Modulation of tea and tea polyphenols on benzo(a)pyrene-induced DNA damage in Chang liver cells

The protective effects of three tea extracts (green tea, GTE; oolong tea, OTE; and black tea, BTE) and five tea polyphenols (epicatechin, EC; epicatechin gallate, ECG; epigallocatechin, EGC; epigallocatechin gallate, EGCG; and theaflavins, THFs) on benzo[a]pyrene (B[a]P)-induced DNA damage in Chang liver cells were evaluated using the comet assay. B[a]P-induced DNA damage in Chang liver cells was significantly (p < 0.05) inhibited by GTE and OTE at a concentration of 10 microg/ml and by BTE at 25 microg/ml. At a concentration of 100 microg/ml, the % tail DNA was reduced from 33% (B[a]P treated only) to 10, 9, 13%, by GTE, OTE and BTE, respectively. EC and ECG did not cause DNA damage in cells according to the results of the comet assay; however, EGC, EGCG and theaflavins caused DNA damage in cells at a concentration of 100 microM. The results indicated that EC and ECG had protective effects against B[a]P-induced DNA damage in cells at a concentration of 10-100 microM. Although EGC, EGCG and the theaflavins caused DNA damage at a high concentration, but they had protective effects against B[a]P-induced DNA damage in cells at a low concentration of 10-50 microM. The results also showed that the DNA damage in cells induced by EGC, EGCG, and the theaflavins was due to the generation of superoxide during incubation with cells at a higher concentration. Therefore, tea catechins and THFs play an important role in enabling tea extracts to inhibit DNA damage in Chang liver cells.     

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.     

Inhibition of beta-catenin/Tcf activity by white tea,green tea,and epigallocatechin-3-gallate (EGCG): minor contribution of H(2)O(2) at physiologically relevant EGCG concentrations

Epigallocatechin-3-gallate (EGCG) is the major polyphenol present in white tea and green tea. Recently, it was reported that the addition of EGCG and other tea polyphenols to cell culture media, minus cells, generated significant levels of H(2)O(2), with the corollary that this might represent an "artifact" in cell culture studies which seek to examine the chemopreventive mechanisms of tea. We show here that in cell growth media with and without serum, and in growth media containing human embryonic kidney 293 (HEK293) cells plus serum, physiologically relevant concentrations of EGCG (< or =25 microM) generated H(2)O(2) with a peak concentration of the order of 10-12 microM. However, addition of 20 microM H(2)O(2) directly to HEK293 cells transiently transfected with wild-type or mutant beta-catenin constructs and TCF-4 had no significant effect on beta-catenin/TCF-4 reporter activity or beta-catenin expression levels. In contrast, 2-25 microM EGCG inhibited beta-catenin/TCF-4 reporter activity in a concentration-dependent fashion and there was a concomitant reduction in beta-catenin protein levels in the cell lysates without changes in TCF-4 expression. The inhibition of reporter activity was recapitulated by white tea and green tea, each tested at a 25 microM EGCG equivalent concentration in the assay, and this was unaffected by the addition of exogenous catalase. The results indicate that physiologically relevant concentrations of tea and EGCG inhibit beta-catenin/TCF-4 reporter activity in HEK293 cells due to reduced expression of beta-catenin and that this is unlikely to be an artifact of H(2)O(2) generation under the assay conditions used here. These data are consistent with the findings from in vivo studies, showing the suppression of intestinal polyps by tea, via an apparent down-regulation of beta-catenin and Wnt target genes.     

Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining

ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD(+) as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD(+) or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 microM H(2)O(2) and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r > or = 0.92, p<0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.     

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.     

