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Hormonal and genotoxic activity of resveratrol
Toxicology Letters 136 (2002) 133 /142 www.elsevier.com/locate/toxlet
Hormonal and genotoxic activity of resveratrol E. Schmitt a, L. Lehmann b, M. Metzler b, H. Stopper a,* a b
Institute of Pharmacology and Toxicology, University of Wu¨rzburg, Versbacherstrasse 9, D-97078 Wurzburg, Germany Institute of Food Chemistry and Toxicology, University of Karlsruhe, Fritz Haber Weg 2, D-76131 Karlsruhe, Germany Received 23 April 2002; received in revised form 24 July 2002; accepted 24 July 2002
Abstract Resveratrol (RES) is a natural polyphenol present in red wines and various human food items. The estrogenic activity of RES was demonstrated in two in vitro assay systems, i.e. binding to human estrogen receptor a and stimulation of MCF-7 cell proliferation. To investigate the inhibition of cell proliferation observed at high concentrations of RES, we analyzed the compound for genotoxic potential. RES induced cellular toxicity, micronuclei, and metaphase chromosome displacement in L5178Y mouse lymphoma cells. Likewise, the induction of micronuclei was observed in Chinese hamster V79 cells. Determination of kinetochore signals in micronuclei and cell cycle analysis suggested that RES did not cause a direct disturbance of mitosis. In support of this notion, cell-free tubulin polymerization studies indicated no direct effect of RES on microtubule assembly. According to an estimation of daily intake and bioavailability, concentrations that were found genotoxic in vitro might be reached in human exposure. On the other side, the estrogenic acitivity might be beneficial. Therefore, further investigations of mechanisms, possibly including animal models, would be desirable to clarifiy a potential risk for humans. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phytoestrogens; Resveratrol; Estrogen receptor; Breast cancer cells; Genotoxicity; Micronuclei; Cell cycle
1. Introduction Resveratrol (RES, Fig. 1) is a natural polyphenol present in red wines and various human foods.
It is synthesized by a variety of plant species in response to injury, UV irradiation and fungal attack. Since it is only synthesized in the skin of the grape berries, red wine contains more RES
Abbreviations: BSA, bovine serum albumin; CREST, calcinosis, Raynaud’s phenomenon, oesophagal motility abnormalities, sclerodactyly and telangiectasia; CYP, cytochrome P450; DAPI, 4?,6?-diamidino-2-phenylindole; DES, diethylstilbestrol, E-3,4-bis(p hydroxyphenyl)-hex-3-ene; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; E2, 17b-estradiol; EDTA, ethylenediaminetetraacetate, sodium salt; EGTA, ethylene glycol-bis(2-aminoethylether)-tetraacetic acid; FBS, fetal bovine serum; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; MN, micronucleus; PBS, phosphate-buffered saline; PBS-CMF, phosphatebuffered saline free of calcium and magnesium; Pipes, piperazine-1,4-bis(2-ethanesulfonic acid); RPMI, Roswell Memorial Park Institute (RPMI 1640 cell culture medium); RES, resveratrol, 3,4?,5-trihydroxystilbene; Tris, tris(hydroxymethyl)aminomethane. * Corresponding author. Tel.: /49-931-201-48427; fax: /49-931-201-48446 E-mail address: [email protected] (H. Stopper). 0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 2 9 0 - 4
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Fig. 1. Chemical structures of RES (1), DES (2) and E2 (3).
than white wine. It has been suggested that RES may be the main active principle involved in the ‘French Paradox’, the inverse correlation between red wine consumption and the incidence of cardiovascular disease (Wu et al., 2001). RES has been shown to exhibit estrogenic activity (Gehm et al., 1997; Ashby et al., 1999; Bowers et al., 2000). The observation that RES is an effective radical-scavenger suggested that it acts as a natural antioxidant against oxidative DNAdamage (Cadenas and Barja, 1999; Lin and Tsai, 1999). In addition to these anti-initiating properties, RES exhibits anti-inflammatory effects. For example, it suppresses cyclooxgenase activity and the induction of the transcription factor NF kappa B by other agents (Bhat and Pezzuto, 2002). RES shows antimutagenic and anticarcinogenic activities. Benzo[a]pyrene-induced cancerogenesis could be reduced by RES by inhibition of CYP1A1 (El Attar and Virji, 1999; Ciolino and Yeh, 1999; Chun et al., 1999). RES also inhibited the 7, 12dimethylbenz[a]anthracene (DMBA)-induced cancerogenesis in a mouse-model (Jang et al., 1997). In another model RES reduced the intestinal tumorigenesis in genetically predisposed mice (Schneider et al., 2001). Furthermore, RES is a known inducer of apoptosis. Interestingly, the proapoptotic effect may be limited to tumor cells,
while normal cells remain unharmed (Lu et al., 2001). Therefore, great interest in RES as a cancer chemopreventive or even cancer therapeutic agent has evolved (Gusman et al., 2001; Savouret and Quesne, 2002). Opposite to this view, we demonstrate here that RES exhibits a genotoxic potential in vitro.
2. Materials and methods 2.1. Test substances RES (3,4?,5-Trihydroxy-trans -stilbene), 17b-estradiol (E2), and diethylstilbestrol (DES) of the highest available purity (E/98%) were obtained from Sigma Chemie GmbH (Deisenhofen, Germany). 2.2. Cell culture MCF-7, MDA-MB-231 and V79 cells were purchased from American Type Culture Collection (ATTC, Hanassas, USA). MCF-7 and MDAMB-231 cells were cultured in phenol red-free RPMI 1640 medium supplemented with antibiotics, L-glutamine (0.25 mg/ml), sodium pyruvate (107 mg/ml), human insulin (0.2 ng/ml), and 5%
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fetal bovine serum (FBS; all from Sigma Chemie GmbH). L5178Y tk / mouse lymphoma cells, clone 3.7.2c were routinely cultured in suspension in RPMI 1640 medium supplemented with antibiotics, L-glutamine (0.25 mg/ml), sodium pyruvate (107 mg/ml) and 10% heat-inactivated horse serum (all from Sigma Chemie GmbH). Chinese hamster V79 cells were cultured in DMEM pH 7.5 containing glucose (4500 mg/l) and supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ ml) and 10% FBS. All cells were grown in a humidified atmosphere with 5% CO2 in air at 37 8C.
