1999     

Dietary polyphenols protect against<Emphasis Type="Italic"> N</Emphasis>-nitrosamines and benzo(a)pyrene-induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 human hepatoma cells

Background  Dietary polyphenols have been reported to have a variety of biological actions, including anticarcinogenic and antioxidant activities. Aim of the study  In the present study we investigated the protective effect of dietary polyphenols against N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR) and benzo(a)pyrene (BaP)-induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 cells. Methods  Human hepatocellular carcinoma (HepG2) cells, which retain many specialized liver functions and drug metabolizing enzyme activities, were used as in vitro model for human hepatocytes. NDMA, NPYR and BaP were employed to induce DNA damage. DNA damage (strand breaks, oxidized pyrimidines and oxidized purines) was evaluated by the alkaline single cell gel electrophoresis or comet assay. Results  None of the polyphenols concentrations tested in presence or absence of Fpg (formamidopyrimidine-DNA glycosylase), or Endo III (Endonuclease III) caused DNA damage per se. Increasing concentrations of BaP (25–100 μM) induced a significant increase of DNA strand breaks, Fpg and Endo III sensitive sites in a dose dependent manner. Myricetin and quercetin decreased DNA strand breaks and oxidized pyrimidines induced by NDMA, but not oxidized purines. However, both flavonoids reduced oxidized pyrimidines and purines induced by NPYR. DNA strand breaks induced by NPYR were prevented by quercetin, but not by myricetin. BaP-induced DNA strand breaks and oxidized pyrimidines were strongly reduced by myricetin and quercetin, respectively. While oxidized purines induced by BaP were reduced by quercetin, myricetin had no protective effect. (+)-Catechin and (−)-epicatechin reduced DNA strand breaks, oxidized pyrimidines and oxidized purines induced by NDMA. DNA strand breaks, and oxidized purines induced by NPYR were also prevented by (+)-catechin and (−)-epicatechin, while the maximum reduction of oxidized pyrimidines was found by (+)-catechin and (−)-epicatechin at 10 μM. (+)-Catechin and (−)-epicatechin decreased also DNA strand breaks and oxidized pyrimidines but not oxidized purines induced by BaP. Conclusions  Our results clearly indicate that polyphenols protect human derived cells against DNA strand breaks and oxidative DNA damage effects of NDMA, NPYR or BaP, three carcinogenic compounds which occur in the environment.     

Effect of complex polyphenols and tannins from red wine (WCPT) on chemically induced oxidative DNA damage in the rat

Summary Background: Flavonoids are polyphenolic antioxidants occuring in vegetables and fruits as well as beverages such as tea and wine which have been thought to influence oxidative damage. Aim of the study: We wanted to verify whether a complex mixture of wine tannins (wine complex polyphenols and tannins, WCPT) prevent chemically-induced oxidative DNA damage in vivo. Methods: Oxidative DNA damage was evaluated by measuring the ratio of 8-hydroxy-2'-deoxyguanosine (80HdG)/ 2-deoxyguanosine (2dG) × 10⁻⁶ in hydrolyzed DNA using HPLC coupled with electrochemical and UV detectors. Results: We treated rats with WCPT (57 mg/kg p.o.) for 14 d, a dose 10-fold higher than what a moderate wine drinker would be exposed to. WCPT administration significantly reduced the ratio of 80HdG/2dG × 10⁻⁶ in liver DNA obtained from rats treated with 2-nitropropane (2NP) relative to controls administered 2NP only (33.3 ± 2.5 vs. 44.9 ± 3.2 × 10⁻⁶ 2dG; μ± SE; p<0.05). On the contrary, pretreatment with WCPT for 10 d did not protect the colon mucosa from oxidative DNA damage induced by 1,2-dimethylhydrazine (DMH). 2NP and DMH are hepatic and colon carcinogens, respectively, capable of inducing oxidative DNA damage. Conclusions: WCPT have protective action against some types of chemically-induced oxidative DNA damage in vivo. Received: 25 January 1999, Accepted: 27 May 1999     

Antioxidant and radical scavenging properties in vitro of polyphenolic extracts from red wine