Protective effect of Crocus sativus stigma extract and crocin (trans-crocin 4) on methyl methanesulfonate-induced DNA damage in mice organs

This study was designed to examine the effect of aqueous extract of Crocus sativus stigmas (CSE) and crocin (trans-crocin 4) on methyl methanesulfonate (MMS)-induced DNA damage in multiple mice organs using the comet assay. Adult male NMRI mice in different groups were treated with either physiological saline (10 mL/Kg, intraperitoneal [ip]), CSE (80 mg/Kg, ip), crocin (400 mg/Kg, ip), MMS (120 mg/Kg, ip), and CSE (5, 20, and 80 mg/Kg, ip) 45 min prior to MMS administration or crocin (50, 200, and 400 mg/Kg, ip) 45 min prior to MMS administration. Mice were sacrificed about 3 h after each different treatment, and the alkaline comet assay was used to evaluate the effect of these compounds on DNA damage in different mice organs. The percent of DNA in the comet tail (% tail DNA) was measured. A significant increase in the % tail DNA was seen in nuclei of different organs of MMS-treated mice. In control groups, no significant difference was found in the % tail DNA between CSE- or crocin-pretreated and saline-pretreated mice. The MMS-induced DNA damage in CSE-pretreated mice (80 mg/Kg) was decreased between 2.67-fold (kidney) and 4.48-fold (lung) compared to those of MMS-treated animals alone (p < 0.001). This suppression of DNA damage by CSE was found to be depended on the dose, which pretreatment with CSE (5 mg/Kg) only reduced DNA damage by 6.97%, 6.57%, 7.27%, and 9.90% in liver, lung, kidney, and spleen, respectively (p > 0.05 as compared with MMS-treated group). Crocin also significantly decreased DNA damage by MMS (between 4.69-fold for liver and 6.55-fold for spleen, 400 mg/Kg), in a dose-dependent manner. These data indicate that there is a genoprotective property in CSE and crocin, as revealed by the comet assay, in vivo.     

Oxidatively modified proteins and DNA in digestive gland cells of the fresh-water mussel Unio tumidus in the presence of tannic acid and its derivatives

The oxidative effect of tannic acid and its two derivatives (ellagic and gallic acid), naturally occurring plant polyphenols, has been studied on digestive gland cells of the fresh-water mussel Unio tumidus. A spectrophotometric method was used to determine the protein thiol groups after incubation of the cells with the polyphenols at concentrations of 1, 15 and 60 microM. The results showed that the oxidative modification of proteins increased in a concentration-dependent manner but no changes were observed at the concentration of 1 microM. The comet assay (single-cell gel electrophoresis assay) with the formamido-pyrimidine glycosylase (FPG) protein was used to assess oxidative DNA base damage. The cells were treated with polyphenols at the concentrations of 30 and 60 microM and post-incubated with FPG. FPG strongly enhanced DNA damage induced by the polyphenols, indicating that N-7 guanine oxidation is responsible for the observed effect. Using the comet assay in combination with proteinase K we were able to demonstrate the presence of DNA-protein cross-links as the probable cause of the decrease in DNA migration. After treatment of the cells with tannic acid and its metabolites at concentrations of 120, 180 and 240 microM, they were post-incubated with proteinase K. After this treatment an increased DNA migration was observed, indicating the presence of DNA-protein cross-links. We have also used a fluorescence method with Hoechst 33258/propidium iodide DNA-binding dyes to study the extent of DNA fragmentation after exposure of the cells to polyphenols at concentrations of 1, 5 and 60 microM. The results demonstrate that the polyphenols can induce apoptosis and necrosis at higher concentrations (5 and 60 microM). All experimental data suggest that tannic, ellagic and gallic acids at concentrations above 1 microM are able to interact with proteins and DNA, which leads to their degradation or changes in their function.     

The Impact of UV Radiation on Paramecium Populations from Alpine Lakes

Paramecium populations from a clear and a glacier‐fed turbid alpine lake were exposed to solar simulated ultraviolet (UVR) and photosynthetically active radiation (PAR) at 8 and 15 °C. The ciliates were tested for DNA damage (comet assay), behavioral changes, and mortality after UVR + PAR exposure. High DNA damage levels (~58% tail DNA) and abnormal swimming behavior were observed, although no significant changes in cell numbers were found irrespective of the lake origin (clear, turbid), and temperatures. We conclude that environmental stressors such as UVR and their effects may influence the adaptation of ciliates living in alpine lakes.     