2.3. Estrogen receptor binding assay 1.5 nM isolated human estrogen receptor a (PanVera, Madison, USA) were solved in binding buffer (1.5 mM EDTA, 10 mM Tris, 1 mM EGTA, 1mM NaVO4, 10% glycerol, 10 mg/ml gglobulin, 0.5 mM phenylmethylsulfonylfluoride, 0.2 mM leupeptin) and incubated with 6.5 nM [3H]-E2 (Amersham Biosciences, Freiburg, Germany) and increasing concentrations of unlabeled competitors (10 pM /100 mM). The mixtures were incubated at 4 8C for 18.5 h, and then 250 ml of 16% hydroxyapatite in binding buffer was added to each tube. The resulting pellet was washed twice with 0.9 ml binding buffer, then suspended in scintillation cocktail, and the radioactivity measured in a Packard 2250CA scintillation counter.
2.4. Cell proliferation analysis 80 000 /100 000 MCF-7 or MDA-MB-231 cells/ ml were seeded in 25 ml-culture flasks. Twentyfour hours later medium was exchanged. Fresh medium contained charcoal/dextrane-treated FBS (5%) and various concentrations of test compounds. At 96 h medium was exchanged again and cells were harvested at 144 h (MCF-7) and 120 h (MDA-MB-231). Cell numbers were determined by Coulter Counter analysis.
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2.5. In vitro micronucleus assay and kinetochore staining Exponentially growing mouse lymphoma L5178Y cells were treated for 4 h with the test substances dissolved in dimethylsulfoxide (DMSO). The maximum DMSO concentration during treatment was 1%. After removing the test compounds by medium replacement, the cells were incubated for another 20 h, then washed with PBS buffer, collected by centrifugation (5 min at 1000 rpm), and placed on glass slides by cytospin centrifugation. Fixation was performed with methanol at /20 8C for 1 h. For staining, the slides were washed with PBS buffer, incubated for 3 min with a solution of 62.5 mg/ml acridine orange in Soerensen buffer (67 mM Na2HPO4/KH2PO4, pH 6.8), and washed again with PBS buffer. Numbers of nuclei and micronuclei (MN) were scored using fluorescence microscopy. For each concentration of test compound, three slides with 1000 nuclei per slide were evaluated. Kinetochore staining in L5178Y cells was achieved by incubating the fixed cell preparations after rinsing with PBS-CMF/0.1% Tween 20 (v/v) with CREST serum for 60 min in a humidified chamber at 37 8C. After rinsing with PBS-CMF/0.5% Tween 20 (v/v), the cells were incubated for 30 min with FITC-conjugated goat anti-human antibody (diluted 1:100 in PBS), rinsed again with PBS-CMF/ 0.1% Tween 20 (v/v) and counterstained with bisbenzimide 33258 (1 mg/ml, 5 min). Slides were mounted for fluorescence microscopy using antifade solution (Oncor, Heidelberg, Germany). At least 100 MN were analyzed for the presence of kinetochore signals. V79 cells were grown on glass slides in quadriperm vials (25 000 /50 000 cells per slide) for 24 h prior to incubation with the test compound for 6 h. The cells were further incubated in fresh DMEM for various time periods up to 18 h, fixed for 5 min in 3.5% freshly depolymerized paraformaldehyde in CB buffer (137 mM NaCl, 5 mM KCl, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 2 mM MgCl2, 2 mM EGTA, 5 mM Pipes, 5.5 mM glucose, pH 6.1; Schultz and Onfelt, 2000) and postfixed with methanol at /20 8C for at least 1 h. Cells were then stained with anti-a-tubulin antibodies for the
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mouse antibody (diluted 1:100 in PBS) according to the procedure given above for kinetochore staining of L5178Y cells.
evaluation of cell morphology, with CREST antibodies for the presence of kinetochore signals, and with DAPI antifade solution for the scoring of nuclei and MN: slides after fixation were first washed three times with PBS-CMF pH 8.0 for 5 min each. Nonspecific binding was blocked by incubation with goat serum for 1 h at 37 8C. Slides were then incubated with 100 ml of a 1% aqueous BSA solution containing monoclonal mouse anti-a-tubulin antibody (Sigma, diluted 1:500) and goat polyvalent anti-human antibody (FITC-conjugated, Sigma, diluted 1:200) for 1 h at 37 8C, followed by incubation with 100 ml of 1% aqueous BSA containing the secondary antibodies goat anti-mouse antibody (CY3-conjugated, Jackson Immune Research, diluted 1:250) and CREST antibodies (DPC Biermann, Bad Nauheim, Germany, diluted 1:15) for 1 h at 37 8C. Between and after incubations, slides were washed with PBSCMF pH 8.0 three times for 5 min each. Finally, slides were mounted in antifade solution containing 1 mg/ml DAPI. On each slide, nuclei and MN were visualized using ultraviolet excitation, and cytoplasmic microtubule complex and kinetochore signals were analyzed under green and blue illumination. By means of triple bandpass filter, all signals could be assessed simultaneously, scoring at least 2000 cells per slide for MN, morphology of mitosis, cell divisions and signs of cytotoxicity, e.g. karyorrhectic cells and apoptotic bodies. Cells in the process of cleavage exhibit an appearance that could by classified as interphase according to the morphology of the reformed nuclei, i.e. early G1 cells, yet are still connected by an elongated cytoplasmic thread with remnants of the midbody staining with anti-a-tubulin antibodies.
L5178Y cells were incubated with the test compounds for 5 h. The cells were harvested, fixed in 70% aqueous ethanol for 1 h at 4 8C, washed with PBS, and stained with propidium iodide (0.25 mg/ml) containing RNAase (25 units/ml) for 1 h at room temperature. Cells were analyzed for their DNA content with FACScan using CELLQEST software (BD, Heidelberg, Germany). If not analyzed immediately after staining, they were stored at 4 8C.
