Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA

Phenolic phytochemicals are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against free radicals. The aims of this study were to assess the free radical-scavenging activities of several common phenolic phytochemicals, and then, the effects of the most potent phenolic phytochemicals on oxidative damage to DNA in cultured cells. Epigallocatechin gallate (EGCG) scavenged the stable free radical, alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH), most effectively, while quercetin was about half as effective. Genistein, daidzein, hesperetin, and naringenin did not scavenge DPPH appreciably. Jurkat T-lymphocytes that were pre-incubated with relatively low concentrations of either EGCG or quercetin were less susceptible to DNA damage induced by either a reactive oxygen species or a reactive nitrogen species, as evaluated by the comet assay. More specifically, control cells had a comet score of only 17+/-5, indicating minimal DNA damage. Cells challenged with 25 microM hydrogen peroxide (H(2)O(2)) or 100 microM 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) had comet scores of 188+/-6 and 125+/-12, respectively, indicating extensive DNA damage. The H(2)O(2)-induced DNA damage was inhibited with 10 microM of either EGCG (comet score: 113+/-23) or quercetin (comet score: 82+/-7). Similarly, the SIN-1-mediated DNA damage was inhibited with 10 microM of either EGCG (comet score: 79+/-13) or quercetin (comet score: 72+/-17). In contrast, noticeable DNA damage was induced in Jurkat T-lymphocytes by incubating with 10-fold higher concentrations (i.e., 100 microM) of either EGCG (comet score: 56+/-17) or quercetin (comet score: 64+/-13) by themselves. Collectively, these data suggest that low concentrations of EGCG and quercetin scavenged free radicals, thereby inhibiting oxidative damage to cellular DNA. But, high concentrations of either EGCG or quercetin alone induced cellular DNA damage.     

Assay and analysis for anti- and pro-oxidative effects of ascorbic acid on DNA with the bulk acoustic wave impedance technique

A bulk acoustic wave (BAW) impedance sensor has been applied for in situ monitoring of the whole process of DNA oxidative damage induced by the vitamin C (Vc)-Fe (III) system, based on its real-time responses to the density-viscosity change of the tested solution due to the damages occurring on the DNA molecules. The results showed that Vc exhibited two conflicting effects, i.e., pro-oxidation and anti-oxidation on the DNA at different Vc concentrations in the damage system, and the "threshold" concentration of Vc for these two effects was estimated to be about 100 micromol/L. The end-point frequency change of the sensor (Deltaf(m)) was found to be linearly related to the initial concentration of the soybean DNA (C(DNA)) in the range of 40-1000 microg/mL, and the exponential relationship between the frequency change (Deltaf(0)) vs damaging time suggested that the Fe (III)-mediated DNA damage by Vc could be described as a first-order kinetics reaction. The effects of variations in concentrations of Vc and Fe3+ on the DNA oxidative damage were discussed, and based on investigations for the enhancing influence of H2O2 and inhibiting influence of HO* scavengers on the DNA damage, the nature and physiological toxicity of the damage in biological system were also examined. In addition, UV-vis spectra and electrophoresis analysis were also used, and the experimental observations were in good agreement with the above results.     

Protection by quercetin and quercetin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes

Flavonoids are claimed to protect against cardiovascular disease, certain forms of cancer and ageing, possibly by preventing initial DNA damage. Therefore, we investigated the protective effects of the flavonoid quercetin against the formation of oxidative DNA damage and bulky DNA adducts in human lymphocytes, both in vitro and ex vivo. First, human lymphocytes were pre-incubated with various concentrations of quercetin, followed by incubation with hydrogen peroxide; protection against oxidative DNA damage was evaluated by use of the single-cell gel electrophoresis (Comet) assay. Second, quercetin-treated human lymphocytes were challenged by treatment with benzo(a)pyrene (B(a)P), and BPDE-DNA adduct formation was measured by (32)P-postlabelling. Third, in a pilot study, lymphocytes from female volunteers who consumed a quercetin-rich blueberry/apple juice mixture for four weeks, were treated ex vivo with an effective dose of H(2)O(2) and benzo(a)pyrene, respectively, at three different time points, i.e. before (t=0 weeks), during (t=2 weeks) and after (t=4 weeks) the intervention. Results in vitro: a significant dose-dependent protection by quercetin against both the formation of oxidative DNA damage (p<0.01) and of BPDE-DNA adducts (p<0.05) was observed. Results in vivo: four weeks of juice intervention led to a significant increase in the total antioxidant capacity of plasma, as reflected by the increase of the TEAC value from 773 microM trolox equivalent at t=0 to 855 microM at t=4 weeks (p=0.04) and an increase in plasma quercetin content from 5.0 to 10.6 nM (p=0.03). After intervention, the level of oxidative damage upon ex vivo exposure to H(2)O(2) was non-significantly (p=0.07) decreased by 41%, and the BPDE-DNA adduct level induced ex vivo was non-significantly decreased by 11%. The combination of our findings in vitro and ex vivo provides evidence that quercetin is able to protect against chemically induced DNA damage in human lymphocytes, which may underlie its suggested anticarcinogenic properties.     

Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis

As part of a randomized placebo-controlled study to evaluate the effect of lycopene supplementation on DNA damage in men with prostate cancer, a nonrandomized 5th arm using tomato sauce was included and reported here. Thirty-two patients with localized prostate adenocarcinoma consumed tomato sauce-based pasta dishes for 3 weeks (30 mg of lycopene/day) before their scheduled radical prostatectomy. Prostate tissue was obtained as biopsies at baseline and as resected tissue at the time of the prostatectomy. Serum and prostate lycopene, serum prostate specific antigen (PSA) concentrations, and leukocyte DNA 8-OH-deoxyguanosine/deoxyguanosine (8OHdG) were measured at baseline and at the end of the intervention. Cancer cells in paraffin sections of prostate biopsies and postintervention resected tissue were compared for 8OHdG staining and for apoptosis. Adherence to the daily consumption of tomato-based entrees was 81.6% of the intended dose, and serum and prostate lycopene concentrations increased 1.97- and 2.92-fold (P < 0.001), respectively. Mean serum PSA concentrations decreased by 17.5% (P < 0.002) and leukocyte 8OHdG decreased by 21.3% (P < 0.005) after tomato sauce consumption. Resected tissues from tomato sauce-supplemented patients had 28.3% lower prostate 8OHdG compared with the nonstudy control group (P < 0.03). Cancer cell 8OHdG staining of Gleason Score-matched resected prostate sections was reduced by 40.5% in mean nuclear density (P < 0.005) and by 36.4% in mean area (P < 0.018) compared with the presupplementation biopsy. Apoptotic index was higher in hyperplastic and neoplastic cells in the resected tissue after supplementation. These data taken as a whole indicate significant uptake of lycopene into prostate tissue and a reduction in DNA damage in both leukocyte and prostate tissue. Whether reduction in DNA damage to prostate cancer cells is beneficial awaits further research, although reduction in serum PSA concentrations is promising.     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid

Lipid hydroperoxide (LOOH)-dependent lipid peroxidation was induced in alpha-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 microM. The effects of structurally related flavonoids at concentrations from 10 to 500 microM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid-metal complexes.     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with α-linolenic acid

Lipid hydroperoxide (LOOH)–dependent lipid peroxidation was induced in α-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 μM. The effects of structurally related flavonoids at concentrations from 10 to 500 μM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid–metal complexes.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

Exposure of mammalian cells to 60-Hz magnetic or electric fields: analysis of DNA repair of induced, single-strand breaks

DNA damage was induced in isolated human peripheral lymphocytes by exposure at 5 Gy to 60Co radiation. Cells were permitted to repair the DNA damage while exposed to 60-Hz fields or while sham-exposed. Exposed cells were subjected to magnetic (B) or electric (E) fields, alone or in combination, throughout their allotted repair time. Repair was stopped at specific times, and the cells were immediately lysed and then analyzed for the presence of DNA single-strand breaks (SSB) by the alkaline-elution technique. Fifty to 75 percent of the induced SSB were repaired 20 min after exposure, and most of the remaining damage was repaired after 180 min. Cells were exposed to a 60-Hz ac B field of 1 mT; an E field of 1 or 20 V/m; or combined E and B fields of 0.2 V/m and 0.05 mT, 6 V/m and 0.6 mT, or 20 V/m and 1 mT. None of the exposures was observed to affect significantly the repair of DNA SSB.     