DNA repair of hydrogen peroxide-induced damage in human lymphocytes in the presence of four antimutagens. A study with alkaline single cell gel electrophoresis (comet assay).

We assessed four antimutagenic compounds' influences on DNA repair in human lymphocytes exposed in vitro to hydrogen peroxide (20 microM, 5 min, at 4 degrees C). DNA damage and repair were estimated by means of alkaline single cell gel electrophoresis (comet assay). It was noticed that the enhancement of DNA repair was relatively strongest when fluphenazine was present in the cell culture medium. In the cases of anthocyanins and alkylresorcinols, the effects were almost 6-9 times weaker than that of FPh. The effect of todralazine on DNA repair was relatively weakest. Further study should be done on fluphenazine as a potential DNA repair-enhancing compound.     

Green tea polyphenol (−)-epigallocatechin gallate reduces matrix metalloproteinase-9 activity following transient focal cerebral ischemia

Green tea polyphenol (?)-epigallocatechin gallate (EGCG) has been reported to reduce neuronal damage after cerebral ischemic insult. EGCG is known to reduce matrix metalloproteinase (MMP) activity. MMP can play an important role in the pathophysiology of neurological disorders including cerebral ischemia. The purpose of the current study was to investigate whether EGCG shows an inhibitory effect on MMP activity and neural tissue damage following transient focal cerebral ischemia. In the present study, C57BL/6 mice were subjected to 80 min of focal ischemia induced by middle cerebral artery occlusion (MCAO). Animals were killed 24 h after ischemia. EGCG (50 mg/kg) was administered intraperitoneally immediately after ischemia. Gelatin gel zymography showed an increase in the active form of MMP-9 after ischemia. EGCG reduced ischemia-induced up-regulation of the active form of MMP-9. In in situ zymography, EGCG reduced up-regulation of gelatinase activity induced by cerebral ischemia. Co-incubation with EGCG reduced gelatinase activity directly in postischemic brain section. In 2,3,5-triphenyltetrazolium chloride (TTC) assay, brain infarction was remarkable in the middle cerebral artery territory after focal cerebral ischemia. In EGCG-treated mice, infarct volume was significantly reduced compared with vehicle-treated mice. These results demonstrate that EGCG, a green tea polyphenol, may reduce up-regulation of MMP-9 activity and neuronal damage following transient focal cerebral ischemia. In addition to its antioxidant effect, MMP-9 inhibition might be a possible mechanism potentially involved in the neuroprotective effect of a green tea polyphenol, EGCG.     

Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure

A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.     

Green tea polyphenol (-)-epigallocatechin-3-gallate induces neurorescue of long-term serum-deprived PC12 cells and promotes neurite outgrowth

Our previous studies have shown that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) prevents neuronal cell death caused by several neurotoxins. The present study sought to determine the neuroprotective effect of EGCG when it is administered after the induction of cell damage ('neurorescue'). In an attempt to imitate a progressive mode of death, PC12 cells were initially subjected to serum-starvation conditions for a period of 1 or 3 days before administration of EGCG (0.1-10 microM) for up to 3 days. In spite of the high percentage of cell death, single or repetitive administration of EGCG (1 microM) significantly attenuated cell death. The neurorescue effect of EGCG was abolished by pre-treatment with the protein kinase C inhibitor GF109203X (2.5 microM), suggesting the involvement of the protein kinase C pathway in neurorescue by the drug. This is consistent with the rapid (15 min) translocation of the protein kinase C alpha isoform to the cell membrane in response to EGCG. The correlative neurite outgrowth activity of EGCG on PC12 cells may also contribute to its neurorescue effect. The present findings suggest that EGCG may have a positive impact on aging and neurodegenerative diseases to retard or perhaps even reverse the accelerated rate of neuronal degeneration.     