2.6. Analysis of metaphase ring arrangement
2.9. Tubulin polymerisation assay
L5178Y cells were treated for 4 h with the substances and fixed at various postincubation times. Fixation and staining were performed as described for micronuclei above. Chromosomes that were dislocated from the metaphase ring arrangement were registered. Spindle staining was achieved with mouse anti-a-tubulin antibody (diluted 1:250 in PBS) and FITC-labeled goat anti-
Microtubule proteins were prepared from bovine brain by two cycles of assembly and disassembly according to the method of Shelanski et al. (1973). The cell-free polymerization of microtubules was carried out as described by Pfeiffer and Metzler (1996). Briefly, the test compounds dissolved in DMSO were added to assembly buffer pH 6.4 containing 10 mM freshly thawed micro-
2.7. Cell viability test L5178Y cells were treated with various concentrations of the test compounds for 10 h. Cell numbers were determined by Coulter Counter analysis. After that, the cell suspension was stained with ethidium bromide (12 ng/ml) and fluorescein diacetate (30 ng/ml), cells were placed on glass slides in PBS without fixation, and the numbers of vital cells (green fluorescence) and dead cells (red fluorescence) were scored by fluorescence microscopy; 200 cells were evaluated per slide. 50 000 V79 cells each were seeded in 35 mm petri dishes and incubated until exponential growth was achieved (24 h). Cells were then treated with various concentrations of RES for 6 h and subsequently kept in fresh medium for 18 h. The numbers of dead and of viable cells were determined prior to treatment with RES and after postincubation by electronic cell counting (Casy). 2.8. Cell cycle analysis
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tubule proteins to yield various concentrations of the compounds and 2% DMSO in a final volume of 0.5 ml. After 20 min at 35 8C, microtubule assembly was started by adding 0.5 mM guanosine triphosphate, and the absorbance at 350 nm was measured for 30 min. Disassembly was then achieved by lowering the temperature to 4 8C, followed by a second polymerization cycle at 35 8C. The control incubations contained all components plus 2% DMSO but no test compound.
3. Results and discussion By using two different in vitro assays, our studies demonstrate that the phytochemical RES (Fig. 1) is estrogenic. In agreement with data published in the literature (Gehm et al., 1997; Ashby et al., 1999; Bowers et al., 2000) RES competed with [3H]-E2 for binding to the human estrogen receptor a under cell-free conditions (Fig. 2) and induced proliferation of the estrogen receptor-positive cell line MCF-7 in a dose-dependent manner at concentrations ranging from 10 nM to 10 mM (Fig. 3); at higher concentrations, RES inhibited the proliferation of MCF-7 cells. Estradiol (1 nM), which was included as a positive control for hormone-induced cell proliferation, yielded a relative proliferation of 2.9. In the estrogen receptor-negative MDA-MB-231 cells,
Fig. 2. Binding affinity to the human estrogen receptor a: competition of bound [3H]-E2 with various concentrations of RES and E2. Data are means9/standard deviations from at least four independent experiments.
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RES inhibited cell growth at all tested concentrations (10 nM and higher, Fig. 3). Relative proliferation after estradiol treatment (at various concentrations up to 1 nM) was within the standard deviation of the negative controls (data not shown). These results for RES are in accordance with the studies of Lu and Serrero (1999) and Mgbonyebi et al. (1998). The inhibitory activity of RES on cell proliferation may be due to an estrogen receptor-independent effect. Therefore, we investigated the genotoxic potential of RES. In L5178Y mouse lymphoma cells we found a reduction in cell proliferation and in cell viability, as well as an induction of micronuclei (MN, Fig. 4). The structurally related synthetic estrogen DES (Fig. 1) was included in our study as a compound with a known mechanism of genotoxic action, i.e. disturbing the assembly of spindle microtubules and thus interfering with mitosis. At equal concentration, DES inhibited cell proliferation slightly more than RES, was similar in cellular toxicity, and appeared slightly less effective in inducing MN (Fig. 4). Chromosome displacement and a dose-dependent increase in distorted spindle morphology could be seen in RES-treated L5178Y cells (Table 1). This is considered typical for spindle disturbing substances like DES, which accordingly exhibited effects in these endpoints (Table 1). These observations seem to suggest that RES might act like DES by inhibiting microtubule assembly and thereby causing mitotic disturbances. It is typical for such compounds to induce MN containing whole chromosomes, which can be detected using anti-kinetochore antibodies. In fact, DES induced 81% kinetochore-positive MN (Table 1). However, MN induced by RES were only up to 46% kinetochore-positive (Table 1). Another typical feature of compounds interfering with mitosis is inhibition of the cell cycle in the M or G2/M-phase, as can also be seen with DES (Stopper et al., 1994; Table 2). In contrast to DES, RES caused a delay in the G1-phase but not of the G2/M-phase at high concentrations (10 mM and higher; Table 2). The failure of RES to disturb spindle assembly was also concluded from studies on the effects of RES on cultured V79 cells. In this cell system, inhibition of cell proliferation was noted at RES
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Fig. 3. Induction of cell proliferation by various concentrations of RES in the human estrogen-sensitive breast cancer cell line MCF-7 (top) and in the human estrogen-insensitive breast cancer cell line MDA-MB-231(bottom). Control, untreated cells; solvent control, 0.1% ethanol (vehicle). Estradiol (1 nM, positive control) yielded a relative proliferation of 2.9 in MCF-7 cells. Data are means9/ standard deviations from at least three independent experiments.
concentrations of 100 mM and higher (Fig. 5); the number of dead cells was about the same at all concentrations. When V79 cells were treated with 100 mM RES for 6 h and the numbers of MN and cell divisions determined at various times thereafter, it was observed that RES gave rise to a surge of freshly divided cells which peaked 11 h after removing the compound from the medium, followed by a marked increase in the number of MN (Fig. 6). The frequency of kinetochore-positive MN was very low at any time and never exceeded 8 per 1000 cells. For example, the MN determined at postincubation times 14, 15 and 18 h contained 11, 5 and 7% kinetochore-positive MN, respectively. Virtually no cells exhibiting apoptotic bodies and few karyorrhectic cells were observed. When the same experiment was carried out with 50 mM RES, the surge of freshly divided cells peaked at postincubation time 7 h and the MN at 10 h
with a MN rate about half of that observed with 100 mM RES (data not shown). Direct evidence that RES lacks the ability to disturb microtubule assembly was obtained from a cell-free polymerization assay (Fig. 7). Even at 200 mM concentration, RES was unable to elicit any effect on the assembly of microtubule proteins, in contrast to DES, which marked inhibited this assembly at 20 mM. Taken together, our results do not support the notion that the genotoxicity of RES is due to interference with the assembly of the mitotic spindle, despite the close structural similarity of RES and DES. In other studies (Hsieh et al., 1999; Matsuoka et al., 2001; Kuwajerwala et al., 2002) it was shown that RES delayed the cells in the Sphase of cell cycle, but also, that this influence on cell cycle progression depended on the cell type (Kuwajerwala et al., 2002). The difference between
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Table 2 Effect of RES on the cell cycle of mouse lymphoma cells L5178Y Treatment
G1-phase
S-phase
G2/M-phase
Control DMSO DES
/ 0.1% 40 mM
27.29/0.5 28.89/1.1 6.89/0.7
54.79/1.1 54.19/1.7 58.69/3.2
18.19/1.6 17.19/0.8 34.69/2.4
RES
60 mM 40 mM 20 mM 10 mM 1 mM
38.19/0.8 38.49/1.5 36.89/0.8 32.09/0.6 29.19/0.7
57.59/1.3 54.69/1.6 55.19/0.5 58.29/0.5 51.19/3.2
4.49/0.5 7.09/0.1 8.19/0.2 9.89/1.2 19.89/2.5
Data are means of two independent experiments.