Summary Background & Aims Red wine polyphenols inhibit chemically-induced oxidative DNA damage in vivo in experimental animals through a mechanism which is still unclear. On this basis, we tried to clarify the mechanisms of inhibition of DNA oxidation in vitro by wine extracts containing monomeric and polymeric phenols (WE) and monomer-free complex polyphenols and tannins (WCPT) from red wine. Methods Oxidative DNA damage was induced by incubating DNA with GSH/Fe³⁺ or cumene hydroperoxide (CumOOH) in vitro and using 8-OH–2-deoxyguanosine (8-OHdG) levels as a measure of DNA oxidation. Levels of 8-OHdG were determined by HPLC coupled with electrochemical detector (ESA). Results and conclusions WCPT and WE, at μM concentrations, reduced concentration-dependently oxidative DNA damage induced by GSH/Fe³⁺. WCPT and WE also reduced DNA oxidation by CumOOH. In conclusion, complex polyphenols and tannin extracts from red wine, with or without small molecular phenols, prevent oxidative DNA damage through a dual mechanism, iron binding and direct free radical scavenging. Received: 27 November 2000 / Accepted: 11 April 2001     

Spinach and tomato consumption increases lymphocyte DNA resistance to oxidative stress but this is not related to cell carotenoid concentrations

Summary Background The increased consumption of fruit and vegetables has been linked to protection against different chronic diseases, but the dietary constituents responsible for this association have not been clearly identified. Aim of the study We evaluated the effect of spinach and spinach+tomato puree consumption on cell DNA resistance to an oxidative stress. Methods To this aim, in a dietary controlled intervention study, 9 healthy female volunteers consumed a basal diet low in carotenoids (< 600 μg/day) enriched with daily portions (150 g) of spinach (providing about 9 mg lutein, 0.6 mg zeaxanthin, 4 mg β-carotene) for 3 weeks (from day 0 to day 21) followed by a 2 week wash-out period (basal diet) and finally another 3 weeks (from day 35 to day 56) of diet enriched with daily portions of spinach (150 g) + tomato puree (25 g, providing about 7 mg lycopene, 0.3 mg β-carotene). At the beginning and the end of each period of vegetable intake, blood samples were collected for lymphocyte separation. Carotenoid concentrations of lymphocytes were determined by HPLC and DNA damage was evaluated by the comet assay following an ex vivo treatment with H₂O₂. Results During the first period of spinach consumption, lymphocyte lutein concentration did not increase significantly (from 1.6 to 2.2 μmol/10¹² cells) while lycopene and β-carotene concentrations decreased significantly (from 1.0 to 0.1 μmol/10¹² cells, P < 0.001, and from 2.2 to 1.2 μmol/10¹² cells, P < 0.05, respectively). Lutein and lycopene concentrations increased after spinach+tomato puree consumption (from 1.2 to 3.5 μmol/10¹² cells, P < 0.01, and from 0.1 to 0.7 μmol/10¹² cells, P < 0.05, respectively). The increase may be attributed to the addition of tomato puree to spinach; however, the different concentrations of carotenoids in lymphocytes registered at the beginning of the two intervention periods may have affected the results. DNA resistance to H₂O₂ insult increased significantly after both the enriched diets (P < 0.01); however, no “additive effect” was seen after spinach + tomato puree consumption. In the spinach + tomato intervention period an inverse correlation was observed between lymphocyte lycopene concentration and DNA damage, but this seems not able to explain the protection observed. Conclusions The consumption of carotenoid-rich foods even for a short period of time gives protection against oxidative stress. The results obtained seem to suggest that this protective role is not specifically related to carotenoids. However they may contribute together with other substances present in vegetables to lymphocyte resistance to oxidative damage. Received: 20 September 2001, Accepted: 18 February 2002     

In Vitro Studies on the Inhibition of Colon Cancer by Butyrate and Polyphenolic Compounds

