Antigenotoxic effect of kefir and ayran supernatants on fecal water-induced DNA damage in human colon cells

Fermented dairy products and their component bacteria have been shown to possess health-promoting functions in consumers and recently have been suggested to reduce the risk of colorectal cancer. Kefir and ayran are two popular fermented milk drinks that have their origins in the Caucasus region of Russia. The present study aimed to evaluate their potential anticancer properties in colon cells in vitro. The comet assay and transepithelial resistance assay were used to assess the effect of kefir and ayran supernatants on genotoxicity of fecal water samples and on intestinal tight junction integrity. Their antioxidant capacity was measured by trolox equivalent antioxidant capacity assay and compared with that of unfermented milk. The results showed that DNA damage induced by 2 of 4 fecal water samples was significantly decreased by kefir and ayran supernatants and with ayran the effect was dose-dependent. However no effect on intestinal tight junctions was observed. The supernatants of kefir and ayran contained high amounts of acetic and lactic acid but only a very small quantity of caproic and butyric acid, and they showed significantly greater antioxidant capacity than milk. These findings suggest kefir and ayran can reduce DNA damage, which might be due to their antioxidant capacities.

Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress

Prostate cancer is one of the most frequent cancer types in Western societies and predominately occurs in the elderly male. The strong age-related increase of prostate cancer is associated with a progressive accumulation of oxidative DNA damage which is presumably supported by a decline of the cellular antioxidative defence during ageing. Risk of developing prostate cancer is much lower in many Asian countries where soy food is an integral part of diet. Therefore, isoflavones from soy were suggested to have chemopreventive activities in prostate cells. Here, we have investigated the hypothesis that the soy-isoflavone genistein could protect DNA of LAPC-4 prostate cells from oxidative stress-related damage by enhancing the expression of antioxidative genes and proteins. A 24 h preincubation with genistein (1-30 microM) protected cells from hydrogen peroxide-induced DNA damage, as determined by the comet assay. Analysis of two cDNA macroarrays, each containing 96 genes of biotransformation and stress response, revealed a modulated expression of 3 genes at 1 microM and of 19 genes at 10 microM genistein. Real-time PCR confirmed the induction of three genes encoding products with antioxidant activities, namely glutathione reductase (2.7-fold), microsomal glutathione S-transferase 1 (1.9-fold) and metallothionein 1X (6.3-fold), at 1-30 microM genistein. 17Beta-estradiol, in contrast, decreased the expression of metallothionein 1X at 0.3 microM (2.0-fold), possibly pointing to an estrogen receptor-mediated regulation of this gene. Immunocytochemical staining revealed an induction of metallothionein proteins at 30 microM genistein, while their intracellular localization was unaffected. Metallothioneins were previously found to protect cells from hydrogen peroxide-induced DNA damage. Hence, our findings indicate that genistein protects prostate cells from oxidative stress-related DNA damage presumably by inducing the expression of antioxidative products, such as metallothioneins. Genistein, therefore, might counteract the age-related decline of important antioxidative defence systems which in turn maintain DNA integrity.

Antigenotoxicity of probiotics and prebiotics on faecal water-induced DNA damage in human colon adenocarcinoma cells

Six strains of lactic acid producing bacteria (LAB) were incubated (1 x 10(8)cfu/ml) with genotoxic faecal water from a human subject. HT29 human adenocarcinoma cells were then challenged with the resultant samples and DNA damage measured using the single cell gel electrophoresis (comet) assay. The LAB strains investigated were Bifidobacterium sp. 420, Bifidobacterium Bb12, Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus bulgaricus and Enterococcus faecium. DNA damage was significantly decreased by all bacteria used with the exception of Strep. thermophilus. Bif. Bb12 and Lact. plantarum showed the greatest protective effect against DNA damage. Incubation of faecal water with different concentrations of Bif. Bb12 and Lact. plantarum revealed that the decrease in genotoxicity was related to cell density. Non-viable (heat treated) probiotic cells had no effect on faecal water genotoxicity. In a second study, HT29 cells were cultured in the presence of supernatants of incubations of probiotics with various carbohydrates including known prebiotics; the HT29 cells were then exposed to faecal water. Overall, incubations involving Lact. plantarum with the fructooligosaccharide (FOS)-based prebiotics Inulin, Raftiline, Raftilose and Actilight were the most effective in increasing the cellular resistance to faecal water genotoxicity, whereas fermentations with Elixor (a galactooligosaccharide) and Fibersol (a maltodextrin) were less effective. Substantial reductions in faecal water-induced DNA damage were also seen with supernatants from incubation of prebiotics with Bif. Bb12. The supernatant of fermentations involving Ent. faecium and Bif. sp. 420 generally had less potent effects on genotoxicity although some reductions with Raftiline and Elixor fermentations were apparent.

Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser(428). This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser(15)) and Chk1 (Ser(296)) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo. (c) 2007 Elsevier Inc. All rights reserved.

Role of oxygen radicals in DNA damage and cancer incidence

The development of cancer in humans and animals is a multistep process. The complex series of cellular and molecular changes participating in cancer development are mediated by a diversity of endogenous and exogenous stimuli. One type of endogenous damage is that arising from intermediates of oxygen (dioxygen) reduction - oxygen-free radicals (OFR), which attacks not only the bases but also the deoxyribosyl backbone of DNA. Thanks to improvements in analytical techniques, a major achievement in the understanding of carcinogenesis in the past two decades has been the identification and quantification of various adducts of OFR with DNA. OFR are also known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations. The most extensively studied lesion is the formation of 8-OH-dG. This lesion is important because it is relatively easily formed and is mutagenic and therefore is a potential biomarker of carcinogenesis. Mutations that may arise from formation of 8-OH-dG involve GC. TA transversions. In view of these findings, OFR are considered as an important class of carcinogens. The effect of OFR is balanced by the antioxidant action of non-enzymatic antioxidants as well as antioxidant enzymes. Non-enzymatic antioxidants involve vitamin C, vitamin E, carotenoids (CAR), selenium and others. However, under certain conditions, some antioxidants can also exhibit a pro-oxidant mechanism of action. For example, beta-carotene at high concentration and with increased partial pressure of dioxygen is known to behave as a pro-oxidant. Some concerns have also been raised over the potentially deleterious transition metal ion-mediated (iron, copper) pro-oxidant effect of vitamin C. Clinical studies mapping the effect of preventive antioxidants have shown surprisingly little or no effect on cancer incidence. The epidemiological trials together with in vitro experiments suggest that the optimal approach is to reduce endogenous and exogenous sources of oxidative stress, rather than increase intake of anti-oxidants. In this review, we highlight some major achievements in the study of DNA damage caused by OFR and the role in carcinogenesis played by oxidatively damaged DNA. The protective effect of antioxidants against free radicals is also discussed.

Effect of processed and red meat on endogenous nitrosation and DNA damage

Haem in red meat (RM) stimulates the endogenous production of mutagenic nitroso compounds (NOC). Processed (nitrite-preserved red) meat additionally contains high concentrations of preformed NOC. In two studies, of a fresh RM versus a vegetarian (VEG) diet (six males and six females) and of a nitrite-preserved red meat (PM) versus a VEG diet (5 males and 11 females), we investigated whether processing of meat might increase colorectal cancer risk by stimulating nitrosation and DNA damage. Meat diets contained 420 g (males) or 366 g (females) meat/per day. Faecal homogenates from day 10 onwards were analysed for haem and NOC and asso- ciated supernatants for genotoxicity. Means are adjusted for differ- ences in male to female ratios between studies. Faecal NOC concentrations on VEG diets were low (2.6 and 3.5 mmol/g) but significantly higher on meat diets (PM 175 ± 19 nmol/g versus RM 185 ± 22 nmol/g; P 5 0.75). The RM diet resulted in a larger pro- portion of nitrosyl iron (RM 78% versus PM 54%; P < 0.0001) and less nitrosothiols (RM 12% versus PM 19%; P < 0.01) and other NOC (RM 10% versus PM 27%; P < 0.0001). There was no statis- tically significant difference in DNA breaks induced by faecal water (FW) following PM and RM diets (P 5 0.80). However, PM re- sulted in higher levels of oxidized pyrimidines (P < 0.05). Surpris- ingly, VEG diets resulted in significantly more FW-induced DNA strand breaks than the meat diets (P < 0.05), which needs to be clarified in further studies. Meats cured with nitrite have the same effect as fresh RM on endogenous nitrosation but show increased FW-induced oxidative DNA damage.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Watercress supplementation in diet reduces lymphocyte DNA damage and alters blood antioxidant status in healthy adults

