- Email: [email protected]
Effect of genistein-8-C-glucoside from Lupinus luteus on DNA damage assessed using the comet assay in vitro
Cell Biology International 33 (2009) 247e252 www.elsevier.com/locate/cellbi
Short communication
Effect of genistein-8-C-glucoside from Lupinus luteus on DNA damage assessed using the comet assay in vitro Agata Rucinska, Teresa Gabryelak* Department of General Biophysics, University of Lodz, Banacha 12/16, Lodz 90-237, Poland Received 9 April 2008; revised 17 June 2008; accepted 12 November 2008
Abstract Genistein-8-C-glucoside (G8CG) belongs to natural isoflavones phytoestrogens, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants, with possible anticarcinogenic effects in various in vitro systems and in vivo animal models. We used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.) to study its cytotoxic and genotoxic effects on mouse embryonic fibroblast (line NIH 3T3). The MTT assay to assess cytotoxicity and comet assay for the detection of DNA damage were used. The cells were exposed to various concentrations of genistein-8-C-glucoside (2.5e110 mM) and hydrogen peroxide (5e90 mM). The effect of G8CG alone or in combination with H2O2 was determined. G8CG at concentrations >20 mM significantly reduced cell viability and induced DNA damage. In contrast, lower concentrations of (2.5e10 mM) G8CG showed antioxidant properties against H2O2-induced DNA damage with no associated toxicity. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Genistein-8-C-glucoside; DNA damage; Comet assay; Hydrogen peroxide
1. Introduction Genistein is a major phytoestrogen of plants such as soybeans and is commonly found in a variety of human foods (Liggins et al., 2000). Akiyama et al. (1987) first demonstrated that genistein isolated from Pseudomonas species inhibited the tyrosine-specific protein kinases of the epidermal growth factor (EGF) receptor. Thereafter, studies have focused on the pharmacological activities of genistein as a tyrosine kinase inhibitor, its chemoprotectant activities against cancer and cardiovascular disease, and its phytoestrogen activity. Genistein is a major subject of discussion in the context of nutraceuticals and functional foods, and may provide a case-study for evaluating the delivery of health-promoting compounds through genetically modified plants (Dixon and Ferreira, 2002). These isoflavones are relatively safe, but they are known to exert multiple effects, including antioxidant activity, * Corresponding author. Tel./fax: þ48 42 635 44 74. E-mail address: [email protected] (T. Gabryelak).
and can also induce apoptosis (Fioravanti et al., 1998; Po et al., 2002) and/or genetic damage (Boos and Stopper, 2000). Most isoflavones are present in plants mainly as glycosidic conjugates located mainly in the cell vacuoles. Genistein exists in nature in its glycoside form rather than in its aglycone form. Whether the flavonoids are biologically active in their glycosidic forms or require hydrolysis to their aglycone forms for activity is controversial. Choi et al. (2007) demonstrated that 7-glycoside form of genistein, possesses an anti-proliferative effect on human ovarian cancer SK-OV-3 cells similar to that of genistein. However, the authors demonstrated different effects of genistein and genistin on the cell cycle and induction of apoptosis. Thus both isoflavones can act via different mechanisms or multiple pathways, with possible differences between genistein and genistin (glucose is attached to genistein in genistin) on cell damage being notably associated with absorption velocity in metabolism process (Andlauer et al., 2000). On these and other studies (Kato et al., 2000; Allred et al., 2001), isoflavone glycosides such as genistin have a biological
1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.11.003
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
248
activity that does not require initial hydrolysis to genistein. Genistein-8-C-glucoside induced genotoxicity has not yet been reported, but other studies in vitro suggest that its aglycone form is active (Fig. 1). We have investigated whether the genotoxic activity of G8CG is similar to the activity of a free form of genistein. The work demonstrates cytotoxic and genotoxic potential of genistein-8-C-glucoside isolated from Lupinus luteus L. in estrogen receptor-negative mouse embryo fibroblasts (NIH 3T3 cells) to avoid stimulatory effects of the tested compound on cell proliferation. We also examined the effect of G8CG on DNA damage induced by hydrogen peroxide.
column (3 80 cm) packed with polyamine. G8CG was eluted by ethanolewater gradient. Genistein-8-C-glucoside was a beige powder with a purity of 97.5% and was stored in a refrigerator, protected from light, and under nitrogen. G8CG was taken from stock solution (500 mM) in 1% dimethyl sulfoxide (DMSO), which had no detectable effect on NIH 3T3 cell viability. G8CG was added to suspensions of cultured cells at from 2.5 to 110 mM. Hydrogen peroxide was added to the cell suspension in the range of 5e90 mM. Control cells were treated with PBS (pH 7.4). Cells were incubated with the G8CG for 1 h at 37 C. The effect of G8CG alone or in combination with H2O2 was determined.