Mechanism of oxidative DNA damage induced by quercetin in the presence of Cu(II)

Quercetin, one of flavonoids, has been reported to be carcinogenic. There have been no report concerning carcinogenicity of kaempferol and luteolin which have structure similar to quercetin. DNA damage was examined by using DNA fragments obtained from the human p53 tumor suppressor gene. Quercetin induced extensive DNA damage via reacting with Cu(II), but kaempferol and luteolin induced little DNA damage even in the presence of Cu(II). Excessive quercetin inhibited copper-dependent DNA damage induced by quercetin. Bathocuproine, a Cu(I)-specific chelator, catalase and methional inhibited the DNA damage by quercetin, whereas free hydroxyl radical scavengers did not. Site specificity of the DNA damage was thymine and cytosine residues. The site specificity and the inhibitory effects suggested that DNA-copper-oxygen complex rather than free hydroxyl radical induced the DNA damage. Formation of 8-oxodG by quercetin increased extensively in the presence of Cu(II), whereas 8-oxodG formation by kaempferol or luteolin increased only slightly. This study suggests a good relationship between carcinogenicity and oxidative DNA damage of three flavonoids. The mechanism of DNA damage by quercetin was discussed in relation to the safety in cancer chemoprevention by flavonoids.     

Detection and analysis of Bacillus subtilis growth with piezoelectric quartz crystal impedance based on starch hydrolysis

A piezoelectric quartz crystal (PQC) impedance method based on the alpha-amylase-catalyzed hydrolysis of starch present in a culture medium has been developed for in situ monitoring of the whole growth process of Bacillus subtilis and the variation in the activity of alpha-amylase during bacterial growth. An S-shaped response behavior was observed for Deltaf(0), and simultaneously inverse S-shaped responses were found for DeltaR(1) and DeltaL(1). The ratio of DeltaR(1) to Deltaf(0) or DeltaL(1) coincided well with that calculated from Martin's equations reflecting the solution density-viscosity effect, suggesting that the continuing change in liquid loading onto the PQC surface causes significant variation in Deltaf(0), DeltaR(1), and DeltaL(1). Bacterial growth equations were derived from the kinetics of the enzyme-catalyzed hydrolysis of starch, which fit well with the experimental responses of Deltaf(0), DeltaR(1), and DeltaL(1). Kinetic parameters of bacterial growth, including the asymptote (A), the maximum specific growth rate (microm), and the lag time (lambda), were obtained and were in good agreement with those obtained from the pour plate count method. The variation in the activity of alpha-amylase exhibited peak-type behavior with its maximum value at the later stage of the log phase. In addition, the influence of initial bacterial concentration was also investigated.     

Study on the interaction between DNA and protein induced by anticancer drug carboplatin

The interaction of DNA and human serum albumin (HSA) in the presence of anticancer drug carboplatin was studied with piezoelectric quartz crystal impedance (PQCI) and electrochemistry techniques. In the PQCI analysis, the correlative parameters including the frequency (f0), the motional resistance (R(m)), and the static capacitance (C0) in the experiment were obtained and discussed in detail. Additionally, the kinetics parameters of the cross-linking process were investigated and a response kinetics model was deduced. The values of association rate constant k1, dissociation rate constant k(-1) and the reaction equilibrium constant K were estimated to be 1.895 mg/ml(-1) s(-1), 4.7 x 10(-5) s(-1) and 4.03 x 10(4) (mg/ml)-1, respectively. Furthermore, cyclic voltammetry (CV) and electrochemical AC impedance techniques were employed to testify the cross-linking process.     

Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A

Dietary iron may contribute to colon cancer risk via production of reactive oxygen species (ROS). The aim of the study was to determine whether physiological ferric/ferrous iron induces oxidative DNA damage in human colon cells. Therefore, differentiated human colon tumour cells (HT29 clone 19A) were incubated with ferric-nitrilotriacetate (Fe-NTA) or with haemoglobin and DNA breaks and oxidised bases were determined by microgelelectrophoresis. The effects of Fe-NTA were measured with additional H(2)O(2) (75microM) and quercetin (25-100microM) treatment. Analytic detection of iron in cell cultures, treated with 250microM Fe-NTA for 15 min to 24h, showed that 48.02+/-5.14 to 68.31+/-2.11% were rapidly absorbed and then detectable in the cellular fraction. Fe-NTA (250-1000microM) induced DNA breaks and oxidised bases, which were enhanced by subsequent H(2)O(2) exposure. Simultaneous incubation of HT29 clone 19A cells with Fe-NTA and H(2)O(2) for 15 min, 37 degrees C did not change the effect of H(2)O(2) alone. The impact of Fe-NTA and H(2)O(2)-induced oxidative damage is reduced by the antioxidant quercetin (75-67% of H(2)O(2)-control). Haemoglobin was as effective as Fe-NTA in inducing DNA damage. From these results we can conclude that iron is taken up by human colon cells and participates in the induction of oxidative DNA damage. Thus, iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor. Inhibition of damage by quercetin reflects the potential of antioxidative compounds to influence this risk factor. Quantitative data on the genotoxic impact of ferrous iron (e.g. from red meat) relative to the concentrations of antioxidants (from plant foods) in the gut are now needed to determine the optimal balance of food intake that will reduce exposure to this type of colon cancer risk factor.     

The DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity

T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed.     

Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Effects of dietary antioxidants on human DNA ex vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and alpha-tocopherol, were investigated, at concentrations up to 200 microM, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 min exposure to hydrogen peroxide (final concentrations H2O2: 30, 45, or 60 microM). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H2O2 from these polyphenolics. Neither ascorbic acid nor alpha-tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.     

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67+/-19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations > or =100 microM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations > or 300 microM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by dl-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (> or =1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H₂O₂-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H₂O₂-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H₂O₂-mediated DNA damage may be related to the carcinogenic effects of quercetin.     

Antioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus

Bivalve molluscs, particularly mussels, are sensitive biomarkers of aquatic ecosystem pollution. The tannins, water-soluble plant polyphenols, may play an important role in this environment and, mainly as a consequence of interaction with pollutants, their toxicity may change. We studied three naturally occurring compounds, tannic acid, ellagic acid and gallic acid, for their ability to modulate DNA damage produced by these tannins alone and in the presence of the oxidative stress inducer H(2)O(2), in cells of the digestive gland of mussels (Unio tumidus). After the treatment of the cells with polyphenols at different concentrations (1, 5, 15, 30, 60, 80, 100, 120, 180, 240 microM) and with hydrogen peroxide in the range of 0.04 and 0.1mM, single-strand breaks (ssb) in DNA were investigated, using the comet assay. The ability of phenolic acids to decrease DNA damage through their antioxidant properties was also assessed. The results show that the phenols, which are known as antioxidative agents, could also act as pro-oxidants. They induced ssb in DNA of the digestive gland at concentrations higher that 10 microM, but lower doses (1 and 5 microM) did not contribute to the DNA damage. This study was also designed to evaluate the protective effect of these tannins against H(2)O(2)-mediated DNA damage in the cells. In this treatment, the two concentrations (1 and 5 microM) significantly decreased the amount of lesions induced by H(2)O(2) (0.04 and 0.1mM). In conclusion, our results demonstrate that antioxidative properties of tannins may change to pro-oxidative activities at the higher concentrations. This suggests that the biologic actions of these compounds may be rather complicated.