Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Mutation Research 513 (2002) 113–120

Different effects of genistein and resveratrol on oxidative DNA damage in vitro William Win a , Zhuoxiao Cao a , Xingxiang Peng a , Michael A. Trush b , Yunbo Li a,∗ a

b

Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. Albert Hall, St. John’s University, 8000 Utopia Parkway, Jamaica, NY 11439, USA Division of Toxicological Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA Received 5 July 2001; received in revised form 28 August 2001; accepted 29 August 2001

Abstract Previous studies have demonstrated that phenolic compounds, including genistein (4 ,5,7-trihydroxyisoflavone) and resveratrol (3,4 ,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in ␾X-174 plasmid DNA. H2 O2 /Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H2 O2 /Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H2 O2 suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H2 O2 /Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H2 O2 were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Genistein; Resveratrol; DNA strand breaks; Reactive oxygen species; Copper; Hydrogen peroxide; Hydroquinone

1. Introduction Genistein and resveratrol (Fig. 1) are naturally occurring phenolic antioxidants present in high levels in soybeans and grapes, respectively [1,2]. Recent studies have indicated that these two phenolic chemAbbreviations: ROS, reactive oxygen species; HQ, hydroquinone; BCS, bathocuproinedisulfonic acid; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; SOD, superoxide dismutase; HRP, horseradish peroxidase ∗ Corresponding author. Tel.: +1-718-990-5253; fax: +1-718-990-1877. E-mail address: [email protected] (Y. Li).

icals exert anticarcinogenic effects in various in vitro systems and in vivo animal models [1–5]. Although, the exact molecular mechanisms involved in the anticarcinogenic effects of these two compounds are not fully understood, scavenging ROS is believed to be responsible, at least partially, for their anticarcinogenic effects [1–5]. Production of ROS, including superoxide anion radical, hydrogen peroxide, singlet oxygen and hydroxyl radical is associated with normal cellular metabolism [6]. There are many potential cellular sources of ROS production. These include cytosolic xanthine oxidase, transition metal ions, plasma membrane NADPH

1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 3 0 3 - 5

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and resveratrol on oxidative DNA strand breaks induced by H2 O2 /Cu(II) and hydroquinone (HQ)/Cu(II). Both H2 O2 /Cu(II) and HQ/Cu(II) have been previously shown to cause DNA strand breaks in plasmid DNA [17,18]. Our results showed that genistein at micromolar concentrations markedly inhibited oxidative DNA strand breaks, whereas, resveratrol at similar concentrations increased the oxidative DNA strand breaks induced by H2 O2 /Cu(II). In addition, resveratrol + Cu(II), but not genistein + Cu(II), also caused DNA strand breaks in this plasmid DNA system.

2. Materials and methods 2.1. Materials Fig. 1. Structures of genistein and resveratrol.

oxidase, lysosomes, peroxisomes, the endoplasmic reticulum and the mitochondrial electron transport systems [6]. ROS are also generated by exogenous sources, such as ultraviolet (UV) light, gamma radiation, and a number of xenobiotics [7,8]. Studies over the last two decades have demonstrated that ROS may play an important role in the pathogenesis of a variety of diseases and disorders, including aging, neurodegenerative diseases, cardiovascular diseases, and cancer [8–12]. Induction of cancer is a multistage process, which has been experimentally defined as initiation, promotion and progression [13,14]. Accumulating evidence suggests that ROS produced by either endogenous or exogenous sources are critically involved in all three stages of carcinogenesis [13,14]. ROS are able to cause damage to genomic DNA leading to mutation, activation of protooncogenes and inactivation of tumor suppressor genes [13,14]. ROS can also interfere with normal cell signaling through modifying transcription factors and protein kinase cascades [15,16]. Oxidative modification of cell signal transduction by ROS may result in dysregulated cell growth, differentiation and death, together with the DNA mutations, ultimately leading to the development of cancer [13,14]. To further understand the mechanisms by which the phenolic antioxidants, genistein and resveratrol inhibit carcinogenesis, in this study with ␾X-174 plasmid DNA, we examined the effects of genistein