2. Materials and methods
2.4. In vitro proliferation inhibition assay
2.1. Chemical and reagents
Proliferation of cells was measured by the method of Mossman (1983), based on the cleavage and conversion of the soluble yellow dye MTT to water-insoluble purple formazan in living cells. Cells were placed in 96-well microtiter plates at 4 104 cells in 200 ml per well. Cells were treated with genistein (2.5e110 mM) at 37 C in a 5% carbon dioxidee 95% air atmosphere for 24 h, and recovered after two gentle washes with PBS (pH 7.4). MTT (3-[4, 5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) was dissolved in PBS at 5 mg/ml. Briefly, 50 ml of MTT solution was added to each well, followed by 3 h of incubation. MTT-containing medium was removed, and 100 ml of DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 545 nm with a correction at 630 nm using an ELISA plate reader (Awarness Technology Inc.) Stat Fax Type. Cell proliferation was calculated as the percent ratio of absorbance of the samples to the referent control.
Normal melting-point (NMP) and low melting-point (LMP) agarose, phosphate-buffered saline (PBS), 40 ,6-diamidino-2phenylindole (DAPI), dimethyl sulfoxide (DMSO), 3-[4,5-2yl]-2-5-diphenyltetrazolium bromide (MTT), and hydrogen peroxide were purchased from Sigma (St. Louis, MO, USA). DMEM minimal essential medium, trypsin, L-glutamine, new born calf serum and gentamycin were obtained from Gibco (BRL). Tissue culture dishes and flasks were purchased from Nuclon (Roskilde, Denmark). All other reagents and solvents were of analytical grade. 2.2. Cell culture Mouse embryonic fibroblasts (cell line NIH 3T3), used as cellular model, were purchased from Child Health Centre in Warsaw (Poland). Cells were grown as monolayer in DMEM medium supplemented with 10% new born calf serum with 100 U/ml gentamycin. The cultures were incubated at 37 C in an atmosphere of 5% CO2 and 95% air with >95% humidity. Exponentially growing cells were dispersed by a scraper or by treatment with 0.1% trypsin, and diluted with the growth medium for further experiments. 2.3. Isolation of genistein-8-C-glucoside and treatment of cells Genistein-8-C-glucoside was isolated from flowers of L. luteus L. by the method of Laman and Volynets (1974). Isoflavone was subsequently extracted by methanol, ethylacetate, n-butanol and purified using a chromatographic
Fig. 1. Chemical structures of genistein-8-C-glucoside.
2.5. Detection of DNA damage by comet assay DNA strand breaks were evaluated using single-cell gel electrophoresis e the comet assay of Singh et al. (1988) and Duthie et al. (1996), with some modifications of B1asiak and Kowalik (2000). Cells (2.5e3 105) were seeded into 10 cm Petri dishes 24 h before they were exposed to G8CG alone (2.5e110 mM) or in combination with H2O2 (5e90 mM in PBS) at 37 C for 1 h and 30 min. After incubation, the culture medium was removed, the cells washed twice with ice-cold PBS (pH 7.4), trypsinized and resuspended in fresh culture medium. An aliquot of 30 ml of the cells was mixed with 50 ml of 0.75% low melting-point (LMP) agarose at 37 C and rapidly spread on microscope slides pre-coated with 0.5% normal melting-point (NMP) agarose. The slides were covered with a coverslip and allowed to solidify on ice. After 10 min, the slides were placed in a freshly made cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 which 10% DMSO and 1% Triton X-100 had been added immediately prior to use) for 1 h at 4 C. After lysis, the slides were placed in electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 min to allow unwinding of the DNA and DNA breakage at alkali-labile sites. Electrophoresis was conducted in the same buffer by applying an electric current of 0.73 V/cm
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
249
whereas 30 mM or higher significantly reduced the percentage of viable fibroblasts was in a dose-dependent manner (IC50 ¼ 71 mM). The results show this phytoestrogen has cytotoxic properties in NIH 3T3 cells, which is a hormonesensitive cell line.