Our aim was to investigate the effect of several dietary polyphenols on uptake of ¹⁴C-butyrate (¹⁴C-BT) by Caco-2 cells and try to correlate this effect with the modulation of the anticarcinogenic effect of BT in these cells. Acutely, uptake of ¹⁴C-BT (10 μM) was decreased by resveratrol, quercetin, myricetin, and chrysin, and increased by xanthohumol, catechin, and epicatechin; and uptake of ¹⁴C-BT (20 mM) was reduced by resveratrol, quercetin, myricetin, chrysin, EGCG, and epicatechin. Resveratrol acts as a competitive inhibitor of ¹⁴C-BT uptake. Chronically, quercetin and EGCG increased uptake of ¹⁴C-BT (10 μM), whereas myricetin, rutin, chrysin, and xanthohumol decreased it. Moreover, catechin (1 μM), quercetin, myricetin, rutin, EGCG, and chrysin increased uptake of ¹⁴C-BT (20 mM), whereas catechin (0.1 μM) decreased it. EGCG, myricetin, and catechin decreased MCT1 mRNA expression, while chrysin increased it; quercetin, rutin, and xanthohumol had no effect. BT (5 mM; 48 h) markedly decreased cellular viability and proliferation and increased cell differentiation and apoptosis. In general, combination of polyphenolic compounds with BT did not significantly modify these changes. In conclusion, changes in uptake of BT induced by polyphenols do not correlate with changes on the effect of BT upon cell viability, cell proliferation, differentiation, and apoptosis.     

Researching on new species of “Mate”: <Emphasis Type="Italic">Ilex brevicuspis</Emphasis>

Summary. Background: Ilex paraguariensis St. Hilaire (Aquifoliaceae) (“Mate” or “Yerba mate”) is one of the most commercialized plants of South America which grows naturally in NE Argentina, Uruguay, SE Brazil and E Paraguay, where it is also cultivated. It is used to prepare a tea-like beverage (infusions or decoctions) appreciated for its peculiar flavor, stimulation and nutritional properties. Ilex brevicuspis Reisseck grows in the same habitat and is widely used as a substitute or adulterant of Ilex paraguariensis. In a previous work, methylxanthines (caffeine, theobromine and theophylline) were not detected in it by HPLC. Aim of the study: This study was undertaken in order to isolate, identify and quantify the polyphenolic compounds (caffeoyl derivatives and flavonoids) and to investigate some of the pharmacological activities of I. brevicuspis, related with the traditional use of the “Mate” (choleretic, intestinal propulsion and antioxidant activities). Acute toxicity was also investigated. Methods: Decoctions, like extracts, were prepared in order to compare the results with preparations commonly used by the local people. For the phytochemical analysis, the extracts were analyzed by HPLC with a diode array detector. Choleretic and intestinal propulsion activities were assayed in rats. Sodium dehydrocholate (DHC) was used as a choleretic reference standard. Antioxidant activity was tested in liposomes that were oxidized by the free radical generator 2,2'-azobis [amidinopropane] chloride (AAPH). Results: For the first time in I. brevicuspis the following compounds were isolated and quantified: A) caffeoyl derivative compounds (chlorogenic acid; caffeic acid; 3,4-dicaffeoylquinic acid; 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid. B) flavonoids (rutin, quercetin and kaempferol). Biological activity assays demonstrated that I. brevicuspis extracts produced a significant increase of bile flow (BF) in rats in the first 30 min period and in the percentage of BF increase accumulated at 120 min. It also produced an increase in the intestinal propulsion activity. Moreover, this species showed a high antioxidant activity. The acute toxicity test showed that Ilex brevicuspis did not produce any sign of toxicity at the analyzed doses. Conclusions: An Argentinean Ilex specie (I. brevicuspis) has choleretic, intestinal propulsion, antioxidant activities and these results may lead to the potential development of a new “Yerba Mate” and/or phytopharmaceutical products, without central nervous system (CNS) stimulant activity. Received: 10 July 2002, Accepted: 5 November 2002 Correspondence to: R. Filip     

Does a vegetarian diet influence genomic stability?