Resveratrol, genistein, hydrogen peroxide (H2 O2 ), hydroquinone (HQ), bathocuproinedisulfonic acid (BCS), cupric sulfate, superoxide dismutase (SOD) from bovine erythrocytes, catalase from bovine liver, mannitol, and dimethyl sulfoxide (DMSO) were obtained from Sigma (St. Louis, MO). The ␾X-174 RF I plasmid DNA was purchased from New England Biolabs (Beverly, MA). Dulbecco’s phosphate-buffered saline (PBS) was obtained from Gibco (Grand Island, NY). 2.2. Assay for oxidative DNA strand breaks The ROS-mediated DNA strand breaks were measured by the conversion of supercoiled ␾X-174 RF I double-stranded DNA to open circular and linear forms, according to the procedure described previously [18]. Briefly, 0.2 ␮g DNA was incubated with the indicated concentrations of H2 O2 , Cu(II), genistein, resveratrol, or other chemicals in PBS at 37◦ C at a final volume of 24 ␮l in 1.5 ml brown microcentrifuge tubes. Following incubation, the samples were immediately loaded in a 1% agarose gel containing 40 mM Tris, 20 mM sodium acetate and 2 mM EDTA, and electrophoresed in a horizontal slab gel apparatus in Tris/acetate/EDTA gel buffer. After electrophoresis, the gels were stained with 0.5 ␮g/ml solution of ethidium bromide for 30 min, followed by another 30 min destaining in water. The gels were then photographed under UV light.

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2.3. Reduction of Cu(II) to Cu(I) The reduction of Cu(II) to Cu(I) by H2 O2 or resveratrol was determined by using the Cu(I)-specific reagent, BCS as described previously [19]. In brief, Cu(II) was incubated with H2 O2 or resveratrol in 1 ml PBS containing 0.3 mM BCS at 37◦ C for 30 min. The stable BCS-Cu(I) complex was determined by measuring its absorbance at 480 nm. The concentration of Cu(I) was calculated from a BCS-Cu(I) standard curve [19]. 2.4. Measurement of O2 consumption O2 consumption caused by HQ/Cu(II) was monitored with a Clark oxygen electrode (YSI 5300, Yellow Springs, OH) upon mixing HQ with Cu(II) in the presence or absence of genistein in 3 ml air-saturated PBS at 37◦ C, as described before [20]. 2.5. Measurement of H2 O2 The presence of H2 O2 was indirectly determined by O2 release upon adding catalase (250 units/ml) to the reaction mixture of H2 O2 and genistein in air-saturated PBS at 37◦ C [21]. The O2 generation was monitored with a Clark oxygen electrode as described above. The concentration of H2 O2 in the reaction mixture was calculated from an H2 O2 standard curve. 3. Results and discussion 3.1. Inhibition of H2 O2 /Cu(II)- and HQ/Cu(II)-mediated DNA strand breaks by genistein Induction of single-strand breaks to supercoiled double-stranded plasmid DNA leads to formation of open circular DNA, while the formation of a linear form of DNA is indicative of double-strand breaks [18]. It has been demonstrated previously that in the presence of Cu(II), H2 O2 is able to cause strand breaks in isolated DNA [17]. Although, the exact reactive species remain to be chemically defined, a bound hydroxyl radical or its equivalent derived from the reaction of H2 O2 and Cu has been suggested to mediate the DNA strand breaks [17]. As shown in Fig. 2, incubation of DNA with 25 ␮M H2 O2