(280 mA) for 20 min. Finally, the slides were washed in neutralization buffer (0.4 M Tris, pH 7.5) 3 times for 5 min each, dried and stained with DAPI (2 mg/ml). To assess DNA repair, the slides were placed in tubes with PBS (pH 7.4) before lysis and incubated for 2 h at 4 C. After incubation, they were washed in PBS and staining processes were carried out as above. The comets were analyzed at 200 magnification with an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (Cohu, San Diego, CA), and equipped with a UV-1 filter block (excitation filter of 359 nm and barrier filter of 461 nm). This was connected to a personal computer-based image analysis system, Lucia-Comet 4.51 (Laboratory Imaging, Prague, Czech Republic). Fifty cells were randomly selected from each sample and the fraction of DNA in the tail was measured. Tail DNA was determined to assess the extent of DNA damage.
3.2. Genistein-8-C-glucoside and DNA strand breaks Cell suspensions exposed for 1 h to genistein-8-C-glucoside over the range from 2.5 to 110 mM were also incubated with hydrogen peroxide at from 5 to 90 mM in PBS. The incubation with H2O2 lasted 0.5 h after pre-incubating cells with G8CG. The doseeresponse relationship of G8CG (Fig. 3A and B) indicates the levels of DNA strand breaks expressed as the percentage of total fluorescence migrating in the tail for each nucleus (% DNA in tail). G8CG contributed to DNA damage at doses 2.5, 5, 7.5 and 10 mM, but there were no significant changes in DNA in comparison with untreated control cells (Fig. 3A). G8CG exhibits genotoxic properties and the level of strand breaks increased with the growing concentrations of phytoestrogen. Genistein-8-C-glucoside significantly reduced DNA damage caused by hydrogen peroxide at 2.5e10 mM (Fig. 3B). The data show that for 5 and 7.5 mM, G8CG gave the most protection against oxidative properties of H2O2. The possibility of DNA repair of the changes caused by the phytoestrogens at 7 concentrations (15e110 mM) was checked. Fig. 4 demonstrates that although strand breaks in nucleic acid can be repaired 3 h after removing genotoxic agent, the mechanism is most effective 12 h after G8CG removal, with an 18 h repair period being no better.
2.6. Statistics The results are presented as mean SEM for the comet assay and mean SD for the other methods. The statistical difference between the control and treated groups was evaluated by the Student0 s t-test. A P value <0.05, 0.01 and 0.001 was accepted as statistically significant. 3. Results 3.1. Cell proliferation inhibition by genistein-8-Cglucoside The cytotoxic effect of genistein-8-C-glucoside was determined directly after 1 h of incubation with G8CG, which showed a concentration-dependent inhibition of cell growth (Fig. 2). Low-dose G8CG (2.5e20 mM) did not result in proliferation of NIH 3T3 cells when compared to control,
4. Discussion The structure of genistein resembles that of endogenous estrogens and can bind to the estrogen b receptor (ERb),
120
cell viability (
of control)
100
*
80
* 60
** ***
40
*** 20
0 control
2.5
5
7.5
10
15
20
30
50
70
9
0
110
G8CG [µM] Fig. 2. Cell viability after 1 h exposure at 37 C of NIH 3T3 cells to genistein-8-C-glucoside (G8CG). The data are expressed as percentage of the control value (value obtained for untreated cells immediately after 1 h incubation). The results were obtained from 4 individual experiments. Error bars denote SD; *P < 0.05; **P < 0.01; ***P < 0.001.
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
250
A 40
** *
* DNA in tail
30
* * *
20
*
10
0 control
2.5
5
7.5
10
15
20
30
50
70
90
110
G8CG [µM]
B 60 0 µM G8CG 50
*
2.5 µM G8CG
DNA in tail
5 µM G8CG 40
7.5 µM G8CG
*
*
10 µM G8CG
***
*
30
*
20
* 10
* *
* **
*
*
*
*
*
* *
*
*
0 control
5
10
15
30
60
90
H2O2 [µM] Fig. 3. DNA damage of NIH 3T3 cells exposed for 1 h at 37 C to: (A) genistein-8-C-glucoside (G8CG); (B) genistein-8-C-glucoside (G8CG) and hydrogen peroxide (H2O2) for 30 min. The level of DNA breaks is expressed as the percentage of DNA migrating in the tail of the comet (percentage of DNA in the tail). The number of cells in each of 4 individual treatments was 50. Error bars denote SEM.; *P < 0.05; **P < 0.01.
