Generation Dependent Effects and Entrance to Mitochondria of Hybrid Dendrimers on Normal and Cancer Neuronal Cells In Vitro.

Dendrimers as drug carriers can be utilized for drugs and siRNA delivery in central nervous system (CNS) disorders, including various types of cancers, such as neuroblastomas and gliomas. They have also been considered as drugs per se, for example as anti-Alzheimer's disease (AD), anti-cancer, anti-prion or anti-inflammatory agents. Since the influence of carbosilane-viologen-phosphorus dendrimers (SMT1 and SMT2) on the basic cellular processes of nerve cells had not been investigated, we examined the impact of two generations of these hybrid macromolecules on two murine cell lines-cancer cell line N2a (mouse neuroblastoma) and normal immortalized cell line mHippoE-18 (embryonic mouse hippocampal cell line). We examined alterations in cellular responses including the activity of mitochondrial dehydrogenases, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, and morphological modifications and fractions of apoptotic and dead cells. Our results show that both dendrimers at low concentrations affected the cancer cell line more than the normal one. Also, generation-dependent effects were found: the highest generation induced greater cytotoxic effects and morphological modifications. The most promising is that the changes in mitochondrial membrane potential and transmission electron microscopy (TEM) images indicate that dendrimer SMT1 can reach mitochondria. Thus, SMT1 and SMT2 seem to have potential as nanocarriers to mitochondria or anti-cancer drugs per se in CNS disorders.

Cytotoxic activity of genistein-8-C-glucoside form Lupinus luteus L. and genistein against human SK-OV-3 ovarian carcinoma cell line.

Genistein belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. Numerous in vitro studies suggest that isoflavones, particularly genistein, have both chemopreventive and chemotherapeutic potential in multiple tumor types. However, the molecular and cellular mechanisms of genistein effects on human ovarian cancer cells are still little known. In the present study, we investigated anticancer activity of genistein and its natural glucoside, genistein-8-C-glucoside isolated from flowers of Lupinus luteus L. We examined the effects of the two isoflavones alone or in combination on cultured human SK-OV-3 ovarian carcinoma cells. The cells were exposed to genistein and genistein-8-C-glucoside at various concentrations (1-90 µM) for 24 and 48 h. The cytotoxic and apoptotic properties of compounds were studied by the colorimetric 3-[4,5-2-yl]-2-5-diphenyltetrazolium bromide assay and the acridine orange/ethidium bromide staining technique. The morphological features of SK-OV-3 cells were examined by Nomarski differential interference contrast combined with a confocal laser scanning microscope. The level of ROS was evaluated with fluorescence probes: dichlorofluorescein-diacetate by flow cytometry. Changes in mitochondrial membrane potential were determined using 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide. Genistein-treatment and genistein-8-C-glucoside-treatment resulted in the inhibition of cell proliferation, induction of apoptotic cell death and loss of mitochondrial membrane potential. The present data provide the first evidence in vitro that genistein-8-C-glucoside and combination genistein-genistein-8-C-glucoside could be a potential chemotherapeutic candidate for ovarian cancer therapy.

[Applications of electromagnetic radiation in medicine]. / Zastosowanie promieniowania elektromagnetycznego w medycynie.

Recent decades have been devoted to the intense search for the response to questions related to the impact of radiation on the human body. Due to the growing fashion for a healthy lifestyle, increasing numbers of works about the alleged dangers of electromagnetic waves and diseases that they cause appeared. However, the discoveries of 20th century, and knowledge of the properties of electromagnetic radiation have allowed to broaden the horizons of the use of artificial sources of radiation in many fields of science and especially in medicine. The aim of this paper is to show that although excessive radiation or high doses are dangerous to the human body, its careful and controlled use, does not pose a threat, and it is often necessary in therapy. The possibility of using ionizing radiation in radiotherapy, isotope diagnostics or medical imaging, and non-ionizing radiation in the treatment for dermatological disorders and cancers will be presented. The unique properties of synchrotron radiation result in using it on a large scale in the diagnosis of pathological states by imaging methods.