Fig. 2. Effects of genistein (panel A) and the hydroxyl radical scavengers, mannitol and DMSO on H2 O2 /Cu(II)-mediated DNA strand breaks. The ␾X-174 plasmid DNA was incubated with 25 ␮M H2 O2 + 10 ␮M Cu(II) in the presence or absence of the indicated concentrations of genistein (panel A), mannitol or DMSO (panel B) in PBS at 37◦ C for 30 min. The DNA strand breaks were determined as described in Section 2. In this figure and the following ones (Figs. 3–7), the marker used was Lambda DNA-Hind III digest (New England Biolabs, Beverly, MA) with the sizes (from top to bottom) being 23,130, 9416, 6557, 4361, 2322, and 2027 bp, respectively. The control was ␾X-174 plasmid DNA incubated in PBS in the absence of any added agents.

and 10 ␮M Cu(II) for 30 min resulted in increased formation of open circle and linear forms of DNA, indicating that both single-strand and double-strand DNA breaks can be induced by H2 O2 /Cu(II) at the

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indicated concentrations and incubation time. Addition of genistein at 25, 50 and 100 ␮M to H2 O2 /Cu(II) resulted in a partial inhibition of the conversion of supercoiled DNA to open circle and linear forms, indicating that genistein is able to protect against H2 O2 /Cu(II)-mediated DNA damage. The inhibition of H2 O2 /Cu(II)-mediated DNA strand breaks by genistein exhibited a concentration-dependent relationship (Fig. 2A). To compare the inhibitory effects of genistein on H2 O2 /Cu(II)-mediated DNA damage with other ROS scavengers, both mannitol and DMSO were used. Mannitol and DMSO are typical hydroxyl radical scavengers. As shown in Fig. 2B, neither DMSO nor mannitol at up to 10 mM inhibited H2 O2 /Cu(II)-mediated DNA strand breaks. These results indicate that as compared with mannitol and DMSO genistein is a more potent inhibitor of H2 O2 /Cu(II)-mediated DNA strand breaks in this plasmid DNA system. To further examine the inhibitory effect of genistein on oxidative DNA damage, HQ + Cu(II) was used. It has been previously shown that HQ/Cu(II) is able to induce DNA strand breaks, with both Cu(II)/Cu(I) redox cycle and H2 O2 being critically involved [18]. Similar to what was observed with H2 O2 /Cu(II), the presence of genistein at 25–100 ␮M also markedly inhibited the DNA strand breaks induced by HQ/Cu(II) (Fig. 3).

3.2. Failure of genistein to inhibit Cu(II)/Cu(I) redox cycle and to react with H2 O2 With either H2 O2 /Cu(II) or HQ/Cu(II) system, a Cu(II)/Cu(I) redox cycle is critically involved in the production of the reactive species that mediate DNA strand breaks [17,18]. Therefore, the inhibition of H2 O2 /Cu(II)- or HQ/Cu(II)-mediated DNA strand breaks by genistein might occur as a result of the direct effect of genistein on the Cu(II)/Cu(I) redox cycle. To examine this possibility, the reduction of Cu(II) to Cu(I) by H2 O2 was measured in the presence of genistein. As shown in Table 1, the presence of 100 ␮M genistein did not affect the H2 O2 -mediated reduction of Cu(II) to Cu(I) as detected by the BCS assay. Since, a Cu(II)/Cu(I) redox cycle is critically involved in Cu-mediated oxidation of HQ, resulting in oxygen utilization and concomitant production of ROS [19], we further examined the effect of genistein on the oxygen utilization caused by HQ/Cu(II). As shown in Fig. 4, addition of Cu(II) to HQ resulted in a marked consumption of oxygen. However, the HQ/Cu(II)-mediated oxygen consumption was not significantly altered by the presence of 100 ␮M genistein. In contrast, the presence of 50 ␮M BCS, a Cu(I) specific chelator [18,19], completely blocked the HQ/Cu(II)-induced oxygen consumption. These observations strongly suggest that genistein does not affect Cu(II)/Cu(I) redox cycle in either H2 O2 /Cu(II) or HQ/Cu(II) system. Another possibility for the inhibition by genistein of H2 O2 /Cu(II)- or HQ/Cu(II)-mediated DNA strand breaks was that genistein might directly react with H2 O2 . To examine this possibility, 25 ␮M H2 O2 Table 1 Effects of genistein on H2 O2 -mediated reduction of Cu(II) to Cu(I)a

Fig. 3. Effects of genistein on HQ/Cu(II)-mediated DNA strand breaks. The ␾X-174 plasmid DNA was incubated with 5 ␮M HQ + 10 ␮M Cu(II) in the presence or absence of the indicated concentrations of genistein in PBS at 37◦ C for 30 min. The DNA strand breaks were determined as described in Section 2.

