Resveratrol affects in a different way primary versus fixed DNA damage induced by H2O2 in mammalian cells in vitro

Toxicology Letters 135 (2002) 1 – 9 www.elsevier.com/locate/toxlet

Resveratrol affects in a different way primary versus fixed DNA damage induced by H2O2 in mammalian cells in vitro Rosella De Salvia a, Fabiola Festa b, Ruggero Ricordy a, Paolo Perticone a, Renata Cozzi b,* a

Centro di Genetica E6oluzionistica del CNR c/o Dip. Genetica e Biologia Molecolare, Uni6ersita` Roma ‘La Sapienza’, Rome, Italy b Dipartimento di Biologia, Uni6ersita` degli Studi ‘Roma Tre’, Viale Marconi 446, 00146 Rome, Italy Received 20 February 2002; received in revised form 25 April 2002; accepted 25 April 2002

Abstract Resveratrol (3,5,4%-trihydroxystilbene) is a natural occurring molecule, synthesized by plants in response to different stresses. Recent literature data seem to converge in indicating Resveratrol as an agent possessing protective effects against oxidative stresses through different mechanisms. Furthermore conflicting data are present in relation to its activity of free radical scavenger. Here we studied the antioxidant activity actually exerted by the agent against reactive oxygen species induced by H2O2 treatments in CHO cells. Our attention has been focused on two major potential mechanisms: scavenging activity and interference with oxidative metabolism, by the analysis of three important targets: intracellular oxidation (Dichlorofluorescein Test), primary DNA damage (Comet Assay) and fixed DNA damage (chromosomal aberrations). Cells were treated with a single H2O2 dose (2 × 10 − 4 M) in order to induce Reactive Oxygen Species and than challenged with Resveratrol to test its ability in modulating damage. Two experimental protocols have been applied: (i) simultaneous treatment and (ii) a 3 h Resveratrol pre-treatment. In our experimental conditions Resveratrol does not appear able, ‘per se’, to induce primary DNA damage whereas a slight increase in endogenous oxidation and chromosomal aberrations at the highest dose have to be noticed. In combined treatments the molecule appears to differently affect primary and fixed DNA damage. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Resveratrol; Oxidative stress; DNA damage; Comet assay; Chromosomal aberrations; DCFH

1. Introduction Several classes of agents active as antimutagens and anticarcinogens have been found in many

* Corresponding author. Tel.: + 39-06-551763330; fax: + 39-06-55176321 E-mail address: [email protected] (R. Cozzi).

foods and beverages of natural origin (for a review, see Weisburger, 2001). Recently a wide variety of polyphenolic compounds [flavonoids (anthocyanins, catechins) and non-flavonoids (stilbenes)], found mainly in vegetables and fruits (overall grapes and their derivatives), attracted attention as promising antitumor agents (Fauconnneau et al., 1997; Frankel et al., 1993a). Between them Resveratrol (3,4%,5,-trihydroxystil-

0378-4274/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-4274(02)00151-0

2

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

bene) present in a significant amount both in usual mediterranean consumption (red wine, grapes, peanuts, green vegetables and other edible spermatophytes) (Fremont, 2000), and in many oriental herbal beverages (green tea) and medicines (Casper et al., 1999), has recently focused attention for its high health protective activity. Early interest on the agent started from the observation that a reduced risk of heart disease through inhibition of cholesterol lipoproteins oxidation was associated with moderate wine consumption (Gaziano et al., 1993; Frankel et al., 1993b); moreover data on inhibition of all stages of tumor induction greatly increased researches on this field (Clifford et al., 1996; Jang et al., 1997). Recently attention has been focused on the mechanisms possibly underlying these protective activities, producing a rich field of researches, as revised by Fremont (Fremont, 2000). More in detail, literature data seem to converge in indicating Resveratrol as an agent possessing protective effects against oxidative stresses. Different hypotheses have been suggested in order to elucidate the mechanisms involved in this effect: inhibition of lipid peroxidation (Frankel et al., 1993b; Chanvitayapongs and Sun, 1997; Tadolini et al., 2000) and of cellular production of reactive oxygen species (Sgambato et al., 2000; Jan and Surh, 2001), interference with the metabolic pathway(s) of reactive oxidants (Jang and Pezzuto 1998; Olas et al., 2001). Conflicting data are present in relation to its activity of free radical scavenging, dealing with the ability to scavenge hydroxyl radical (Burkhardt et al., 2001) or stable free radicals (Fauconnneau et al., 1997), but also to increase oxidative DNA strand breaks through the induction of copper-peroxide complexes (Fukuhara and Miyata, 1998; Hadi et al., 2000; Win et al., 2002). The aim of our work was to study the antioxidant activity eventually exerted by Resveratrol against reactive oxygen species (ROS) induced by H2O2 treatment, in Chinese hamster ovary (CHO) cells. We focused our attention on two major potential mechanisms: (a) scavenging activity and interference with oxidative metabolism (b) pro-oxidant effect. The following three targets were analysed: (i) intracellular oxidation (cytofluorimetric analysis of cellular fluorescence), (ii) pri-