acting as ER agonists or antagonists. Although the biological activity of genistein has often been attributed to its competitive affinity for ER, genistein may also modify cancer risk via biochemical mechanisms such as apoptosis, cell growth modification and other signal pathways interactions that might be unrelated to its estrogenic activity. Numerous in vitro studies have reported non-glycosylated genistein to be clastogenic (chromosome breaking), DNA damaging and even mutagenic, in contrast to in vivo studies that generally have shown no genotoxicity (e.g. McClain et al., 2006). Clastogenesis or DNA mutations are thought to be carcinogenic events in cells that are damaged but not killed by exposure to toxic agents. At high concentrations, genistein induces micronucleus formation in Chinese hamster V79 cells, indicative of chromosome breakage (Snyder and Gillies, 2003). Other investigators had also reported micronucleus formation and DNA strand breaks in cultured V79 cells (Kulling and Metzler, 1997), human lymphoblastoid cells exposed to genistein (Morris et al., 1998), and human peripheral blood lymphocytes (Kulling et al., 1999) after in vitro exposure to genistein.
In contrast, other studies suggest that genistein can be beneficial in in vitro and in vivo systems (Dixon and Ferreira, 2002). Genistein possesses antioxidant properties and inhibits DNA topoisomerase II activity (Kaufmann, 1998), angiogenesis, and invasion (Shao et al., 1998; Alhasan et al., 2001), all of which can contribute to chemoprevention (Lamartiniere, 2000). Our data show that phytoestrogens G8CG have cytotoxic properties in vitro. ‘‘Physiologically relevant’’ concentrations (2.5e10 mM) of G8CG were not toxic, while higher concentrations (>20 mM) decreased proliferation of cells in a dosedependent manner (Fig. 2), which is in agreement with other in vitro results (Zava and Duwe, 1997; Hsieh et al., 1998; Chen and Donovan, 2004), which indicated that the high concentrations (>20 mM) showing that non-glycosylated genistein caused cytotoxicity and DNA ladder formation. Gupta et al. (2002) demonstrated that all flavonoids are cytotoxic at high concentrations, but more realistically the effects of flavonoids on cell damage will depend on their structure (Casagrande and Darbon, 2001). Oxidative DNA damage may play an important role in carcinogenesis (Shibutani et al., 1991). In NIH 3T3 cells,
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
251
15 µM G8CG 20 µM G8CG 60
30 µM G8CG 50 µM G8CG
DNA in tail
70 µM G8CG 90 µM G8CG
40
110 µM G8CG * *
*
*
*
*
* *
20
*
*
*
* **
*
*
** **** **
* **
*****
*
** **** **
0 0
0.5
3
6
12
18
[h] Fig. 4. DNA damage of NIH 3T3 cells exposed for 1 h at 37 C to the concentrations of genistein-8-C-glucoside (G8CG): 15, 20, 30, 50, 70, 90 and 110 mM. The level of DNA breaks is expressed as the percentage of DNA migrating in the tail of the comet (percentage of DNA in the tail). The data demonstrated the DNA damage and DNA repair after 0.5, 3, 6, 12 and 18 h after removing the G8CG. The number of cells in each of 4 independent treatments was 50. Error bars denote SEM.; *P < 0.05; **P < 0.01; ***P < 0.001.