In vivo DNA damage in gastric epithelial cells

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.     

DNA-damaging potential and glutathione depletion of 2-cyclohexene-1-one in mammalian cells, compared to food relevant 2-alkenals

2-Cyclohexene-1-one (CHX) occurs as a natural ingredient in some tropical fruits and has been detected as a contaminant in certain artificially sweetened soft drinks. To elucidate its cytotoxic/genotoxic effectiveness, CHX was tested in mammalian cell lines (V79 and Caco-2) and in primary human colon cells in comparison to structurally related 2-alkenals. Inhibition of cell growth (IC(50)) and cytotoxicity (LC(50)) were determined by protein staining with sulforhodamin B (SRB) and by trypan blue exclusion, respectively. DNA damage--both strand breaks and oxidised purines--was quantified by comet assay. Depletion of glutathione was measured in a kinetic assay, based on 5-thio-2-nitrobenzoate (TNB) formation. For CHX, a moderate cytotoxicity was observed after 1h incubation in V79 cells (LC(50): 4.75mM). The 2-alkenals ((E)-2-octenal (OCTE), (2E,4Z)-2,4-hexadienal (HEXDI), (E)-2-nonenal (NONE), (2E,6Z)-2,6-nonadienal (NONDI)) exhibited a distinctly higher cytotoxicity, except for (E)-2-hexenal (HEX) (LC(50): 3.67mM) and cinnamaldehyde (CA) (LC(50): 4.45mM). If the incubation time was prolonged to 24h, an IC(50) of 15microM was obtained for CHX which is well within the range obtained for the 2-alkenals (4 and 17microM). Concentration-dependent DNA damage was observed after 1h incubation with CHX. The respective DC(50) values (concentration inducing DNA damage in 50% of cells) were 272microM (V79) and 455microM (Caco-2). All 2-alkenals were more active under these conditions, except for CA. In primary human colon cells, CHX (800microM, 30min) exhibited a weak, but still significant DNA-damaging potential. Glutathione levels in V79 cells were effectively depleted (down to approximately 20%) by CHX concentrations not yet inducing DNA damage (c < or = 50microM). Incubation with CHX or 2-alkenals (50 and 100microM, 1h), followed by H2O2 treatment (5min, 25microM) resulted in increased levels of oxidised purines in the modified comet assay. CHX and HEX, additionally tested in primary human colon cells, depleted glutathione and increased the sensitivity towards oxidative stress.     

Study of the biological effects and DNA damage exerted by a new dipalladium-Hmtpo complex on human cancer cells

The new dipalladium complex [Pd(2)(mu-mtpo-N(3),N(4))(2)(phen)(2)](NO(3))(2) (where phen=1,10-phenantroline; Hmtpo=5,7-dihydro-7-oxo-5-methyl[1,2,4]triazolopyrimidine), (Pd(2)-Hmtpo, or complex I), interacts effectively with DNA plasmid (pBS), as studied by circular dichroism spectroscopy (CD), causing large helix distortions, altering the direction of the main DNA helix axis and producing unwinding of the DNA double helix. DNA damage induced by complex I was highly significant at 2.81 microM (ovarian carcinoma TG cell line), as assessed by comet assay, a dose at which all treated nuclei showed more than 30% DNA migration to the comet tail. DNA damage effect is a consequence of genotoxicity and not a false positive response caused by cytotoxicity. In vitro cytotoxic assay on the two human tumor cell lines TG and BT-20 (breast carcinoma), shows that doses of 0.47, 1.41 and 2.81 microM produce significant antiproliferative effects after 4 days of treatment compared with control. Complex I was highly cytotoxic at 2.81 microM causing an inhibition of viable cells of 65.5%. Cisplatin (cis-DDP) exhibits lower cytotoxic activity in TG cells than dipalladium complex (a cisplatin dose of 6.67 microM inhibits 30.3%) and does not cause migration of DNA to comet tail.     