mary DNA damage (single cell gel electrophoresis), (iii) fixed DNA damage (chromosomal aberrations). 2. Materials and methods CHO cells are routinely cultivated in our laboratory in Ham F-10 medium (Euroclone) supplemented with 10% foetal calf serum (FCS), 1% L-glutamine, 2% penicillin (5000 IU/ml) and streptomycin (5000 mg). Under these conditions, the average cell cycle lasts 12 h. In all experiments, cells were seeded at a density of 106/5 ml flask. After 4 h, cells were treated according to the various protocols.

2.1. Chemicals Resveratrol (3,4%,5 trihydroxy-stilbene) (Sigma), was dissolved in DMSO to obtain a 100 mM solution, immediately before use and added to the cultures according to the different schedules, at concentrations ranging from 5× 10 − 5 to 2× 10 − 4 M (see figures) The agent was supplied to cells in the dark and washed out by a twice wash in PBS. Hydrogen peroxide (H2O2) was dissolved in NaCl (0.9%) from a 30% stock solution and added to the cultures at the final concentration of 10 − 4 and 2× 10 − 4 M. 2%-7%-Dichlofluorescein diacetate (DCFH) (Kodak) was dissolved in ethanol and added to the cultures at the final concentration of 5×10 − 6 M.

2.2. Experimental schedules Two protocols have been used. In the protocol ‘A’ (simultaneous treatments), the cells were challenged with both Resveratrol and H2O2 at the same time for 30 min; than the cells were twice washed and processed following the specific endpoint In the protocol ‘B’ (Resveratrol pretreatment) the cells were challenged with Resveratrol for 3 h, twice washed and than challenged with H2O2 for 30 min in NaCl (to optimize the H2O2 effect); after a new wash, the cells were processed. For details on single test procedures, please see below.

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

2.3. Dichlorofluorescein test Cells were cultured for 15 min in PBS lacking Ca2 + and Mg2 + (PBS = ) containing DCFH (2,7Dichlorofluorescein diacetate KODAK). The medium was then removed and the cells seeded in NaCl (0.9%). Both protocol treatments were performed in NaCl: (i) protocol ‘A’: both agents supplied together for 30 min, than cells were washed twice; (ii) protocol ‘B’: Resveratrol was supplied for a 3 h pulse in normal medium. After a three times wash, H2O2 was added in NaCl to the cells for 30 min, than cells were washed twice again. The cells were then trypsinized and analysed in a FACSTAR cytometer (Becton Dickinson) equipped with a 5 W argon laser (coherent, 488 nm emission). Ten thousand cells were analysed for each sample and the experiment was repeated three times. The data are expressed in fluorescence arbitrary units9Standard Deviation (S.D.).