Background: Cruciferous vegetable (CV) consumption is associated with a reduced risk of several cancers in epidemiologic studies. Objective: The aim of this study was to determine the effects of watercress (a CV) supplementation on biomarkers related to cancer risk in healthy adults. Design: A single-blind, randomized, crossover study was conducted in 30 men and 30 women (30 smokers and 30 nonsmokers) with a mean age of 33 y (range: 19-55 y). The subjects were fed 85 g raw watercress daily for 8 wk in addition to their habitual diet. The effect of supplementation was measured on a range of endpoints, including DNA damage in lymphocytes (with the comet assay), activity of detoxifying enzymes (glutathione peroxidase and superoxide dismutase) in erythrocytes, plasma antioxidants (retinol, ascorbic acid, a-tocopherol, lutein, and beta-carotene), plasma total antioxidant status with the use of the ferric reducing ability of plasma assay, and plasma lipid profile. Results: Watercress supplementation (active compared with control phase) was associated with reductions in basal DNA damage (by 17%; P = 0.03), in basal plus oxidative purine DNA damage (by 23.9%; P = 0.002), and in basal DNA damage in response to ex vivo hydrogen peroxide challenge (by 9.4%; P = 0.07). Beneficial changes seen after watercress intervention were greater and more significant in smokers than in nonsmokers. Plasma lutein and P-carotene increased significantly by 100% and 33% (P < 0.001), respectively, after watercress supplementation. Conclusion: The results support the theory that consumption of watercress can be linked to a reduced risk of cancer via decreased damage to DNA and possible modulation of antioxidant status by increasing carotenoid concentrations.

Epicatechin and its methylated metabolite attenuate UVA-induced oxidative damage to human skin fibroblasts

The ultraviolet A component of sunlight causes both acute and chronic damage to human skin. In this study the potential of epicatechin, an abundant dietary flavanol, and 3'-O-methyl epicatechin, one of its major in vivo metabolites, to protect against UVA-induced damage was examined using cultured human skin fibroblasts as an in vitro model. The results obtained clearly show that both epicatechin and its metabolite protect these fibroblasts against UVA damage and cell death. The hydrogen-donating antioxidant properties of these compounds are probably not the mediators of this protective response. The protection is a consequence of induction of resistance to UVA mediated by the compounds and involves newly synthesized proteins. The study provides clear evidence that this dietary flavanol has the potential to protect human skin against the deleterious effects of sunlight.

Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-29-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5- HmdC, 5-formyl-29-deoxycytidine (5-FodC), 5-carboxyl-29-deoxycytidine (5-CadC), 5-hydroxymethyl-29-deoxyuridine (5- HmdU), and the (59S) diastereomer of 8,59-cyclo-29-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 106 nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tetfamily dioxygenase enzymes in Arabidopsis.

Anti-proliferative effect of rhein, an anthraquinone isolated from Cassia species, on Caco-2 human adenocarcinoma cells

Objective: In recent years the use of anthraquinone laxatives, in particular senna, has been associated with damage to the intestinal epithelial layer and an increased risk of developing colorectal cancer. In the present study we evaluated the cytotoxicity of rhein, the active metabolite of senna, on human colon adenocarcinoma cells (Caco-2) and its effect on cell proliferation. Methods: Cytotoxicity studies were performed using MTT, NR and TEER assays whereas 3H-thymidine incorporation and western blot analysis were used to evaluate the effect of rhein on cell proliferation. Moreover, for genoprotection studies Comet assay and oxidative biomarkers measurement (malondialdehyde and reactive oxygen species) were used. Results: Rhein (0.1-10μg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco-2 cells. Rhein (0.1 and 1 μg/ml) significantly reduced cell proliferation as well as MAP kinase activation; by contrast, at the high concentration (10μg/ml) rhein significantly increased cell proliferation and ERK phosphorylation. Moreover, rhein (0.1-10μg/ml) (i) did not adversely affect the integrity of tight junctions and hence epithelial barrier function, (ii) did not induce DNA damage rather it was able to reduce H2O2-induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and ROS levels induced by H2O2/Fe2+. Conclusions: Rhein, was devoid of cytotoxic and genotoxic effects in colon adenocarcinoma cells. Moreover, at concentrations present in the colon after a human therapeutic dosage of senna, rhein inhibited cell proliferation via a mechanism which seems to involve directly the MAP kinase pathway. Finally, rhein prevents the DNA damage probably via an anti-oxidant mechanism.