Fig. 4. Effects of RES on cell proliferation (top), number of dead cells (center), and induction of MN (bottom) in mouse lymphoma cells L5178Y. Cells were exposed to the compounds for 10 h for cell proliferation and viability assessment and for 4 h followed by a 20 h recovery period for micronucleus induction. Solvent control, 1% DMSO (vehicle).
delay in S-phase in those studies and in G1-phase in ours thus may be due to cell type differences, or treatment conditions. However, no support for a direct interference with mitosis can be gained from
those studies either. Therefore, an indirect or secondary effect, possibly caused by the compound’s toxic effects may be responsible for the distortion of the metaphase arrangements seen in our investigation, and RES-induced genotoxicity may be caused by other mechanisms. Matsuoka et al. (2001) tested the genotoxicity of RES using several endpoints. In agreement with our studies, RES induced MN and increases in sister-chromatid-exchanges (SCE). However, RES did not induce an increase in chromosomal aberrations or bacterial mutations. An increased MN frequency in the absence of chromosomal aberrations could be explained by MN containing whole chromosomes due to spindle disturbances. However, our data do not support this explanation. In agreement with our data, the increased SCE frequency points towards a chromosome breaking effect. The absence of bacterial mutation rules out direct modification of DNA leading to gene
Table 1 Genotoxicity of RES in mouse lymphoma cells L5178Y Compound
MN/1000 cells
Distorted spindle/ 100 metaphases
Dislocated chromosomes/ 100 metaphases
Kinetochore-positive MN in 100 MN
Solvent DES 40 mM RES 60 mM RES 40 mM RES 20 mM RES 10 mM RES 1 mM
4.39/1.7 13.39/2.1 21.39/1.3 16.39/2.2 13.09/2.9 10.39/1.3 10.09/1.0
3.59/0.5 16.09/0 26.09/0 17.09/1.0 14.09/2.0 13.09/3.0 9.09/0
2.79/0.6 9.39/1.1 24.79/4.2 16.39/5.7 16.09/4.6 15.09/4.8 14.79/1.1
22 81 46 40 35 27 20
Data are means of two independent experiments.
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Fig. 5. Cytotoxicity of RES in Chinese hamster V79 cells. Cells were exposed to RES for 6 h and incubated for another 18 h. Control, 1% DMSO (vehicle).
Fig. 6. MN and cell divisions in V79 cells after exposure to 100 mM RES for 6 h and various postincubation times. MN data are means from two independent experiments. Control cells (1% DMSO) exhibit 489/4 cell divisions (three independent experiments) and 99/2 MN per 1000 cells (nine independent experiments), 3 of which were kinetochore-positive and 6 were kinetochore-negative on average.
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Fig. 7. Effects of RES and DES on cell-free microtubule assembly. Control, 2% DMSO (vehicle).
mutations. Overall, the mechanism of RES induced genotoxicity remains unclear. The daily intake of flavanoids in western countries has been estimated to amount to 0.5 / 1.0 g (Peterson and Dwyer, 1998). Especially red grapes and wines contain concentrations of 0.2 /8 mg/l of RES (Mattivi, 1993; Romero-Perez et al., 1996; Wamhoff et al., 1998). Bioavailability of RES in humans was determined to be more than 50% (Bertelli et al., 1996a,b; Blache et al., 1997). Based on the data given by Bertelli et al. (1996a,b) it can be estimated that */even after moderate wine consumption (0.4 l) */plasma levels of more than 100 nM RES can be reached. This is well in the range where estrogenic effects in vitro could be detected. However, Freyberger et al. (2001) found no estrogenic effects in rats after in vivo exposure, although a plasma level of 1/2 mM was measured. The achievable plasma level for human exposure is also close to the range of genotoxicity. It has been discussed by Bertelli et al. (1996a,b) that RES could accumulate in peripheral tissues after daily ingestion. Therefore, concentrations that were found genotoxic in vitro might be reached in human exposure. Estrogenic activity, if present in vivo, might be beneficial as has been suggested for the uptake of other phytohormones like soy isoflavones. However, a further investigation of
RES-induced genotoxicity including in vivo investigations and elucidation of the mechanism of genotoxicity would be desirable to clarify a potential risk for humans.