Promising low-toxicity of viologen-phosphorus dendrimers against embryonic mouse hippocampal cells.

A new class of viologen-phosphorus dendrimers (VPDs) has been recently shown to possess the ability to inhibit neurodegenerative processes in vitro. Nevertheless, in the Central Nervous Systems domain, there is little information on their impact on cell functions, especially on neuronal cells. In this work, we examined the influence of two VPD (VPD1 and VPD3) of zero generation (G0) on murine hippocampal cell line (named mHippoE-18). Extended analyses of cell responses to these nanomolecules comprised cytotoxicity test, reactive oxygen species (ROS) generation studies, mitochondrial membrane potential (ΔΨm) assay, cell death detection, cell morphology assessment, cell cycle studies, as well as measurements of catalase (CAT) activity and glutathione (GSH) level. The results indicate that VPD1 is more toxic than VPD3. However, these two tested dendrimers did not cause a strong cellular response, and induced a low level of apoptosis. Interestingly, VPD1 and VPD3 treatment led to a small decline in ROS level compared to untreated cells, which correlated with slightly increased catalase activity. This result indicates that the VPDs can indirectly lower the level of ROS in cells. Summarising, low-cytotoxicity on mHippoE-18 cells together with their ability to quench ROS, make the VPDs very promising nanodevices for future applications in the biomedical field as nanocarriers and/or drugs per se.

Mechanism of cationic phosphorus dendrimer toxicity against murine neural cell lines.

The purpose of this manuscript is to study the toxic responses against murine embryonic hippocampal cells (mHippoE-18) and neuroblastoma cells (N2a) to treatment with cationic phosphorus dendrimers (CPD). Two low generations of CPD--generation 2 (G2) and generation 3 (G3)--were applied to cell cultures to monitor events leading to either apoptosis or necrosis. These processes were analyzed using several bioassays, which included the detection of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) alterations, morphology changes, apoptotic and dead cells, cytochrome c (Cyt c) release, caspase 3 activity, DNA fragmentation, as well as changes in cell cycle phases distribution. The results showed that CPD became highly cytotoxic at concentrations above 1 µM and at 0.7 µM in the case of G3 for mHippoE-18 cells. The toxicity was manifested by a pronounced decrease in cell viability, which is correlated with disturbances in cellular activities, such as massive ROS generation. The breakdown of cellular processes leads mainly to the necrotic cell death. Our findings are of high importance in the context of further biomedical studies on CPD.

Viologen-phosphorus dendrimers exhibit minor toxicity against a murine neuroblastoma cell line.

Dendrimers containing viologen (derivatives of 4,4'-bipyridyl) units in their structure have been demonstrated to exhibit antiviral activity against human immunodeficiency virus (HIV-1). It has also recently been revealed that novel dendrimers with both viologen units and phosphorus groups in their structure show different antimicrobial, cytotoxic and hemotoxic properties, and have the ability to influence the activity of cholinesterases and to inhibit α-synuclein fibrillation. Since the influence of viologen-phosphorus structures on basic cellular processes had not been investigated, we examined the impact of such macromolecules on the murine neuroblastoma cell line (N2a). We selected three water-soluble viologen-phosphorus (VPD) dendrimers, which differ in their core structure, number of viologen units and number and type of surface groups, and analyzed several aspects of the cellular response. These included cell viability, generation of reactive oxygen species (ROS), alterations in mitochondrial activity, morphological modifications, and the induction of apoptosis and necrosis. The MTT assay results suggest that all of the tested dendrimers are only slightly cytotoxic. Although some changes in ROS formation and mitochondrial function were detected, the three compounds did not induce apoptosis or necrosis. In light of these results, we can assume that the tested VPD are relatively safe for mouse neuroblastoma cells. Although more research on their safety is needed, VPD seem to be promising nanoparticles for further biomedical investigation.