G8CG induced detectable damage (comets) only at high, cytotoxic concentrations (Fig. 3A). Studies conducted in vitro of non-glycosylated genistein suggest it is mutagenic. The positive findings for genotoxicity have only been observed in in vitro systems, generally being negative in vivo. The mechanism of genistein genotoxicity may involve inhibition of topoisomerase II, an enzyme involved in DNA replication. Genistein binds to and stabilizes the topoisomeraseeDNA complex inhibiting re-ligation, resulting in DNA strand breaks (Markovits et al., 1989; Kaufmann, 1998). Treatment with H2O2 caused significant DNA damage in our NIH fibroblasts. In contrast, cells previously supplemented with G8CG (2.5e 10 mM) were from oxidative injury to their DNA (Fig. 3B). The antioxidant activity of genistein with respect to DNA damage has been reported indicating that it inhibits carcinogenesis induced by H2O2, as well as protecting DNA against UV light and Fenton reaction induced damage (Wei et al., 1996). Our data are also consistent with that of Foti et al. (2005), who showed that the DNA protective effect of genistein is lost at above 10 mM. Genistein is an effective scavenger of free radicals as well as a strong inhibitor of hydrogen peroxide formation, not only in vivo but also in vitro (Wei et al., 1993; Record et al., 1995). Ruiz-Larrea et al. (1997) have shown that genistein is the most potent antioxidant among isoflavones in both the aqueous and lipophilic phases of cells, suggesting the importance of the specific molecular structure of genistein, not only of the 40 hydroxyl group but also the 50 -hydroxyl group. In vitro lipoprotein oxidation studies suggest that some oxidative metabolites of genistein, particularly 3-OH-genistein, may be more potent than genistein itself (Rufer and Kulling, 2006). The possibility of DNA to repair the damage caused by G8CG has been shown in Fig. 4. Twelve hours was the shortest time at which effective repair could be seen. In previous studies (Rucinska et al., 2007), we reported that genistein-8-Cglucoside induces oxidative DNA damage and acts as an antioxidant to potentially prevent such damage. However,
repair process of DNA damage in the present study occurred considerably faster and more efficiently. In conclusion, our study reports a hypothesis that, not only genistein, but also genistein-8-C-glucoside demonstrates a double nature of action in vitro. G8CG at ‘‘physiological concentrations’’ increases cell protection against oxidative stress, while higher concentrations are cytotoxic and genotoxic to NIH 3T3 fibroblasts. Acknowledgements We would like to thank Dr Sergej Kirko (National Academy of Sciences, Belarus) for the isolation of genistein8-C-glucoside. References Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, et al. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987;262:5592e5. Alhasan SA, Aranha O, Sarkar FH. Genistein elicits pleiotropic molecular effects on head and neck cancer cells. Clin Cancer Res 2001;7:4174e81. Allred CD, Ju YH, Allred KF, Chang J, Helferich WG. Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein. Carcinogenesis 2001;22:1667e73. Andlauer W, Kolb J, Furst P. Absorption and metabolism of genistin in the isolated rat small intestine. FEBS Lett 2000;475(2):127e30. B1asiak J, Kowalik J. A comparison of the in vitro genotoxicity of tri- and hexavalent chromium. Mutat Res 2000;469:135e45. Boos G, Stopper H. Genotoxicity of several clinically used topoisomerase II inhibitors. Toxicol Lett 2000;116:7e16. Casagrande F, Darbon JM. Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: regulation of cyclin-dependent kinases CDK2 and CDK1. Biochem Pharm 2001;61:1205e15. Chen AC, Donovan SM. Genistein at a concentration present in soy infant formula inhibits Caco-2BBe cell proliferation by causing G2/M cell cycle arrest. J Nutr 2004;134:1303e8. Choi EJ, Kim T, Lee M. Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells. Life Sci 2007;80: 1403e8.
252
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
Dixon RA, Ferreira D. Genistein. Phytochemistry 2002;60:205e11. Duthie SJ, Ma O, Ross MA, Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res 1996;56:1291e5. Fioravanti L, Cappelletti V, Miodini P, Ronchi E, Brivio M, Di Fronzo G. Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro. Cancer Lett 1998;130:143e52. Foti P, Erba D, Riso P, Spadafranca A, Criscuoli F, Testolin G. Comparison between daidzein and genistein antioxidant activity in primary and cancer lymphocytes. Arch Biochem Biophys 2005;433:421e7. Gupta S, Saha B, Giri AK. Comparative antimutagenic and anticlastogenic effects of green tea and black tea: a review. Mutat Res 2002;512:37e65. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 1998;58:3833e8. Kato K, Takahashi S, Cui L, Toda T, Suzuki S, Futakuchi M, et al. Suppressive effects of dietary genistin and daidzin on rat prostate carcinogenesis. Japan J Cancer Res 2000;91:786e91. Kaufmann SH. Cell death induced by topoisomerase targeted drugs: more questions than answers. Biochim Biophys Acta 1998;1400:195e211. Kulling SE, Metzler M. Induction of micronuclei, DNA strand breaks and HRRT mutations in cultured Chinese hamster V79 cells by the phytoestrogen coumoestrol. Food Chem Toxicol 1997;35:605e13. Kulling SE, Rosenberg B, Jacobs E, Metzler M. The phytoestrogens coumoestrol and genistein induce structural chromosomal aberrations in cultured human peripheral blood lymphocytes. Arch Toxicol 1999;73: 50e4. Laman NA, Volynets AP. Flavonoids in ontogenesis of Lupinus luteus L. Plant Physiol (Rus) 1974;21:737e45. Lamartiniere CA. Protection against breast cancer with genistein: a component of soy. Am J Clin Nutr 2000;71:1705Se7S. Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and genistein contents of vegetables. J Nutr 2000;84:717e25. Markovits J, Linassier C, Fosse P, Couprie J, Pierre J, Jacquemin-Sablon A, et al. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 1989;49:5111e7. McClain RM, Wolz E, Davidovich A, Bausch J. Genetic toxicity studies with genistein. Food Chem Toxicol 2006;44:42e55.