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pro-apoptotic p53/Bax gene expression and inhibits the growth of transformed cells but not their normal counterparts. Carcinogenesis 22 (2), 321 /328. Matsuoka, A., Furuta, A., Ozaki, M., Fukuhara, K., Miyata, N., 2001. Resveratrol, a naturally occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line. Mutat. Res. 494, 107 /113. Mattivi, F., 1993. Solid phase extraction of trans -resveratrol from wines for HPLC analysis. Z. Lebensm. Unters. Forsch. 196, 522 /525. Mgbonyebi, O., Russo, J., Russo, I.H., 1998. Antiproliferative effect of synthetic resveratrol on human breast epithelial cells. Int. J. Oncol. 12, 865 /869. Peterson, J., Dwyer, J., 1998. Flavanoids: dietary occurrence and biochemical activity. Nutr. Res. 18, 1995 /2018. Pfeiffer, E, Metzler, M., 1996. Interaction of p -benzoquinone and p -biphenoquinone with microtubule proteins in vitro. Chem. -Biol. Interact. 102, 37 /53. Romero-Perez, A.I., Lamuela, R.M., Waterhouse, A., de la Torre-Boronat, M., 1996. Direct HPLC analysis of cis - and trans -resveratrol and piceid isomers in spanish red vitis vinifera wines. J. Agric. Food Chem. 43, 281 /283. Savouret, J.F., Quesne, M., 2002. Resveratrol and cancer: a review. Biomed. Pharmacother. 56 (2), 84 /87. Schneider, Y., Duranton, B., Gosse, F., Schleiffer, R., Seiler, N., Raul, F., 2001. Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis. Nutr. Cancer 39, 102 /107. Schultz, N., Onfelt, A., 2000. Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi- and multinucleated cells. Chem. -Biol. Interact. 126, 97 /123. Shelanski, M.L., Gaskin, F., Cantor, C.R., 1973. Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. USA 70, 765 /768. Stopper, H., Kirchner, S., Schiffmann, D., Poot, M., 1994. Cell cycle disturbance in relation to micronucleus formation induced by the carcinogenic estrogen diethylstilbestrol. Pathobiology 62, 180 /185. Wamhoff, H., Richardt, G., Schneider, V., Tulke, A., 1998. Wein und Gesundheit. Chemie in unserer Zeit 32 (Suppl.), 87 /93. Wu, J.M., Wang, Z.R., Hsieh, T.C., Bruder, J.L., Zou, J.G., Huang, Y.Z., 2001. Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine. (Review) Int. J. Mol. Med. 8 (1), 3 /17.
Hormonal and genotoxic activity of resveratrol E. Schmitt a, L. Lehmann b, M. Metzler b, H. Stopper a,* a b
Institute of Pharmacology and Toxicology, University of Wu¨rzburg, Versbacherstrasse 9, D-97078 Wurzburg, Germany Institute of Food Chemistry and Toxicology, University of Karlsruhe, Fritz Haber Weg 2, D-76131 Karlsruhe, Germany Received 23 April 2002; received in revised form 24 July 2002; accepted 24 July 2002
Abstract Resveratrol (RES) is a natural polyphenol present in red wines and various human food items. The estrogenic activity of RES was demonstrated in two in vitro assay systems, i.e. binding to human estrogen receptor a and stimulation of MCF-7 cell proliferation. To investigate the inhibition of cell proliferation observed at high concentrations of RES, we analyzed the compound for genotoxic potential. RES induced cellular toxicity, micronuclei, and metaphase chromosome displacement in L5178Y mouse lymphoma cells. Likewise, the induction of micronuclei was observed in Chinese hamster V79 cells. Determination of kinetochore signals in micronuclei and cell cycle analysis suggested that RES did not cause a direct disturbance of mitosis. In support of this notion, cell-free tubulin polymerization studies indicated no direct effect of RES on microtubule assembly. According to an estimation of daily intake and bioavailability, concentrations that were found genotoxic in vitro might be reached in human exposure. On the other side, the estrogenic acitivity might be beneficial. Therefore, further investigations of mechanisms, possibly including animal models, would be desirable to clarifiy a potential risk for humans. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phytoestrogens; Resveratrol; Estrogen receptor; Breast cancer cells; Genotoxicity; Micronuclei; Cell cycle
1. Introduction Resveratrol (RES, Fig. 1) is a natural polyphenol present in red wines and various human foods.
It is synthesized by a variety of plant species in response to injury, UV irradiation and fungal attack. Since it is only synthesized in the skin of the grape berries, red wine contains more RES
Abbreviations: BSA, bovine serum albumin; CREST, calcinosis, Raynaud’s phenomenon, oesophagal motility abnormalities, sclerodactyly and telangiectasia; CYP, cytochrome P450; DAPI, 4?,6?-diamidino-2-phenylindole; DES, diethylstilbestrol, E-3,4-bis(p hydroxyphenyl)-hex-3-ene; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; E2, 17b-estradiol; EDTA, ethylenediaminetetraacetate, sodium salt; EGTA, ethylene glycol-bis(2-aminoethylether)-tetraacetic acid; FBS, fetal bovine serum; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; MN, micronucleus; PBS, phosphate-buffered saline; PBS-CMF, phosphatebuffered saline free of calcium and magnesium; Pipes, piperazine-1,4-bis(2-ethanesulfonic acid); RPMI, Roswell Memorial Park Institute (RPMI 1640 cell culture medium); RES, resveratrol, 3,4?,5-trihydroxystilbene; Tris, tris(hydroxymethyl)aminomethane. * Corresponding author. Tel.: /49-931-201-48427; fax: /49-931-201-48446 E-mail address: [email protected] (H. Stopper). 0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 2 9 0 - 4
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Fig. 1. Chemical structures of RES (1), DES (2) and E2 (3).
than white wine. It has been suggested that RES may be the main active principle involved in the ‘French Paradox’, the inverse correlation between red wine consumption and the incidence of cardiovascular disease (Wu et al., 2001). RES has been shown to exhibit estrogenic activity (Gehm et al., 1997; Ashby et al., 1999; Bowers et al., 2000). The observation that RES is an effective radical-scavenger suggested that it acts as a natural antioxidant against oxidative DNAdamage (Cadenas and Barja, 1999; Lin and Tsai, 1999). In addition to these anti-initiating properties, RES exhibits anti-inflammatory effects. For example, it suppresses cyclooxgenase activity and the induction of the transcription factor NF kappa B by other agents (Bhat and Pezzuto, 2002). RES shows antimutagenic and anticarcinogenic activities. Benzo[a]pyrene-induced cancerogenesis could be reduced by RES by inhibition of CYP1A1 (El Attar and Virji, 1999; Ciolino and Yeh, 1999; Chun et al., 1999). RES also inhibited the 7, 12dimethylbenz[a]anthracene (DMBA)-induced cancerogenesis in a mouse-model (Jang et al., 1997). In another model RES reduced the intestinal tumorigenesis in genetically predisposed mice (Schneider et al., 2001). Furthermore, RES is a known inducer of apoptosis. Interestingly, the proapoptotic effect may be limited to tumor cells,
while normal cells remain unharmed (Lu et al., 2001). Therefore, great interest in RES as a cancer chemopreventive or even cancer therapeutic agent has evolved (Gusman et al., 2001; Savouret and Quesne, 2002). Opposite to this view, we demonstrate here that RES exhibits a genotoxic potential in vitro.