Interaction between PAMAM-NH2 G4 dendrimer and 5-fluorouracil in aqueous solution.

The formation equilibrium of poly(amidoamine) dendrimer (PAMAM-NH2 G4) complex with an oncologic drug such as 5-fluorouracil (5-FU) in water at room temperature was examined. Using the results of the drug solubility in dendrimer solutions and the method of equilibrium dialysis, the maximal number of drug molecules in the dendrimer-drug complex and its equilibrium constant were evaluated. Solubility results show that PAMAM-NH2 G4 dendrimer can transfer tens 5-fluorouracil molecules in aqueous solution. The number of active sites in a dendrimer macromolecule being capable of combining the drug, determined by the separation method, amounts to n=30 ± 4. The calculated equilibrium constant of the 5-FU-active site bonding is equal to K=(400 ± 120).

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax.

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe3+ ions. Erythrocytes were exposed to various concentrations of CuSO4, Fe(NO3)3, and K3Fe(CN)6 for up to 5h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO3)3. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO4 increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 microM. This effect was decreased by potassium ferricyanide and Fe(NO3)3 only after 1 and 3h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 microM CuSO4 as well as 20 microM Fe(NO3)3 and 10 mM K3Fe(CN)6 induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K3Fe(CN)6. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO3)3.

Effect of genistein-8-C-glucoside from Lupinus luteus on DNA damage assessed using the comet assay in vitro.

Genistein-8-C-glucoside (G8CG) belongs to natural isoflavones phytoestrogens, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants, with possible anticarcinogenic effects in various in vitro systems and in vivo animal models. We used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.) to study its cytotoxic and genotoxic effects on mouse embryonic fibroblast (line NIH 3T3). The MTT assay to assess cytotoxicity and comet assay for the detection of DNA damage were used. The cells were exposed to various concentrations of genistein-8-C-glucoside (2.5-110 microM) and hydrogen peroxide (5-90 microM). The effect of G8CG alone or in combination with H2O2 was determined. G8CG at concentrations > 20 microM significantly reduced cell viability and induced DNA damage. In contrast, lower concentrations of (2.5-10 microM) G8CG showed antioxidant properties against H2O2-induced DNA damage with no associated toxicity.

Cytotoxicity of the isoflavone genistein in NIH 3T3 cells.

Genistein is one of the naturally occurring isoflavones present in plants such as soybeans and is commonly found in a variety of human foods. A number of studies indicated that this class of compounds exerts anticancerogenic and antimutagenic effects in various in vitro systems and in vivo animal models. We studied the effects of genistein on NIH 3T3 cells in in vitro models. The isoflavone genistein has been identified as having antiproliferative and apoptotic effects on various malignant cell types derived from solid tumors. Therefore, the cytotoxic and apoptotic properties of this compound were studied by MTT assay and Hoechst 33258/propidium iodide staining technique. The morphological changes of cells were examined in inverted fluorescent microscope. The oxidation of protein thiol groups and thiobarbituric-acid-reactive species (TBARS) was also determined. The cells were exposed to different concentrations of genistein (0-90 microM) after 24 h of incubation. The results revealed that genistein in concentrations higher than 20 microM significantly reduced cell viability, caused cell morphological changes and induced apoptotic and necrotic cell death. Oxidative modification of protein increased in the cells exposed to genistein in a dose- and time-dependent manner. In conclusion, our preliminary in vitro studies demonstrate the damaging effects of genistein on the mouse embryonic fibroblast cell line.

Effect of the phytoestrogen, genistein-8-C-glucoside, on Chinese hamster ovary cells in vitro.