2.4. Single cell gel electrophoresis (comet assay) The assay was performed basically according to Klaude et al. (1996) with some modifications. In both protocols, immediately after the end of the (last) treatment, cells were collected and processed for the assay, as follow. Briefly, 20 ml of cell suspension (10000– 20000 cells approximately) were mixed with 180 ml of 0.7% low melting agarose in PBS = at 37 °C and immediately pipetted onto a frosted glass microscope slide precoated with a layer of 1% normal melting point agarose, similarly prepared in PBS. The agarose was allowed to set at 4 °C for the necessary time. Slides were then incubated in lysis solution (2,5 M NaCl, 10 mM Tris– HCl, 100 mM EDTA, pH 10, with 1% Triton and 10% DMSO added fresh) for 50 min. After lysis, slides were placed on a horizontal electrophoresis unit containing fresh buffer (1 mM EDTA, 300 mM NaOH pH 13) and incubated for 20 min to allow unwinding of DNA. Electrophoresis was then conducted in fresh electrophoresis buffer (pH 13) for 15 min at 25 V and 300 mA (0.8 V/cm) at 4 °C. Subsequently, the slides were gently washed in neutralization solu-

3

tion (0.4 M Tris–HCl, pH 7,5) for 5 min and fixed in 100% fresh methanol for 3 min. Slides were stained with 90 ml ethidium bromide (20 mg/ml) and covered with a coverslip. Stained nucleoids were scored visually using a fluorescence microscope (Leica) equipped with a camera COHU. Two slides were analysed for each experimental point and 50 comets on each slide were acquired using the ‘IAS’ software automatic image analysis system purchased from Delta Sistemi (Rome, Italy). The comet images were digitized and Tail Length was calculated and expressed in mm 9 S.D.

2.5. Chromosomal aberrations After H2O2 and/or Resveratrol treatments the cells were incubated in fresh medium for further 18 h. Colchicine (5× 10 − 7 M) was added 2 h before the cells were fixed. Chromosome preparation were obtained by standard methods, which includes 13 min hypotonic (tri-sodium citrate 1%) treatment followed by fixation with methanol: acetic acid (3:1). Chromosomal breakage was evaluated after Giemsa staining. Slides were coded and at least 100 metaphases were analysed for each experimental point. Gaps were not included in the total frequency of aberrations. The mitotic index (MI) was defined by the percentage of metaphases over a total of 1000 nuclei analysed at random.

2.6. Statistical analysis Means and standard errors were determined for each experimental point in all the repeated experiments. Control and treated cultures were compared by Student’s t-test. The DCFH assay data were analysed with Winmdi. The computer analysis was performed by determining the channel of green fluorescence for each of the 10000 cells analysed. The means9 S.D. of green fluorescence intensity were calculated to analyse the significance of the fluorescence increase of the positive fraction of cells induced by H2O2 or Resveratrol and to compare the differences between single and double treatments (H2O2 plus Resveratrol).

4

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

The statistical analysis of the Comet assay values was carried out using t-test to compare the mean value9 S.D. of treated cells versus control ones. All the experiments were repeated three to four times and the data presented are the mean of all experiments.

3. Results

3.1. Background Under present culture conditions (please see M. & M.), cell cycle parameters (data not shown) clearly indicate a cell population in logarithmic growth (G1 = 47.3%, S =46.9% and G2 =5.8%) in the time interval starting 4 h after cell seeding up to 48 h, when cells decrease their growth rate due to approaching confluent conditions. In all treatments, logarithmic growing cells were treated and thus examinated in order to avoid the collection of incorrect data due to cells suffering confluent conditions. Data are shown in two series of figures and tables: A and B, in relation to the experimental schedules as described in Section 2.

3.2. DCFH test (intracellular oxidation) (Fig. 1A and B) In our experimental conditions the control value of DCF mean fluorescence is 1.8 in agreement with our previous data (Cozzi et al., 1997; De Salvia et al., 1999), indicating CHO has a normal level of metabolic oxidation pathway. Dichlorodihydrofluorescin (DCF) content, measured as fluorescence intensity in arbitrary units, appears to be significantly affected by the two highest doses (10 − 4 and 2 ×10 − 4 M) of Resveratrol in both protocols (P B0.05). The H2O2 used dose (2× 10 − 4 M), increases this oxidation parameter about ten times, as expected. A combined simultaneous treatment of both agents (H2O2 +Resvertrol) (Fig. 1A) appears to significantly (P0.001) reduce fluorescence intensity at the Resveratrol doses of 10 − 4 and 2 ×10 − 4 M. On the contrary, a previous 3 h-treatment of cells

with Resveratrol (10 − 4 M) (Fig. 1B) appears to significantly (P 0.001) enhances the H2O2 induced fluorescence intensity.