Morris SM, Chen JJ, Domon OE, McGarrity LJ, Bishop ME, Manjanatha MG, et al. p53, mutations, and apoptosis in genistein-exposed human lymphoblastoid cells. Mutat Res 1998;405:41e56. Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity. J Immunol Methods 1983;65: 55e63. Po LS, Wang TT, Chen ZY, Leung LK. Genistein-induced apoptosis in MCF-7 cells involves changes in Bak and Bcl-x without evidence of anti-oestrogenic effects. J Nutr 2002;88:463e9. Record IR, Dreosti IE, McInerney JK. The antioxidant activity of genistein in vitro. J Nutr Biochem 1995;6:481e5. Rucinska A, Kirko S, Gabryelak T. Effect of the phytoestrogen, genistein-8-Cglycoside on Chinese hajster ovary cells in vitro. Cell Biol Int 2007;31: 1371e8. Rufer CE, Kulling SE. Antioxidant activity of isoflavones and their major metabolites using different in vitro assays. J Agric Food Chem 2006;54: 2926e31. Ruiz-Larrea MB, Mohan AR, Paganda G, Miller NJ, Bolwell GP, RiceEvans CA. Antioxidant activity of phytoestrogenic isoflavones. Free Rad Res 1997;26:63e70. Shao ZM, Wu J, Shen ZZ, Barsky SH. Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 1998;58:4851e7. Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 1991; 349:431e4. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184e91. Snyder RD, Gillies PJ. Reduction of genistein clastogenicity in Chinese hamster V79 cells by daidzeiny and other flavonoids. Food Chem Toxicol 2003;41:1291e8. Wei H, Cai Q, Rahn RQ. Carcinogenesis 1996;17:73e7. Wei H, Wie L, Frenkel K, Bowen R, Barnes S. Inhibition of tumor promoterinduced hydrogen peroxide formation in vitro and in vivo by genistein. Nutr Cancer 1993;20:1e12. Zava D, Duwe G. Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer 1997; 27:31e40.
Short communication
Effect of genistein-8-C-glucoside from Lupinus luteus on DNA damage assessed using the comet assay in vitro Agata Rucinska, Teresa Gabryelak* Department of General Biophysics, University of Lodz, Banacha 12/16, Lodz 90-237, Poland Received 9 April 2008; revised 17 June 2008; accepted 12 November 2008
Abstract Genistein-8-C-glucoside (G8CG) belongs to natural isoflavones phytoestrogens, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants, with possible anticarcinogenic effects in various in vitro systems and in vivo animal models. We used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.) to study its cytotoxic and genotoxic effects on mouse embryonic fibroblast (line NIH 3T3). The MTT assay to assess cytotoxicity and comet assay for the detection of DNA damage were used. The cells were exposed to various concentrations of genistein-8-C-glucoside (2.5e110 mM) and hydrogen peroxide (5e90 mM). The effect of G8CG alone or in combination with H2O2 was determined. G8CG at concentrations >20 mM significantly reduced cell viability and induced DNA damage. In contrast, lower concentrations of (2.5e10 mM) G8CG showed antioxidant properties against H2O2-induced DNA damage with no associated toxicity. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Genistein-8-C-glucoside; DNA damage; Comet assay; Hydrogen peroxide
1. Introduction Genistein is a major phytoestrogen of plants such as soybeans and is commonly found in a variety of human foods (Liggins et al., 2000). Akiyama et al. (1987) first demonstrated that genistein isolated from Pseudomonas species inhibited the tyrosine-specific protein kinases of the epidermal growth factor (EGF) receptor. Thereafter, studies have focused on the pharmacological activities of genistein as a tyrosine kinase inhibitor, its chemoprotectant activities against cancer and cardiovascular disease, and its phytoestrogen activity. Genistein is a major subject of discussion in the context of nutraceuticals and functional foods, and may provide a case-study for evaluating the delivery of health-promoting compounds through genetically modified plants (Dixon and Ferreira, 2002). These isoflavones are relatively safe, but they are known to exert multiple effects, including antioxidant activity, * Corresponding author. Tel./fax: þ48 42 635 44 74. E-mail address: [email protected] (T. Gabryelak).