Genistein-8-C-glucoside (G8CG) belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. A number of studies on flavonoids show their cardioprotective and antiosteoporosis properties in in vitro and in vivo models. As a phytoestrogen, genistein has recently generated interest as a potential anticancer and antiatherogenic agent. Several flavonoids are known as antioxidants and scavengers of free oxygen radicals. In the current investigation we used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.). Many authors have found that the action of genistein is not so simple, although many reports conducted in vitro have demonstrated that it is cytotoxic and genotoxic. Therefore, the cytotoxic and genotoxic effects of this compound in Chinese hamster ovary cells (line CHO) were studied. A colorimetric MTT assay to assess cytotoxicity and a Comet assay for the detection of DNA damage were used. Apoptosis was determined by the Hoechst 33258/propidium iodide staining technique. We have also demonstrated antioxidant properties of G8CG. The level of reactive oxygen species generated by G8CG alone and/or H2O2 was evaluated with fluorescence probes: dichlorofluorescein-diacetate (DCFDA) by flow cytometry. The cells were exposed to various concentrations of genistein-8-C-glucoside (1-290 microM) and hydrogen peroxide (10-130 microM) and the effect of G8CG alone or in combination with H2O2 was determined. The results reveal that G8CG at concentrations higher than 10 microM significantly reduced cell viability, induced apoptosis and DNA damage. However at lower concentrations (5 and 7.5 microM), G8CG showed antioxidant properties, but had no cytotoxic or genotoxic activity.

Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo.

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 microM) for 24 and 48 h and their antioxidant and pro-oxidant effects were determined. The number of SH-groups was quantified spectrophotometrically using Ellman's reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose- and time-dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double-strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH-DA (dichlorofluorescein-diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 microM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 microM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water-soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).

Antioxidative and oxidative changes in the digestive gland cells of freshwater mussels Unio tumidus caused by selected phenolic compounds in the presence of H(2)O(2) or Cu(2+) ions.

Research on biomarkers as early bioindicators of perturbation in populations and individuals has received increasing interest during recent decades. These ecotoxicity studies allow us to measure the impact of environmental stressors and to monitor and evaluate the degradation or restoration of systems. In the present study we used bivalve molluscs (mussels), which are sensitive biomarkers of aquatic ecosystem pollution, to assess the effects of three polyphenols: tannic acid, ellagic acid and gallic acid. These compounds were used in the 1-60 microM concentration range, alone and in the presence of H(2)O(2) (40 and 100 microM) or Cu(2+) ions (50 microM). The fluorescence probe dichlorofluorescein-diacetate (DCFH-DA) was used to measure reactive oxygen species (ROS). The oxidation of DCFH-DA to the fluorescent DCF (dichlorofluorescein) by the phenolic compounds was investigated spectrofluorimetrically. The results showed that the polyphenols tested can act as antioxidants against the ROS present in the digestive gland cells, but their activity is decreased after incubation with hydrogen peroxide or copper ions. SH-groups were determined spectrophotometrically using Ellman's reagent. The results showed that oxidative modification of proteins increased in a concentration-dependent manner in cells incubated with polyphenols (above 15 microM) alone. Incubation of the cells with phenolic acids and H(2)O(2) or Cu(2+) ions revealed that the phenolic acids had prooxidant properties in all concentrations used except for 1 microM tannic and ellagic acid and 40 microM H(2)O(2). DNA fragmentation was estimated by a fluorescence method using Hoechst 33258/propidium iodine binding. The data showed that the phenolic acids alone and in the presence of hydrogen peroxide or copper ions can induce apoptosis and necrosis. The methods used and results obtained indicate that the polyphenols selected can act not only as antioxidants but also as prooxidants in digestive gland cells of Unio tumidus.

Study of interactions between phenolic compounds and H2O2 or Cu(II) ions in B14 Chinese hamster cells.