3.3. Comet assay (primary damage) (Fig. 2A and B) Comet assay, measured as tail length in mm, indicates a value of about 13 as expected in the protocol applied. DNA primary damage measured by Comet assay, is expressed as tail length as measured in mm. Resveratrol ‘per se’ does not induce any detectable DNA damage in both experimental schedules, whereas H2O2 strongly induces an increase in tail length reaching the value of 40 mm that is a four times increase in comparison to the control levels. The combined simultaneous treatment (Fig. 2A) appears not to affect the damage induced by H2O2 and its derivative ROS. On the contrary a previous challenging of the cells with Resveratrol appears to reduce up to the control levels the H2O2 induced damage (Fig. 2B).

3.4. Chromosomal aberrations ( fixed damage, as seen 18 h after the treatments) (Table 1A and B) In the case of Chromosomal Aberrations, taking into account that CHO is an immortalized cell line, a small and constant baseline of damage is present of around 2.5%, mainly deriving from gaps and chromatid breaks. In our experimental conditions the percentage of abnormal cells in CHO cultures appears to be around 2.5. Three doses of Resveratrol have been used: 5× 10 − 5, 10 − 4 and 2× 10 − 4 M. The agent does not significantly increase the aberration rate at the lowest doses. At the highest one (2× 10 − 4 M) we obtained a clear increase in the percentage of abnormal cells (12%, see Table 1A). H2O2 at the used dose significantly increases total aberrations: up to 63 in respect to the control value of 1. When cell are treated with both agents, the two supply protocols appear to produce completely different results. In fact, when Resveratrol is supplied in combination with H2O2 (Table 1A), the total aberrations decrease in a dose related way reaching almost the control value at the highest dose;

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

the mitotic index appears not to be negatively affected at all. On the contrary, when Resveratrol is supplied before H2O2, both the percentage of abnormal cells and total aberrations far to show a decrease, appear to increase in a dose dependent

5

way: from 62 total aberrations in H2O2 alone treatment to 124–155 when cells are pretreated with Resveratrol. The mitotic index remains heavily affected irrespective of the different Resveratrol doses employed (Table 1B).

Fig. 1. (a) Effect of Resveratrol (Resv) and H2O2 simultaneous treatment on DCF fluorescence intensity (arbitrary units) in CHO cells. Data are the mean of three separate experiments. Bars represent S.D. c =control; ° = PB 0.005 comparing combined treatments vs. H2O2 alone. ** = P 0.001 comparing Resveratrol alone vs. control. (b) Effect of 3 h Resveratrol (Resv) pretreatment on DCF fluorescence intensity (arbitrary units) induced by H2O2 treatment in CHO cells. Data are the mean of three separate experiments. Bars represent S.D. ° = PB0.005 comparing Resveratrol alone vs. control. ** =P 0.001 comparing combined treatment vs. H2O2 alone.

6

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

Fig. 2. (a) Comet tail length (expressed in mm) induced by Resveratrol (Resv) and H2O2 simultaneous treatment on CHO cells: averaged values based on three pooled experiments. Bars represent S.D. c =Control. (b) Effect of 3 h Resveratrol (Resv.) pretreatment on DNA damage induced by H2O2 expressed as comet tail length (mm). The data are the mean of three pooled experiments. Bars represent S.D. c =control.

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

4. Discussion

4.1. Intracellular oxidation Resveratrol has been applied in both experimental protocols in three doses. This agent alone appears to induce ‘per se’ a slight increase in endogenous oxidation as revealed by DCFH test. This result is not in agreement with literature data (Sgambato et al., 2000; Jan and Surh, 2001) where Resveratrol treatment does not increase the intracellular oxidation, probably due to differences in treatments. In fact in our conditions, the analysis of cellular oxidation is performed after a shorter Resveratrol treatment (only 3 h in respect to 48 h), allowing the immediate detection of highly reactive ROS. Combined treatments produce data apparently contradictory in the sense that while simultaneous supply of both Resveratrol and H2O2 produces a slight but significant reduction in induced endogenous oxidation, a pretreatment by Resveratrol surprisingly appears to enhance the