Treatment

Formation of Cu(I) (␮M)

25 ␮M H2 O2 + 10 ␮M Cu(II) 25 ␮M H2 O2 + 10 ␮M Cu(II) + 100 ␮M genistein

9.88 ± 0.71 10.33 ± 0.08

a Cu(II) of 10 ␮M was incubated with 25 ␮M H O in the pres2 2 ence or absence of 100 ␮M genistein in PBS containing 0.3 mM BCS at 37◦ C for 30 min. The reduction of Cu(II) to Cu(I) during this 30 min incubation was determined by measuring the formation of the BCS-Cu(I) complex at 480 nm, as described in Section 2. Values represent mean ± S.E. from three separate experiments.

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genistein of the bound hydroxyl radical or its equivalent that has been suggested to mediate the DNA damage. Further studies are needed to investigate the radical-scavenging capacity of genistein. 3.3. Potentiation of H2 O2 /Cu(II)-mediated DNA strand breaks by resveratrol

Fig. 4. Effects of genistein on HQ/Cu(II)-mediated oxygen consumption. Incubation mixtures contained 10 ␮M Cu(II), 100 ␮M genistein + 10 ␮M Cu(II), or 50 ␮M BCS + 10 ␮M Cu(II) in PBS at 37◦ C, and the reaction was initiated by the addition of 50 ␮M HQ. Oxygen consumption was recorded continuously for 8 min with a Clark oxygen electrode, as described in Section 2.

was incubated with 100 ␮M genistein at 37◦ C for 30 min, then the remaining H2 O2 in the reaction mixture was measured using an oxygen electrode. Because genistein inhibited horseradish peroxidase (HRP)-mediated reaction (data not shown), the more commonly used HRP-based assays, such as HRP/p-hydroxyphenylacetate or HRP/scopoletin [22,23] could not be employed to assess the reaction of genistein with H2 O2 . As shown in Table 2, incubation of 100 ␮M genistein with 25 ␮M H2 O2 for 30 min did not result in any significant alteration of the H2 O2 concentration in the reaction mixture. This result indicates that genistein does not directly react with H2 O2 . Because genistein at 100 ␮M, neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H2 O2 , the inhibition of H2 O2 /Cu(II)- or HQ/Cu(II)-mediated DNA strand breaks seen with 25–100 ␮M genistein most likely resulted from the direct scavenging by

Because of its in vivo anticarcinogenic property [1,4,5], we also examined the effects of resveratrol on H2 O2 /Cu(II)-mediated DNA strand breaks. As shown in Fig. 5, the presence of resveratrol at 25–100 ␮M did not inhibit H2 O2 /Cu(II)-induced DNA strand breaks. Rather, addition of resveratrol to H2 O2 /Cu(II) resulted in increased DNA damage, reflected by the increased formation of linear form and degradation of DNA (Fig. 5). 3.4. Induction of DNA strand breaks by resveratrol/Cu(II), but not by genistein/Cu(II) It has been shown that a number of phenolic chemicals in the presence of Cu(II) are capable of inducing DNA strand breaks in isolated DNA [24,25]. Next we determined whether resveratrol in the presence of Cu(II) also caused DNA strand breaks in this plasmid

Table 2 Failure of genistein to react with H2 O2 a Treatment

Remaining H2 O2 (␮M)