Rifaximin modulates the colonic microbiota of patients with Crohn's disease: an in vitro approach using a continuous culture colonic model system.

Rifaximin, a rifamycin derivative, has been reported to induce clinical remission of active Crohn's disease (CD), a chronic inflammatory bowel disorder. In order to understand how rifaximin affects the colonic microbiota and its metabolism, an in vitro human colonic model system was used in this study. We investigated the impact of the administration of 1800 mg/day of rifaximin on the faecal microbiota of four patients affected by colonic active CD [Crohn's disease activity index (CDAI > 200)] using a continuous culture colonic model system. We studied the effect of rifaximin on the human gut microbiota using fluorescence in situ hybridization, quantitative PCR and PCR–denaturing gradient gel electrophoresis. Furthermore, we investigated the effect of the antibiotic on microbial metabolic profiles, using 1H-NMR and solid phase microextraction coupled with gas chromatography/mass spectrometry, and its potential genotoxicity and cytotoxicity, using Comet and growth curve assays. Rifaximin did not affect the overall composition of the gut microbiota, whereas it caused an increase in concentration of Bifidobacterium, Atopobium and Faecalibacterium prausnitzii. A shift in microbial metabolism was observed, as shown by increases in short-chain fatty acids, propanol, decanol, nonanone and aromatic organic compounds, and decreases in ethanol, methanol and glutamate. No genotoxicity or cytotoxicity was attributed to rifaximin, and conversely rifaximin was shown to have a chemopreventive role by protecting against hydrogen peroxide-induced DNA damage. We demonstrated that rifaximin, while not altering the overall structure of the human colonic microbiota, increased bifidobacteria and led to variation of metabolic profiles associated with potential beneficial effects on the host.

The effect of cruciferous and leguminous sprouts on genotoxicity, in vitro and in vivo

Vegetable consumption is associated with a reduced risk of colorectal cancer, which is the second most common cancer after lung/breast cancer within Europe. Some putative protective phytochemicals are found in higher amounts in young sprouts than in mature plants. The effect of an extract of mixed cruciferous and legume sprouts on DNA damage induced by H(2)O(2) was measured in HT29 cells using single cell microgelelectrophoresis (comet). Significant antigenotoxic effect (P < or = 0.05) was observed when HT29 cells were pre-incubated with the extract (100 and 200 microL/mL) for 24 hours and then challenged with H(2)O(2). A parallel design intervention study was carried out on 10 male and 10 female healthy adult volunteers (mean age = 25.5 years) fed 113 g of cruciferous and legume sprouts daily for 14 days. The effect of the supplementation was measured on a range of parameters, including DNA damage in lymphocytes (comet), the activity of various detoxifying enzymes (glutathione S-transferase, glutathione peroxidase, and superoxide dismutase), antioxidant status using the ferric reducing ability of plasma assay, plasma antioxidants (uric acid, ascorbic acid, and alpha-tocopherol), blood lipids, plasma levels of lutein, and lycopene. A significant antigenotoxic effect against H(2)O(2)-induced DNA damage was shown in peripheral blood lymphocytes of volunteers who consumed the supplemented diet when compared with the control diet (P = 0.04). No significant induction of detoxifying enzymes was observed during the study, neither were plasma antioxidant levels or activity altered. The results support the theory that consumption of cruciferous vegetables is linked to a reduced risk of cancer via decreased damage to DNA.

Free radicals, metals and antioxidants in oxidative stress-induced cancer

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular. ROS activation of AP-1 (activator protein) and NF-kappa B (nuclear factor kappa B) signal transduction pathways, which, in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu. Zn-SOD. Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of careinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-kappa B, AP-1 are also reviewed. 2006 Elsevier Ireland Ltd. All rights reserved.