7

H2O2 oxidation damage. Recent in vitro plasmid data (Fukuhara and Miyata, 1998; Hadi et al., 2000) suggested that Resveratrol is capable of generating a copper-peroxide complex mainly in the presence of H2O2 (Win et al., 2002); in our experimental conditions where the DCFH measures reactive oxygen species and H2O2, a Resveratrol pretreatment could be able to affect DCFH values because a copper-peroxide complex may be considered as one of the actual oxygen species measured by the test. Therefore in the protocol where Resveratrol and H2O2 are simultaneously present in the cultures, the decrease of the DCF oxidation could be ascribed to a balance between ‘scavenging’ and ‘pro-oxidant’ ( copper-peroxide complex generation) Resveratrol activity.

4.2. Primary DNA damage The alkaline Comet assay is a well known test able to measure single strand breaks immediately

Table 1 Treatment

Ab. cells excl. gaps (%)

Gaps

Breaks

B%

Chr. tid Exchs.

Chr. me Exchs.

Total CA excl. gaps

MI

B¦

(A) Effect of res6eratrol (Res6) on H2O2 -induced chromosomal aberrations in CHO cells (simultaneous treatment) Control 2 1 1 0 0 0 1 Resv1 (5×10−5 M) 3 1 2 0 0 0 2 Resv2 (10−4 M) 4 2 0 4 0 0 4 Resv3 (2×10−4 M) 12 2 4 3 1 0 8 H2O2 (2×10−4 M) 22 0 15 6 38 4 63 H2O2+Resv1 18 1 3 8 6 6 23 H2O2+Resv2 11 2 1 4 1 1 11 H2O2+Resv3 9 2 0 2 4 4 6

1:00 1:00 0:57 0:50 1:13 1:52 1:36 1:31

(B) Effect of 3 h res6eratrol (Res6) pre-treatment om H2O2 -induced chromosomal aberrations in CHO cells Control 3 2 2 0 0 0 2 Resv1 (5×10−5 M) 4 3 2 0 0 0 2 Resv2 (10−4 M) 4 2 3 1 0 0 4 Resv3 (2×10−4 M) 5 4 3 2 0 0 5 -, H2O2 (2×10−4 M) 40 3 3 20 19 20 62 Resv1, H2O2 55 1 10 30 58 26 124 Resv2, H2O2 57 1 12 33 65 45 155 Resv3, H2O2 62 0 6 39 41 45 131

1.00 0.69 0.67 0.65 0.50 0.33 0.31 0.34

Chromosomal aberrations per 100 cells (fixing time = 18 h)% Ab. Cells excl. gaps =% aberrant cells excluded gaps B%, B¦= chromatid breaks, chromosome breaks Chr.tid Exchs. = chromatid exchanges Chr.mE Exchs. = chromosome exchanges Total Ca excl. gaps = total chromosome aberrations excluded gaps M.I. =normalized mitotic index values. Data are the mean of four separate experiments.

8

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

after their induction in single viable cells with a clear and reproducible dose-effect relationship (for a review see Tice et al., 2000). Furthermore, we have previously shown that this system is particularly suitable in studying antioxidant activity of natural molecules (Festa et al., 2001) As a consequence of the test characteristics, H2O2 treatment strongly induces an increases in tail length. Resveratrol, on the contrary, does not affect comet parameter both alone and in simultaneous treatments with hydrogen peroxide. These results must not be considered as unexpected even if previous literature data (Hadi et al., 2000; Win et al., 2002) suggested Resveratrol ability to generate a copper-peroxide complex which increases DNA strand breaks. In fact, these data were obtained in a plasmid-based DNA cleavage assay. In addition, the time lapse between the complex generation and the effective production of breaks could last more than the time interval between the end of the treatment and the start of the comet assay. On the other hand Resveratrol appears not to be able to exert its scavenging action against H2O2 and induced ROS in simultaneous treatment, probably due to the high reactivity of these species which reach DNA molecule before Resveratrol could act. In the protocol where Resveratrol is supplied to the cells for a 3 h pulse, before H2O2, the hydrogen peroxide damage is completely eliminated, showing a tail length similar to the control ones. This apparent need of a significant lapse of time before the appearance of a Resveratrol protective effect, is partially supported by data discussed in the next section where a protective effect is also present in the protocol A (please see below).