Summary.Background: The vegetarian lifestyle is supposedly healthy, and differences between vegetarians and non-vegetarians in biomarkers related to diseases such as cancer might be expected.Aim of the study: To investigate the possible role of different diets in maintaining genomic stability.Methods: The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit.Among vegetarians there were 13 lacto-ovo-vegetarians (8 women, 5 men) with average length of vegetarian diet 10.8 years (ranging from 5 to 26) and 11 lacto-vegetarians (5 women, 6 men) with average length of vegetarian diet 8.2 years (ranging from 3 to15). All volunteers were nonsmokers, non-consumers of alcohol and had similar education and patterns of physical activity. Chromosome aberrations, micronuclei and DNA damage (strand breaks, oxidised bases and H₂O₂-sensitivity) were examined in peripheral blood lymphocytes of vegetarians and non-vegetarians. Plasma antioxidant status was assessed with the FRAP assay.Results: We did not find any differences in percentage of cells with chromosome aberrations or in the frequency of micronuclei between vegetarians and non-vegetarians or between lacto-ovo and lacto-vegetarians. There was no statistically significant difference in total antioxidant capacity between the groups. The group with traditional dietary habits had significantly higher levels of oxidative DNA damage (strand breaks and oxidised purines, P = 0.005) compared with vegetarians. A significant positive correlation between age and oxidative DNA damage (net FPG-sensitive sites) was found in non-vegetarians, while there was an opposite trend towards a negative association in vegetarians. On the other hand chromosome aberrations correlated with age in vegetarians (r = 0.48, P = 0.017) but not in non-vegetarians.Conclusions: Our results indicate that a vegetarian diet can lead to a slight decrease in oxidative DNA damage in lymphocytes, but other markers of genetic stability are not affected. The lowest level of DNA damage was found in lymphocytes of lactovegetarians, (especially oxidised pyrimidines, P = 0.0017), suggesting that this diet provides some protection against oxidative stress.     

Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines

Summary Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin. Their proposed protective role in tumor development may prevail especially in the intestinal tract due to direct exposure of intestinal epithelia to these dietary ingredients. We have screened more than 30 flavonoids for their effects on cell proliferation and potential cytotoxicity in the human colon cancer cell lines Caco-2, displaying features of small intestinal epithelial cells, and HT-29, resembling colonic crypt cells. In addition, for selected compounds we assessed whether they induce apoptosis by determining caspase-3 activation. Studies on the dose dependent effects of the flavonoids showed antiproliferative activity of all compounds with EC₅₀ values ranging between 39.7 ± 2.3 μM (baicalein) and 203.6 ± 15.5 μM (diosmin). In almost all cases, growth inhibition by the flavonoids occured in the absence of cytotoxicity. There was no obvious structure-activity relationship in the antiproliferative effects either on basis of the subclasses (i.e., isoflavones, flavones, flavonols, flavanones) or with respect to kind or position of substituents within a class. In a subset of experiments we examined the antiproliferative activities of the most potent compound of each flavonoid subgroup in addition in LLC-PK₁, a renal tubular cell line, and the human breast cancer cell line MCF-7. Out of four flavonols tested, three displayed almost equal antiproliferative activities in all cell lines but fisetin was less potent in MCF-7 cells. The flavanones bavachinin and flavanone inhibited growth of Caco-2 and HT-29 cells with lower EC₅₀ values than that obtained in LLC-PK₁ and MCF-7 cells. The lower susceptibility of LLC-PK₁ and MCF-7 cells towards growth arrest was even more pronounced in the case of the flavone baicalein. Half maximal growth-inhibition in LLC-PK₁ and MCF-7 required 2.5 and 6.6 fold higher concentrations than that needed in the intestinal cell lines. The flavonoids failed to affect apoptosis in LLC-PK₁ and MCF-7, whereas baicalein and myricetin were able to induce apoptosis in HT-29 and Caco-2 cells. In conclusion, flavonoids of the flavone, flavonol, flavanone, and isoflavone classes possess antiproliferative effects in different cancer cell lines. The capability of flavonoids for growth inhibition and induction of apoptosis can not be predicted on the basis of their chemical composition and structure. Received: 28 December 1998, Accepted: 18 March 1999     

Flavonoids suppress androgen-independent human prostate tumor proliferation

The present studies compared the effects of selected bioflavonoids on the proliferation of androgen-independent human prostatic tumor cells (PC-3). Complete growth retardation was observed in PC-3 cells treated with 100 microM quercetin, kaempferol, and luteolin, while isomolar genistein, apigenin, and myricetin suppressed PC-3 proliferation by 73%, 70%, and 59%, respectively (p < 0.05). Naringenin and rutin were not as effective and inhibited growth by < 25%. Exposure to increasing concentrations of quercetin and kaempferol led to a dose-dependent decrease in proliferation. Refeeding kaempferol-treated cells (50 microM) complete medium without the flavonoid resulted in a return toward control growth rates. Similar growth recovery was not observed in quercetin-treated cells. The antiproliferative response of PC-3 cells to quercetin and kaempferol was additive when supplemented to the medium at 25 microM. A block in G2-to-M phase progression was observed after the addition of 25 microM kaempferol. When quercetin reached 100 microM, an increase in the proportion of cells in the S phase became apparent within 24 hours. Apoptosis was not evident, even when concentrations of quercetin or kaempferol were raised to 100 microM. The present studies suggest that alterations in cell cycle progression contribute significantly to the antiproliferative effects of quercetin and kaempferol in PC-3 cells.     