2. Materials and methods 2.1. Test substances RES (3,4?,5-Trihydroxy-trans -stilbene), 17b-estradiol (E2), and diethylstilbestrol (DES) of the highest available purity (E/98%) were obtained from Sigma Chemie GmbH (Deisenhofen, Germany). 2.2. Cell culture MCF-7, MDA-MB-231 and V79 cells were purchased from American Type Culture Collection (ATTC, Hanassas, USA). MCF-7 and MDAMB-231 cells were cultured in phenol red-free RPMI 1640 medium supplemented with antibiotics, L-glutamine (0.25 mg/ml), sodium pyruvate (107 mg/ml), human insulin (0.2 ng/ml), and 5%
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fetal bovine serum (FBS; all from Sigma Chemie GmbH). L5178Y tk / mouse lymphoma cells, clone 3.7.2c were routinely cultured in suspension in RPMI 1640 medium supplemented with antibiotics, L-glutamine (0.25 mg/ml), sodium pyruvate (107 mg/ml) and 10% heat-inactivated horse serum (all from Sigma Chemie GmbH). Chinese hamster V79 cells were cultured in DMEM pH 7.5 containing glucose (4500 mg/l) and supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ ml) and 10% FBS. All cells were grown in a humidified atmosphere with 5% CO2 in air at 37 8C.
2.3. Estrogen receptor binding assay 1.5 nM isolated human estrogen receptor a (PanVera, Madison, USA) were solved in binding buffer (1.5 mM EDTA, 10 mM Tris, 1 mM EGTA, 1mM NaVO4, 10% glycerol, 10 mg/ml gglobulin, 0.5 mM phenylmethylsulfonylfluoride, 0.2 mM leupeptin) and incubated with 6.5 nM [3H]-E2 (Amersham Biosciences, Freiburg, Germany) and increasing concentrations of unlabeled competitors (10 pM /100 mM). The mixtures were incubated at 4 8C for 18.5 h, and then 250 ml of 16% hydroxyapatite in binding buffer was added to each tube. The resulting pellet was washed twice with 0.9 ml binding buffer, then suspended in scintillation cocktail, and the radioactivity measured in a Packard 2250CA scintillation counter.
2.4. Cell proliferation analysis 80 000 /100 000 MCF-7 or MDA-MB-231 cells/ ml were seeded in 25 ml-culture flasks. Twentyfour hours later medium was exchanged. Fresh medium contained charcoal/dextrane-treated FBS (5%) and various concentrations of test compounds. At 96 h medium was exchanged again and cells were harvested at 144 h (MCF-7) and 120 h (MDA-MB-231). Cell numbers were determined by Coulter Counter analysis.
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2.5. In vitro micronucleus assay and kinetochore staining Exponentially growing mouse lymphoma L5178Y cells were treated for 4 h with the test substances dissolved in dimethylsulfoxide (DMSO). The maximum DMSO concentration during treatment was 1%. After removing the test compounds by medium replacement, the cells were incubated for another 20 h, then washed with PBS buffer, collected by centrifugation (5 min at 1000 rpm), and placed on glass slides by cytospin centrifugation. Fixation was performed with methanol at /20 8C for 1 h. For staining, the slides were washed with PBS buffer, incubated for 3 min with a solution of 62.5 mg/ml acridine orange in Soerensen buffer (67 mM Na2HPO4/KH2PO4, pH 6.8), and washed again with PBS buffer. Numbers of nuclei and micronuclei (MN) were scored using fluorescence microscopy. For each concentration of test compound, three slides with 1000 nuclei per slide were evaluated. Kinetochore staining in L5178Y cells was achieved by incubating the fixed cell preparations after rinsing with PBS-CMF/0.1% Tween 20 (v/v) with CREST serum for 60 min in a humidified chamber at 37 8C. After rinsing with PBS-CMF/0.5% Tween 20 (v/v), the cells were incubated for 30 min with FITC-conjugated goat anti-human antibody (diluted 1:100 in PBS), rinsed again with PBS-CMF/ 0.1% Tween 20 (v/v) and counterstained with bisbenzimide 33258 (1 mg/ml, 5 min). Slides were mounted for fluorescence microscopy using antifade solution (Oncor, Heidelberg, Germany). At least 100 MN were analyzed for the presence of kinetochore signals. V79 cells were grown on glass slides in quadriperm vials (25 000 /50 000 cells per slide) for 24 h prior to incubation with the test compound for 6 h. The cells were further incubated in fresh DMEM for various time periods up to 18 h, fixed for 5 min in 3.5% freshly depolymerized paraformaldehyde in CB buffer (137 mM NaCl, 5 mM KCl, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 2 mM MgCl2, 2 mM EGTA, 5 mM Pipes, 5.5 mM glucose, pH 6.1; Schultz and Onfelt, 2000) and postfixed with methanol at /20 8C for at least 1 h. Cells were then stained with anti-a-tubulin antibodies for the
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mouse antibody (diluted 1:100 in PBS) according to the procedure given above for kinetochore staining of L5178Y cells.
evaluation of cell morphology, with CREST antibodies for the presence of kinetochore signals, and with DAPI antifade solution for the scoring of nuclei and MN: slides after fixation were first washed three times with PBS-CMF pH 8.0 for 5 min each. Nonspecific binding was blocked by incubation with goat serum for 1 h at 37 8C. Slides were then incubated with 100 ml of a 1% aqueous BSA solution containing monoclonal mouse anti-a-tubulin antibody (Sigma, diluted 1:500) and goat polyvalent anti-human antibody (FITC-conjugated, Sigma, diluted 1:200) for 1 h at 37 8C, followed by incubation with 100 ml of 1% aqueous BSA containing the secondary antibodies goat anti-mouse antibody (CY3-conjugated, Jackson Immune Research, diluted 1:250) and CREST antibodies (DPC Biermann, Bad Nauheim, Germany, diluted 1:15) for 1 h at 37 8C. Between and after incubations, slides were washed with PBSCMF pH 8.0 three times for 5 min each. Finally, slides were mounted in antifade solution containing 1 mg/ml DAPI. On each slide, nuclei and MN were visualized using ultraviolet excitation, and cytoplasmic microtubule complex and kinetochore signals were analyzed under green and blue illumination. By means of triple bandpass filter, all signals could be assessed simultaneously, scoring at least 2000 cells per slide for MN, morphology of mitosis, cell divisions and signs of cytotoxicity, e.g. karyorrhectic cells and apoptotic bodies. Cells in the process of cleavage exhibit an appearance that could by classified as interphase according to the morphology of the reformed nuclei, i.e. early G1 cells, yet are still connected by an elongated cytoplasmic thread with remnants of the midbody staining with anti-a-tubulin antibodies.