4.3. Fixed DNA damage Classical cytogenetic tests such as sister chromatid exchanges (SCE) and chromosomal aberrations (CA) are valuable approaches to measure fixed damage. In the literature Resveratrol has been recently challenged by both tests with similar results (Matsuoka et al., 2001): in fact both parameters appear to be affected in a dose-dependent way. In our conditions a similar but slower effect by Resveratrol alone is confirmed as well as the high DNA damaging effect of H2O2. On the contrary,

in combined treatments, whereas the protocol A (both agents supplied in the same time) appears to induce a dose dependent protective effect of Resveratrol on H2O2 induced damage, the B protocol (Resveratrol supplied for 3 h before H2O2) seems to indicate an enhancing effect both considering the total damage and the percentage of abnormal cells. In both protocols the Mitotic Index appears to be consistent with these data, showing in the first case an increase in cell division, and in the second one a dramatical decrease. In an attempt to explain these results, at present we can tentatively suggest that a 3 h pretreatment of Resveratrol induces strand sensibilization/break of DNA molecule due to copper peroxide complexes, which can evolve in chromosomal damage after a consistent lapse of time. On the other hand the agent, affecting cell cycle progression by a lowering of the S phase (Sgambato et al., 2000; and our data not shown), could allow later the appearance of more damaged cells. 5. Conclusions Resveratrol, in our experimental conditions, seems to have both an antioxidant and a pro-oxidant activity. This behaviour is substantially consequent on the type of treatment performed and is common to many natural antioxidants (for example Ascorbic acid). On the other hand the ability of Resveratrol to induce ‘per se’ oxidant complexes is well documented. We can conclude that the mechanism of Resveratrol action appears to be very complex and elusive, depending from doses applied, supply conditions, test systems employed and end-points observed. Especially this last aspect needs to be carefully considered taking into account its capacity in modulating cell cycle and triggering more damaged cells to undergoing apoptosis (Clement et al., 1998; Holmes-McNary et al., 2000). This last activity could eliminate from cell population cells heavily damaged avoiding their detection. Acknowledgements This work was partially supported by a grant from Consiglio Nazionale delle Ricerche (CNR).

R. De Sal6ia et al. / Toxicology Letters 135 (2002) 1–9

References Burkhardt, S., Reiter, R.J., Tan, D.-X., Hardeland, R., Cabrera, J., Karbownik, M., 2001. DNA oxidatively damaged by chromium(III) and H2O2 is protected by the antioxidants melatonin, acetyl-formyl-5-methoxykynuramine, resveratrol and uric acid. Int. J. Biochem. Cell Biol. 33, 775 – 783. Casper, R.F., Quesne, M., Rogers, I.M., Shirota, T., Jolivet, A., Milgrom, E., Savouret, J.F., 1999. Resverstrol has anthagonist activity on the aryl hidrocarbon receptor: implications for prevention of dioxin toxicity. Mol. Pharmacol. 56, 784 – 790. Chanvitayapongs, S., Sun, A.Y., 1997. Amelioration of oxidative stress by antioxidant and resveratrol in PC12 cells. Neuroreport 8, 1499 –1502. Clement, M.V., Hirpara, J.L., Chawdhury, S.H., Pervaiz, S., 1998. Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells. Blood 92, 996 – 1002. Clifford, A.J., Ebeler, S.E., Ebeler, J.D., Bils, N.D., Hinrichs, S.H., Teissedre, P.L., Watherhouse, A.L., 1996. Delayed tumor onset in transgenic mice fed an aminoacid based diet supplemented with red wine solids. Am. J. Clin. Nutr. 64, 748 – 756. Cozzi, R., Ricordy, R., Aglitti, T., Gatta, V., Perticone, P., De Salvia, R., 1997. Ascoric acid and b-carotene as modulators of oxidative damage. Carcinogenesis 18 (1), 223 –228. De Salvia, R., Fiore, M., Aglitti, T., Festa, F., Ricordy, R., Cozzi, R., 1999. Inhibitory action of melatonin on H2O2and cyclophosphamide-induced DNA damage. Mutagenesis 14 (1), 107 – 112. Fauconnneau, B., Waffo-Teguo, P., Huguet, F., Barrier, L., Decendit, A., Merillon, J.M., 1997. Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis 6inifera cell cultures using in vitro tests. Life Sci. 61, 2103 –2110. Festa, F., Aglitti, T., Duranti, G., Ricordy, R., Perticone, P., Cozzi, R., 2001. Strong antioxidant activity of Ellagic acid in mammalian cells in vitro revealed by the Comet assay. Anticancer Res. 21 (6), 3903 –3908. Frankel, E.N., Kanner, J., German, J.B., Parks, E., Kinsella, J.E., 1993a. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 341, 454– 457. Frankel, E.N., Waterhouse, A.L., Kinsella, J.E., 1993b. Inhibition of human LDL oxidation by resveratrol. Lancet 341, 1103 – 1104. Fremont, L., 2000. Biological effects of resveratrol. Life Sci. 66, 663 – 673. Fukuhara, K., Miyata, N., 1998. Resveratrol as a new type of DNA-cleaving agent. Bioorg. Med. Chem. Lett. 8, 3187 – 3193.