and can also induce apoptosis (Fioravanti et al., 1998; Po et al., 2002) and/or genetic damage (Boos and Stopper, 2000). Most isoflavones are present in plants mainly as glycosidic conjugates located mainly in the cell vacuoles. Genistein exists in nature in its glycoside form rather than in its aglycone form. Whether the flavonoids are biologically active in their glycosidic forms or require hydrolysis to their aglycone forms for activity is controversial. Choi et al. (2007) demonstrated that 7-glycoside form of genistein, possesses an anti-proliferative effect on human ovarian cancer SK-OV-3 cells similar to that of genistein. However, the authors demonstrated different effects of genistein and genistin on the cell cycle and induction of apoptosis. Thus both isoflavones can act via different mechanisms or multiple pathways, with possible differences between genistein and genistin (glucose is attached to genistein in genistin) on cell damage being notably associated with absorption velocity in metabolism process (Andlauer et al., 2000). On these and other studies (Kato et al., 2000; Allred et al., 2001), isoflavone glycosides such as genistin have a biological
1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.11.003
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
248
activity that does not require initial hydrolysis to genistein. Genistein-8-C-glucoside induced genotoxicity has not yet been reported, but other studies in vitro suggest that its aglycone form is active (Fig. 1). We have investigated whether the genotoxic activity of G8CG is similar to the activity of a free form of genistein. The work demonstrates cytotoxic and genotoxic potential of genistein-8-C-glucoside isolated from Lupinus luteus L. in estrogen receptor-negative mouse embryo fibroblasts (NIH 3T3 cells) to avoid stimulatory effects of the tested compound on cell proliferation. We also examined the effect of G8CG on DNA damage induced by hydrogen peroxide.
column (3 80 cm) packed with polyamine. G8CG was eluted by ethanolewater gradient. Genistein-8-C-glucoside was a beige powder with a purity of 97.5% and was stored in a refrigerator, protected from light, and under nitrogen. G8CG was taken from stock solution (500 mM) in 1% dimethyl sulfoxide (DMSO), which had no detectable effect on NIH 3T3 cell viability. G8CG was added to suspensions of cultured cells at from 2.5 to 110 mM. Hydrogen peroxide was added to the cell suspension in the range of 5e90 mM. Control cells were treated with PBS (pH 7.4). Cells were incubated with the G8CG for 1 h at 37 C. The effect of G8CG alone or in combination with H2O2 was determined.
2. Materials and methods
2.4. In vitro proliferation inhibition assay
2.1. Chemical and reagents
Proliferation of cells was measured by the method of Mossman (1983), based on the cleavage and conversion of the soluble yellow dye MTT to water-insoluble purple formazan in living cells. Cells were placed in 96-well microtiter plates at 4 104 cells in 200 ml per well. Cells were treated with genistein (2.5e110 mM) at 37 C in a 5% carbon dioxidee 95% air atmosphere for 24 h, and recovered after two gentle washes with PBS (pH 7.4). MTT (3-[4, 5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) was dissolved in PBS at 5 mg/ml. Briefly, 50 ml of MTT solution was added to each well, followed by 3 h of incubation. MTT-containing medium was removed, and 100 ml of DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 545 nm with a correction at 630 nm using an ELISA plate reader (Awarness Technology Inc.) Stat Fax Type. Cell proliferation was calculated as the percent ratio of absorbance of the samples to the referent control.
Normal melting-point (NMP) and low melting-point (LMP) agarose, phosphate-buffered saline (PBS), 40 ,6-diamidino-2phenylindole (DAPI), dimethyl sulfoxide (DMSO), 3-[4,5-2yl]-2-5-diphenyltetrazolium bromide (MTT), and hydrogen peroxide were purchased from Sigma (St. Louis, MO, USA). DMEM minimal essential medium, trypsin, L-glutamine, new born calf serum and gentamycin were obtained from Gibco (BRL). Tissue culture dishes and flasks were purchased from Nuclon (Roskilde, Denmark). All other reagents and solvents were of analytical grade. 2.2. Cell culture Mouse embryonic fibroblasts (cell line NIH 3T3), used as cellular model, were purchased from Child Health Centre in Warsaw (Poland). Cells were grown as monolayer in DMEM medium supplemented with 10% new born calf serum with 100 U/ml gentamycin. The cultures were incubated at 37 C in an atmosphere of 5% CO2 and 95% air with >95% humidity. Exponentially growing cells were dispersed by a scraper or by treatment with 0.1% trypsin, and diluted with the growth medium for further experiments. 2.3. Isolation of genistein-8-C-glucoside and treatment of cells Genistein-8-C-glucoside was isolated from flowers of L. luteus L. by the method of Laman and Volynets (1974). Isoflavone was subsequently extracted by methanol, ethylacetate, n-butanol and purified using a chromatographic
Fig. 1. Chemical structures of genistein-8-C-glucoside.