Fasting plasma concentrations of selected flavonoids as markers of their ordinary dietary intake

Summary. Background: Dietary flavonoids, especially flavonols, are discussed as potentially preventive agents in the etiology of diseases such as coronary heart disease, stroke, and cancer. However, their consideration in epidemiologic studies is hampered by difficulties in exposure assessment. Aim of the study: By comparison with dietary intake estimates, fasting plasma flavonoid concentrations should be evaluated as possible biomarkers of the ordinary dietary intake. Methods>: 7-d dietary records were completed by 48 healthy female students. Flavonoid intake was estimated by means of available literature data on the flavonoid content of foods. Fasting plasma samples were taken at the end of the record period for flavonoid determination (HPLC). Results: The mean intake estimates (7-d period) of quercetin, kaempferol, naringenin, and hesperetin amounted to 17.9, 4.7, 12.1, and 17.4 mg/d, respectively; the corresponding mean plasma concentrations were 22.9, 10.7, 8.2, and 22.2 nmol/l. For all four flavonoids significant correlations between 7d-intake results and fasting plasma concentrations (r = 0.30–0.46, p < 0.05) existed. As expected from the known short elimination half-life of some plasma flavonoids, distinctly higher correlation coefficients were found for the relationship between intake estimates for the last day before blood sampling and the fasting plasma concentrations (r = 0.42–0.64; p < 0.01). The intraindividual variation in fasting plasma flavonoid concentrations during ad libitum intake was found to be rather high (mean coefficient of variation between 82 and 91 %; n = 4). Conclusions: The flavonoid content in fasting plasma samples seems to be a suitable biomarker of short-term intake and a possible biomarker of the medium-term intake. Due to the high intraindividual variation the combined use of plasma flavonoid concentrations and dietary intake estimates may be the best choice in epidemiologic studies. Received: 16 February 2002, Accepted: 11 July 2002 Correspondence to: J. Radtke     

Role of glutamine on the de novo purine nucleotide synthesis in Caco-2 cells

Summary Background: The body's nucleotide pool derives from three potential sources: de novo synthesis, salvage of preformednucleosides/bases or the diet. The relative contributions of these pathways of assimilation are poorly understood in vivo. Dietary nucleotides have been suggested to have beneficial effects an the development and repair of the gastrointestinal tract. Tissues with a rapid turnover, such as the gut and the immune system cells, may utilise preformed nucleotides (coming from the diet), in situation in which there is a high demand of nucleotides for nucleic acid synthesis. Therefore, nucleotides could be onsidered as conditionally essential nutrients. Aim of the work and methods: -Development of a method to measure synthesis de novo of RNA-purine nucleotides in Caco-2 cells, relying an the incorporation of ¹⁴C-glycine into the purine ring of the nucleotide. To establish the fractional synthesis rate of RNA purine nucleotides in Caco-2 cells, grown in culture medium containing different concentrations of glutamine, in the presence or abscence of added nucleotides. To investigate the degree to which tissue ribonucleosides are derived from the culture medium or from de novo synthesis in the presence of different concentrations of glutamine, using undifferentiated Caco-2 cells, stressed or not by the addition of IL-1β to the medium. Results and conclusions:The presence of high levels of glutamine in the culture medium is essential for cell proliferation (estimated by measurement of the fractional synthesis rate of purine nucleotides) and the presence of nucleotides cannot replace the glutaminedependence of Caco-2 cell proliferation. The incorporation or exogenous purine nucleotides into RNA of Caco-2 cells is rather limited, and it becomes important when cells are stressed by glutamine deprivation. Stress by addition of interleukin-1β resulted in the maintenance or the increase in de novo synthesised RNA-purine nucleotides, even in the presence of exogenous nucleotides. However, the addition of interleukin-1β to the culture medium led to an enhanced salvage of preformed pyrimidine nucleotides for nucleic acid synthesis when glutamine was present in the medium at a concentration of 0.5 mmol/L. Received: 10 December 1998, Accepted: 8 March 2000