25 ␮M H2 O2 25 ␮M H2 O2 + 100 ␮M genistein

25.7 ± 0.5 25.1 ± 1.0

H2 O2 of 25 ␮M was incubated with or without 100 ␮M genistein in PBS at 37◦ C for 30 min, and then the remaining H2 O2 was measured with a Clark oxygen electrode, as described in Section 2. Values represent mean ± S.E. from three separate experiments. a

Fig. 5. Effects of resveratrol on H2 O2 /Cu(II)-mediated DNA strand breaks. The ␾X-174 plasmid DNA was incubated with 25 ␮M H2 O2 + 10 ␮M Cu(II) in the presence or absence of the indicated concentrations of resveratrol in PBS at 37◦ C for 30 min. The DNA strand breaks were determined as described in Section 2.

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presence of Cu were dramatically different (Fig. 6). In data not shown, the absorption spectrum of genistein did not significantly change upon addition of Cu(II), whereas, the spectrum of resveratrol markedly changed upon incubation with Cu(II). The ability of Cu(II) to oxidize resveratrol, resulting in the changes of the absorption spectrum of resveratrol was also described by others [27,28]. These observations indicate that unlike resveratrol, genistein does not appear to react chemically with Cu to a significant extent. 3.5. Involvement of Cu(II)/Cu(I) redox cycle and H2 O2 in resveratrol/Cu(II)-mediated DNA strand breaks Previously, both a Cu(II)/Cu(I) redox cycle and H2 O2 have been shown to be critically involved in the induction of plasmid DNA strand breaks by phenolic chemicals in the presence of Cu(II) [24]. As shown in Fig. 7, addition of either BCS (50 ␮M) or catalase (100 units/ml), but not SOD (100 units/ml) markedly inhibited resveratrol/Cu(II)-mediated DNA strand breaks. This observation indicates that both Cu(II)/ Cu(I) redox cycle and H2 O2 are critical components that lead to production of DNA-damaging species by

Fig. 6. DNA strand breaks induced by genistein + Cu(II) (panel A) and resveratrol + Cu(II) (panel B). The ␾X-174 plasmid DNA was incubated with the indicated concentrations of either genistein (panel A) or resveratrol (panel B) in the presence of 10 ␮M Cu(II) in PBS at 37◦ C for 30 min. The DNA strand breaks were determined as described in Section 2.

system. As shown in Fig. 6, in the presence of 10 ␮M Cu(II), genistein at 25–100 ␮M did not cause any DNA strand breaks, while resveratrol at similar concentrations resulted in a concentration-dependent induction of DNA strand breaks. During the course of this study, two groups also reported the DNA-cleaving effects of resveratrol + Cu in thymus DNA [26,27]. It is interesting to note that although both resveratrol and genistein are phenolic compounds with similar in vivo anticarcinogenic effects [1–5], the in vitro DNA-cleaving effects of these two compounds in the

Fig. 7. Effects of BCS, SOD and catalase on resveratrol/ Cu(II)-mediated DNA strand breaks. The ␾X-174 plasmid DNA was incubated with 100 ␮M resveratrol+10 ␮M Cu(II) in the presence or absence of BCS (50 ␮M), SOD (100 units/ml), or catalase (100 units/ml) in PBS at 37◦ C for 30 min. The DNA strand breaks were determined as described in Section 2.

W. Win et al. / Mutation Research 513 (2002) 113–120 Table 3 Resveratrol-mediated reduction of Cu(II) to Cu(I)a Treatment Resveratrol Resveratrol Resveratrol Resveratrol

Formation of Cu(I) (␮M) + 10 ␮M Cu(II) + 25 ␮M Cu(II) + 50 ␮M Cu(II) + 100 ␮M Cu(II)

9.55 24.33 50.72 98.46

± ± ± ±

0.38 1.15 1.36 1.75

a Resveratrol of 100 ␮M was incubated with the indicated concentrations of Cu(II) in PBS containing 0.3 mM BCS at 37◦ C for 30 min. The reduction of Cu(II) to Cu(I) during this 30 min incubation was determined by measuring the formation of the BCS-Cu(I) complex at 480 nm, as described in Section 2. Values represent mean ± S.E. from three separate experiments.