L5178Y cells were incubated with the test compounds for 5 h. The cells were harvested, fixed in 70% aqueous ethanol for 1 h at 4 8C, washed with PBS, and stained with propidium iodide (0.25 mg/ml) containing RNAase (25 units/ml) for 1 h at room temperature. Cells were analyzed for their DNA content with FACScan using CELLQEST software (BD, Heidelberg, Germany). If not analyzed immediately after staining, they were stored at 4 8C.
2.6. Analysis of metaphase ring arrangement
2.9. Tubulin polymerisation assay
L5178Y cells were treated for 4 h with the substances and fixed at various postincubation times. Fixation and staining were performed as described for micronuclei above. Chromosomes that were dislocated from the metaphase ring arrangement were registered. Spindle staining was achieved with mouse anti-a-tubulin antibody (diluted 1:250 in PBS) and FITC-labeled goat anti-
Microtubule proteins were prepared from bovine brain by two cycles of assembly and disassembly according to the method of Shelanski et al. (1973). The cell-free polymerization of microtubules was carried out as described by Pfeiffer and Metzler (1996). Briefly, the test compounds dissolved in DMSO were added to assembly buffer pH 6.4 containing 10 mM freshly thawed micro-
2.7. Cell viability test L5178Y cells were treated with various concentrations of the test compounds for 10 h. Cell numbers were determined by Coulter Counter analysis. After that, the cell suspension was stained with ethidium bromide (12 ng/ml) and fluorescein diacetate (30 ng/ml), cells were placed on glass slides in PBS without fixation, and the numbers of vital cells (green fluorescence) and dead cells (red fluorescence) were scored by fluorescence microscopy; 200 cells were evaluated per slide. 50 000 V79 cells each were seeded in 35 mm petri dishes and incubated until exponential growth was achieved (24 h). Cells were then treated with various concentrations of RES for 6 h and subsequently kept in fresh medium for 18 h. The numbers of dead and of viable cells were determined prior to treatment with RES and after postincubation by electronic cell counting (Casy). 2.8. Cell cycle analysis
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tubule proteins to yield various concentrations of the compounds and 2% DMSO in a final volume of 0.5 ml. After 20 min at 35 8C, microtubule assembly was started by adding 0.5 mM guanosine triphosphate, and the absorbance at 350 nm was measured for 30 min. Disassembly was then achieved by lowering the temperature to 4 8C, followed by a second polymerization cycle at 35 8C. The control incubations contained all components plus 2% DMSO but no test compound.
3. Results and discussion By using two different in vitro assays, our studies demonstrate that the phytochemical RES (Fig. 1) is estrogenic. In agreement with data published in the literature (Gehm et al., 1997; Ashby et al., 1999; Bowers et al., 2000) RES competed with [3H]-E2 for binding to the human estrogen receptor a under cell-free conditions (Fig. 2) and induced proliferation of the estrogen receptor-positive cell line MCF-7 in a dose-dependent manner at concentrations ranging from 10 nM to 10 mM (Fig. 3); at higher concentrations, RES inhibited the proliferation of MCF-7 cells. Estradiol (1 nM), which was included as a positive control for hormone-induced cell proliferation, yielded a relative proliferation of 2.9. In the estrogen receptor-negative MDA-MB-231 cells,
Fig. 2. Binding affinity to the human estrogen receptor a: competition of bound [3H]-E2 with various concentrations of RES and E2. Data are means9/standard deviations from at least four independent experiments.
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RES inhibited cell growth at all tested concentrations (10 nM and higher, Fig. 3). Relative proliferation after estradiol treatment (at various concentrations up to 1 nM) was within the standard deviation of the negative controls (data not shown). These results for RES are in accordance with the studies of Lu and Serrero (1999) and Mgbonyebi et al. (1998). The inhibitory activity of RES on cell proliferation may be due to an estrogen receptor-independent effect. Therefore, we investigated the genotoxic potential of RES. In L5178Y mouse lymphoma cells we found a reduction in cell proliferation and in cell viability, as well as an induction of micronuclei (MN, Fig. 4). The structurally related synthetic estrogen DES (Fig. 1) was included in our study as a compound with a known mechanism of genotoxic action, i.e. disturbing the assembly of spindle microtubules and thus interfering with mitosis. At equal concentration, DES inhibited cell proliferation slightly more than RES, was similar in cellular toxicity, and appeared slightly less effective in inducing MN (Fig. 4). Chromosome displacement and a dose-dependent increase in distorted spindle morphology could be seen in RES-treated L5178Y cells (Table 1). This is considered typical for spindle disturbing substances like DES, which accordingly exhibited effects in these endpoints (Table 1). These observations seem to suggest that RES might act like DES by inhibiting microtubule assembly and thereby causing mitotic disturbances. It is typical for such compounds to induce MN containing whole chromosomes, which can be detected using anti-kinetochore antibodies. In fact, DES induced 81% kinetochore-positive MN (Table 1). However, MN induced by RES were only up to 46% kinetochore-positive (Table 1). Another typical feature of compounds interfering with mitosis is inhibition of the cell cycle in the M or G2/M-phase, as can also be seen with DES (Stopper et al., 1994; Table 2). In contrast to DES, RES caused a delay in the G1-phase but not of the G2/M-phase at high concentrations (10 mM and higher; Table 2). The failure of RES to disturb spindle assembly was also concluded from studies on the effects of RES on cultured V79 cells. In this cell system, inhibition of cell proliferation was noted at RES
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Fig. 3. Induction of cell proliferation by various concentrations of RES in the human estrogen-sensitive breast cancer cell line MCF-7 (top) and in the human estrogen-insensitive breast cancer cell line MDA-MB-231(bottom). Control, untreated cells; solvent control, 0.1% ethanol (vehicle). Estradiol (1 nM, positive control) yielded a relative proliferation of 2.9 in MCF-7 cells. Data are means9/ standard deviations from at least three independent experiments.