9

Gaziano, J.M., Buring, J.E., Breslow, J.L., Goldhaber, S.Z., Rosner, B., Van Denburgh, M., Willett, W., Hennekens, C.H., 1993. Moderate alchool intake, increased levels of low density lipoprotein and its subfractions and decreased risk of miocardial infarction. New Engl. J. Med. 329, 1829 – 1834. Hadi, S.M., Asad, S.F., Singh, S., Ahmad, A., 2000. Putative mechanism for anticancer and apoptosis-inducing properties of plant-derived polyphenolic compounds. Life 50, 171 – 176. Holmes-McNary, M., Baldwin, A.S. Jr, 2000. Chemopreventive properties of trans-resveratrol are associated with inhibition of activation of the IkB kinase. Cancer Res. 60, 3477 – 3483. Jan, J.H., Surh, Y.J., 2001. Protective effects of resveratrol on hydrogen peroxide-induced apoptosis in rat pheocromocitoma (PC12) cells. Mutat. Res. 496, 181 – 190. Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beeker, C.W.W., Fong, H.H.S., Farnsworth, N.R., Kinghorn, A.D., Manta, R.G., Moon, R.C., Pezzuto, J.M., 1997. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275, 218 – 220. Jang, M., Pezzuto, J.M., 1998. Effects of resveratrol on 12-Otetradecanoylphorbol-13-acetate induced oxidative events and gene expression in mouse skin. Cancer Lett. 134, 81 – 89. Klaude, M., Eriksson, S., Nygren, J., Ahnstrom, G., 1996. The comet assay: menchanisms and thecnical considerations. Mutat. Res. 363, 89 – 96. Matsuoka, A., Furata, A., Ozaki, M., Fukuhara, K., Miyata, N., 2001. Resveratrol, a natural occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line. Mutat. Res. 494, 107 – 113. Olas, B., Wachowicz, B., Saluk-Juszczak, J., Zielinski, T., Kaca, W., Buczynski, A., 2001. Antioxidant activity of resveratrol in endotoxin-stimulated blood platelets. Cell Biol. Toxicol. 17, 117 – 125. Sgambato, A., Ardito, R., Faraglia, B., Boninsegna, A., Wolf, F.I., Cittadini, A., 2000. Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage. Mutat. Res. 496, 171 – 180. Tadolini, B., Juliano, C., Piu, L., Franconi, F., Cabrini, L., 2000. Resveratrol inhibition of lipid peroxidation. Free Radic. Res. 33, 105 – 114. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. Single cell gel/comet assay guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206 – 221. Weisburger, J.H., 2001. Antimutagenesis and anticarcinogenesis, from the past to the future. Mutat. Res. 480 – 481, 23 – 35. Win, W., Cao, Z., Peng, X., Trush, M.A., Li, Y., 2002. Different effects of genistein and Resveratrol on oxidative DNA damage in vitro. Mutat. Res. 513, 113 – 120.