the resveratrol/Cu(II) system. The involvement of a Cu(II)/Cu(I) redox cycle in resveratrol/Cu(II)-induced DNA strand breaks was further supported by the observation that resveratrol was able to reduce Cu(II) to Cu(I) (Table 3). In summary, although both genistein and resveratrol have similar in vivo anticarcinogenic effects [1–5], these two phenolic compounds exhibited different effects on oxidative DNA damage in isolated plasmid DNA. Genistein at micromolar concentrations markedly inhibited H2 O2 /Cu(II)- or HQ/Cu(II)-mediated DNA strand breaks. Since, it does not appear to either affect the Cu(II)/Cu(I) redox cycle or directly react with H2 O2 , genistein is most likely to act as a scavenger of the ROS that participate in the induction of DNA strand breaks by the H2 O2 /Cu(II) or HQ/Cu(II) system. Previously, studies have suggested that a free hydroxyl radical is not responsible for the induction of DNA damage by H2 O2 /Cu(II) or HQ/Cu(II) system [17,18]. Instead, a bound hydroxyl radical or its equivalent appears to participate in the induction of DNA damage by both H2 O2 /Cu(II) and HQ/Cu(II) [17,18]. Whatever the exact reactive species may be involved in the H2 O2 /Cu(II)- or HQ/Cu(II)-induced DNA strand breaks, it appears that genistein can scavenge these reactive species and thereby protect against the subsequent oxidative DNA damage. The ability of genistein to inhibit oxidative DNA damage in vitro may contribute to its anticarcinogenic effects observed in vivo [2,3]. In contrast to genistein, resveratrol at similar concentrations (25–100 ␮M) did not inhibit H2 O2 /Cu(II)-mediated DNA strand breaks. Rather, the DNA strand breaks were increased by addition of resveratrol to the H2 O2 /Cu(II). This

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was because resveratrol + Cu(II) also induced DNA strand breaks. Similar to many other phenolic chemicals [24], both Cu(II)/Cu(I) redox cycle and H2 O2 are involved in the resveratrol/Cu(II)-mediated DNA strand breaks. The significance of the DNA cleaving effects of resveratrol/Cu(II) in resveratrol-mediated anticarcinogenis effects in vivo [1,4,5] remains to be determined. Recent studies have demonstrated that resveratrol is able to induce apoptosis in several types of tumor cells, which is believed to be involved in the in vivo anticancer effects of resveratrol [29,30]. Because induction of DNA strand breaks in cells may result in apoptosis, and Cu exists naturally in the nuclei and is associated with DNA [31], the DNA cleaving effects of resveratrol in the presence of Cu observed in the present study may thus contribute to its in vivo anticancer activity. In this context, cancer cells have been shown to contain elevated levels of Cu [32]. The different effects of genistein and resveratrol on oxidative DNA damage observed in this study may be attributable to the structural differences between these two phenolic chemicals. As shown in Fig. 1, although both compounds contain one resorcinol and one phenol moieties, these two moieties are linked by two different structures: a pyran-4-one ring in genistein and an ethylene group in resveratrol. Thus, it is plausible that the pyran-4-one structure might be responsible for the different effects of genistein and resveratrol on in vitro oxidative DNA damage demonstrated in this study. Acknowledgements This work was supported by a seed grant from St. John’s University (Y.L.) and the National Institutes of Health grant CA91895 (Y.L.). M.A.T. was supported by the National Institutes of Health grants ES03819 and ES08078. William Win was a fifth-year pharmacy student at St. John’s University. References [1] L. Fremont, Biological effects of resveratrol, Life Sci. 66 (2000) 663–673. [2] S. Safe, M.J. Wargovich, G.A. Lamartiniere, H. Mukhtar, Symposium on mechanisms of action of naturally occurring anticarcinogens, Toxicol. Sci. 52 (1999) 1–8.

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