concentrations of 100 mM and higher (Fig. 5); the number of dead cells was about the same at all concentrations. When V79 cells were treated with 100 mM RES for 6 h and the numbers of MN and cell divisions determined at various times thereafter, it was observed that RES gave rise to a surge of freshly divided cells which peaked 11 h after removing the compound from the medium, followed by a marked increase in the number of MN (Fig. 6). The frequency of kinetochore-positive MN was very low at any time and never exceeded 8 per 1000 cells. For example, the MN determined at postincubation times 14, 15 and 18 h contained 11, 5 and 7% kinetochore-positive MN, respectively. Virtually no cells exhibiting apoptotic bodies and few karyorrhectic cells were observed. When the same experiment was carried out with 50 mM RES, the surge of freshly divided cells peaked at postincubation time 7 h and the MN at 10 h
with a MN rate about half of that observed with 100 mM RES (data not shown). Direct evidence that RES lacks the ability to disturb microtubule assembly was obtained from a cell-free polymerization assay (Fig. 7). Even at 200 mM concentration, RES was unable to elicit any effect on the assembly of microtubule proteins, in contrast to DES, which marked inhibited this assembly at 20 mM. Taken together, our results do not support the notion that the genotoxicity of RES is due to interference with the assembly of the mitotic spindle, despite the close structural similarity of RES and DES. In other studies (Hsieh et al., 1999; Matsuoka et al., 2001; Kuwajerwala et al., 2002) it was shown that RES delayed the cells in the Sphase of cell cycle, but also, that this influence on cell cycle progression depended on the cell type (Kuwajerwala et al., 2002). The difference between
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Table 2 Effect of RES on the cell cycle of mouse lymphoma cells L5178Y Treatment
G1-phase
S-phase
G2/M-phase
Control DMSO DES
/ 0.1% 40 mM
27.29/0.5 28.89/1.1 6.89/0.7
54.79/1.1 54.19/1.7 58.69/3.2
18.19/1.6 17.19/0.8 34.69/2.4
RES
60 mM 40 mM 20 mM 10 mM 1 mM
38.19/0.8 38.49/1.5 36.89/0.8 32.09/0.6 29.19/0.7
57.59/1.3 54.69/1.6 55.19/0.5 58.29/0.5 51.19/3.2
4.49/0.5 7.09/0.1 8.19/0.2 9.89/1.2 19.89/2.5
Data are means of two independent experiments.
Fig. 4. Effects of RES on cell proliferation (top), number of dead cells (center), and induction of MN (bottom) in mouse lymphoma cells L5178Y. Cells were exposed to the compounds for 10 h for cell proliferation and viability assessment and for 4 h followed by a 20 h recovery period for micronucleus induction. Solvent control, 1% DMSO (vehicle).
delay in S-phase in those studies and in G1-phase in ours thus may be due to cell type differences, or treatment conditions. However, no support for a direct interference with mitosis can be gained from
those studies either. Therefore, an indirect or secondary effect, possibly caused by the compound’s toxic effects may be responsible for the distortion of the metaphase arrangements seen in our investigation, and RES-induced genotoxicity may be caused by other mechanisms. Matsuoka et al. (2001) tested the genotoxicity of RES using several endpoints. In agreement with our studies, RES induced MN and increases in sister-chromatid-exchanges (SCE). However, RES did not induce an increase in chromosomal aberrations or bacterial mutations. An increased MN frequency in the absence of chromosomal aberrations could be explained by MN containing whole chromosomes due to spindle disturbances. However, our data do not support this explanation. In agreement with our data, the increased SCE frequency points towards a chromosome breaking effect. The absence of bacterial mutation rules out direct modification of DNA leading to gene
Table 1 Genotoxicity of RES in mouse lymphoma cells L5178Y Compound
MN/1000 cells
Distorted spindle/ 100 metaphases
Dislocated chromosomes/ 100 metaphases
Kinetochore-positive MN in 100 MN
Solvent DES 40 mM RES 60 mM RES 40 mM RES 20 mM RES 10 mM RES 1 mM
4.39/1.7 13.39/2.1 21.39/1.3 16.39/2.2 13.09/2.9 10.39/1.3 10.09/1.0
3.59/0.5 16.09/0 26.09/0 17.09/1.0 14.09/2.0 13.09/3.0 9.09/0
2.79/0.6 9.39/1.1 24.79/4.2 16.39/5.7 16.09/4.6 15.09/4.8 14.79/1.1
22 81 46 40 35 27 20
Data are means of two independent experiments.
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Fig. 5. Cytotoxicity of RES in Chinese hamster V79 cells. Cells were exposed to RES for 6 h and incubated for another 18 h. Control, 1% DMSO (vehicle).
Fig. 6. MN and cell divisions in V79 cells after exposure to 100 mM RES for 6 h and various postincubation times. MN data are means from two independent experiments. Control cells (1% DMSO) exhibit 489/4 cell divisions (three independent experiments) and 99/2 MN per 1000 cells (nine independent experiments), 3 of which were kinetochore-positive and 6 were kinetochore-negative on average.
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Fig. 7. Effects of RES and DES on cell-free microtubule assembly. Control, 2% DMSO (vehicle).
mutations. Overall, the mechanism of RES induced genotoxicity remains unclear. The daily intake of flavanoids in western countries has been estimated to amount to 0.5 / 1.0 g (Peterson and Dwyer, 1998). Especially red grapes and wines contain concentrations of 0.2 /8 mg/l of RES (Mattivi, 1993; Romero-Perez et al., 1996; Wamhoff et al., 1998). Bioavailability of RES in humans was determined to be more than 50% (Bertelli et al., 1996a,b; Blache et al., 1997). Based on the data given by Bertelli et al. (1996a,b) it can be estimated that */even after moderate wine consumption (0.4 l) */plasma levels of more than 100 nM RES can be reached. This is well in the range where estrogenic effects in vitro could be detected. However, Freyberger et al. (2001) found no estrogenic effects in rats after in vivo exposure, although a plasma level of 1/2 mM was measured. The achievable plasma level for human exposure is also close to the range of genotoxicity. It has been discussed by Bertelli et al. (1996a,b) that RES could accumulate in peripheral tissues after daily ingestion. Therefore, concentrations that were found genotoxic in vitro might be reached in human exposure. Estrogenic activity, if present in vivo, might be beneficial as has been suggested for the uptake of other phytohormones like soy isoflavones. However, a further investigation of
RES-induced genotoxicity including in vivo investigations and elucidation of the mechanism of genotoxicity would be desirable to clarify a potential risk for humans.
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