Protective effects of green tea polyphenols and their major component, (-)-epigallocatechin-3-gallate (EGCG), on 6-hydroxydopamine-induced apoptosis in PC12 cells

Green tea polyphenols exert a wide range of biochemical and pharmacological effects, and have been shown to possess antimutagenic and anticarcinogenic properties. Oxidative stress is involved in the pathogenesis of Parkinson's disease. However, although green tea polyphenols may be expected to inhibit the progression of Parkinson's disease on the basis of their known antioxidant activity, this has not previously been established. In the present study, we evaluated the neuroprotective effects of green tea polyphenols in the Parkinson's disease pathological cell model. The results show that the natural antioxidants have significant inhibitory effects against apoptosis induced by oxidative stress. 6-Hydroxydopamine (6-OHDA)-induced apoptosis in catecholaminergic PC12 cells was chosen as the in vitro model of Parkinson's disease in our study. Apoptotic characteristics of PC12 cells were assessed by MTT assay, flow cytometry, fluorescence microscopy and DNA fragmentation. Green tea polyphenols and their major component, EGCG at a concentration of 200 microM, exert significant protective effects against 6-OHDA-induced PC12 cell apoptosis. EGCG is more effective than the mixture of green tea polyphenols. The antioxidant function of green tea polyphenols may account for this neuroprotective effect. The present study supports the notion that green tea polyphenols have the potential to be effective as neuropreventive agents for the treatment of neurodegenerative diseases.     

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.     

Modified comet assay as a biomarker of sodium dichromate-induced oxidative DNA damage: optimization and reproducibility.

Hexavalent chromium (Cr[VI]) is a genotoxic carcinogen that has been associated with an increased risk of nasal and respiratory tract cancers following occupational exposure. Although the precise mechanism(s) remain to be elucidated, there is evidence for a role of oxidative DNA damage in the genotoxicity of Cr(VI). In the current study, human white blood cells were treated in vitro with non-cytotoxic concentrations of sodium dichromate (1-100 microM) for 1 h. Analysis by immunocytochemistry indicated the presence of elevated levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine at concentrations of sodium dichromate greater than 10 microM. In contrast, the lowest concentration of dichromate that resulted in a statistically significant increase in levels of formamidopyrimidine DNA glycosylase (FPG)-dependent DNA strand breaks was 100 nM (p<0.05). In addition, levels of both control and dichromate-induced FPG-dependent strand breaks from blood samples taken from the same individuals over 10 months proved remarkably reproducible in the individuals studied. The coefficients of variation over three different times of the year in control and dichromate-induced oxidative DNA damage for the four individuals were 54, 1, 37 and 4, and 45, 6, 21 and 18%, respectively. In summary, these results indicate that physiologically relevant, nanomolar concentrations of sodium dichromate cause DNA base oxidation in human white blood cells in vitro as assessed by the FPG-modified comet assay. Furthermore, comet assay data from an individual are reproducible over an extended period. This consistency is sufficient to suggest that the modified comet assay might prove to be a useful and sensitive biomonitoring tool for individuals occupationally exposed to hexavalent chromium.     

Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.2 to 2 microM induced DNA damage in human leukemic K562 and BV173 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes and leukemic CCRF-CEM cells without the expression of BCR/ABL. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the circular plasmid relaxation assay. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. K562 cells were unable to repair H(2)O(2)-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with Vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Therefore, the mechanism of the anti-leukemic action of STI571 may involve not only the inhibition of BCR/ABL, but also DNA damage in the cells expressing this fusion protein. DNA damage induced by STI571 may follow from oxidative and alkylative base modifications.     

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb2+) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 microM to 200 microM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 microM Pb2+. In tobacco plants growing for 6 weeks in soil polluted with Pb2+ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb2+-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb2+, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb2+ accumulation in the above-ground parts may explain the lower levels or the absence of Pb2+-induced DNA damage in leaves.