The antioxidant and prooxidant effects of tannic, ellagic and gallic acids (1-60 microM) in the presence of hydrogen peroxide (40 and 100 microM) or copper ions (50 microM) on B14 Chinese hamster cells were examined. The fluorescence probe DCFH-DA (dichlorofluorescein-diacetate) was used to analyse the levels of reactive oxygen species. This method showed the reduction in oxidation of DCF (dichlorofluorescein). It indicates that antioxidant capacity of tested polyhenols is decreased in the presence of hydrogen peroxide or copper ions. Spectrophotometric assay with Ellman's reagent was used to determine SH-groups. The experimental results revealed the oxidative modification of proteins after exposure to polyphenols at concentrations above 15 microM. Additional incubation with H2O2 or Cu(2+) ions showed the prooxidant activity of tested complexes also for polyphenols used at a concentration of 1 microM. Fluorescence method with Hoechst 33258/propidium iodide dyes was used to study apoptotic and necrotic cell death. The obtained data demonstrated that polyphenols alone, as well as in the presence of hydrogen peroxide or copper ions, can induce DNA fragmentation.

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.

Interactions of tannic acid and its derivatives (ellagic and gallic acid) with calf thymus DNA and bovine serum albumin using spectroscopic method.

In the present investigation, an attempt has been made to study the interaction of chosen polyphenols (tannic, ellagic and gallic acids) with calf thymus DNA and bovine serum albumin (BSA) employing spectrofluorimetric technique. The fluorescence quenching of DNA-bound ethidium bromide (EB) and BSA-bound 1-anilinonaphthalene-8-sulfonic acid (ANS) by phenolic acids has been examined. As BSA contains two tryptophan residues, the polyphenols influence on protein by measuring the changes in the fluorescence of BSA in the presence of phenolic acids was also evaluated. Our experiments prove that there is a direct interaction between phenols and DNA or BSA. The obtained data suggest that used acids can intercalate to DNA and interact strongly with BSA. The strongest interactions were observed between DNA and ellagic acid and between BSA and tannic acid. The conformational changes were revealed in DNA and BSA after incubation with tested phenolic acids and the extent depended on the phenol structure and the used concentration.

Na+K+-ATPase activity as a biomarker of toxaphene toxicity in Unio tumidus.

In this study, the effect of toxaphene (camphechlor) on ATPase activity in the microsomal fraction of the Unio tumidus's digestive gland was determined. Toxaphene is a man-made mixture consisting of polychlorinated monoterpens, predominantly bornanes. This compound was primarily used as an insecticide, but in 1982 was officially banned because of its destructive effects on human and animal health. Toxaphene can be transported in the air at long distances and can persist in air, soil and water for years revealing acute and chronic toxicity towards aquatic organisms and wildlife, the increasing risk of cancer in both humans and animals. The microsomal fraction isolated from digestive glands was exposed to 1 x 10(-3) M, 1 x 10(-5) M and 1 x 10(-7) M of toxaphene. The obtained data showed that toxaphene induced a loss of ATPase activity in all used concentrations. The Lineweaver-Burk plots for microsomal Na+K+-ATPase in the presence or the absence of toxaphene as an inhibitor indicated a competitive type of inhibition.

Measurement of DNA damage and protein oxidation after the incubation of B14 Chinese hamster cells with chosen polyphenols.

The spectrophotometric method with 2,4-dinitrophenylhydrazine (DNPH) and the alkaline single-cell gel electrophoresis (comet assay) were used to assess the possibility of three polyphenolic compounds (tannic, ellagic and gallic acid) to induce the changes in proteins and DNA. B14 Chinese hamster cells were exposed to various concentrations of used polyphenols (1-60 microM), hydrogen peroxide (10 and 40 microM) copper ions (50 microM), and the effect of phenols alone and in combination with Cu2+ ions and H2O2 was determined. The results reveal that polyphenolic acids in higher concentrations than 1 microM contribute to the changes in the tested molecules and cause protein and DNA damage. The combined treatment of polyphenols with Cu2+ ions and with H2O2 demonstrates that the lowest dose of phenols (1 microM) can act as an antioxidant agent against used chemicals. The DNA repair process was also studied. The data obtained in this experiment demonstrate that the most effective DNA repair occurs 1h after the removal of phenolic compounds. The used methods and obtained results provide additional information about the potential use of these phenols not only as antioxidants but also as pro-oxidants in biological systems.

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.