2.5. Detection of DNA damage by comet assay DNA strand breaks were evaluated using single-cell gel electrophoresis e the comet assay of Singh et al. (1988) and Duthie et al. (1996), with some modifications of B1asiak and Kowalik (2000). Cells (2.5e3 105) were seeded into 10 cm Petri dishes 24 h before they were exposed to G8CG alone (2.5e110 mM) or in combination with H2O2 (5e90 mM in PBS) at 37 C for 1 h and 30 min. After incubation, the culture medium was removed, the cells washed twice with ice-cold PBS (pH 7.4), trypsinized and resuspended in fresh culture medium. An aliquot of 30 ml of the cells was mixed with 50 ml of 0.75% low melting-point (LMP) agarose at 37 C and rapidly spread on microscope slides pre-coated with 0.5% normal melting-point (NMP) agarose. The slides were covered with a coverslip and allowed to solidify on ice. After 10 min, the slides were placed in a freshly made cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 which 10% DMSO and 1% Triton X-100 had been added immediately prior to use) for 1 h at 4 C. After lysis, the slides were placed in electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 min to allow unwinding of the DNA and DNA breakage at alkali-labile sites. Electrophoresis was conducted in the same buffer by applying an electric current of 0.73 V/cm
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
249
whereas 30 mM or higher significantly reduced the percentage of viable fibroblasts was in a dose-dependent manner (IC50 ¼ 71 mM). The results show this phytoestrogen has cytotoxic properties in NIH 3T3 cells, which is a hormonesensitive cell line.
(280 mA) for 20 min. Finally, the slides were washed in neutralization buffer (0.4 M Tris, pH 7.5) 3 times for 5 min each, dried and stained with DAPI (2 mg/ml). To assess DNA repair, the slides were placed in tubes with PBS (pH 7.4) before lysis and incubated for 2 h at 4 C. After incubation, they were washed in PBS and staining processes were carried out as above. The comets were analyzed at 200 magnification with an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (Cohu, San Diego, CA), and equipped with a UV-1 filter block (excitation filter of 359 nm and barrier filter of 461 nm). This was connected to a personal computer-based image analysis system, Lucia-Comet 4.51 (Laboratory Imaging, Prague, Czech Republic). Fifty cells were randomly selected from each sample and the fraction of DNA in the tail was measured. Tail DNA was determined to assess the extent of DNA damage.
3.2. Genistein-8-C-glucoside and DNA strand breaks Cell suspensions exposed for 1 h to genistein-8-C-glucoside over the range from 2.5 to 110 mM were also incubated with hydrogen peroxide at from 5 to 90 mM in PBS. The incubation with H2O2 lasted 0.5 h after pre-incubating cells with G8CG. The doseeresponse relationship of G8CG (Fig. 3A and B) indicates the levels of DNA strand breaks expressed as the percentage of total fluorescence migrating in the tail for each nucleus (% DNA in tail). G8CG contributed to DNA damage at doses 2.5, 5, 7.5 and 10 mM, but there were no significant changes in DNA in comparison with untreated control cells (Fig. 3A). G8CG exhibits genotoxic properties and the level of strand breaks increased with the growing concentrations of phytoestrogen. Genistein-8-C-glucoside significantly reduced DNA damage caused by hydrogen peroxide at 2.5e10 mM (Fig. 3B). The data show that for 5 and 7.5 mM, G8CG gave the most protection against oxidative properties of H2O2. The possibility of DNA repair of the changes caused by the phytoestrogens at 7 concentrations (15e110 mM) was checked. Fig. 4 demonstrates that although strand breaks in nucleic acid can be repaired 3 h after removing genotoxic agent, the mechanism is most effective 12 h after G8CG removal, with an 18 h repair period being no better.
2.6. Statistics The results are presented as mean SEM for the comet assay and mean SD for the other methods. The statistical difference between the control and treated groups was evaluated by the Student0 s t-test. A P value <0.05, 0.01 and 0.001 was accepted as statistically significant. 3. Results 3.1. Cell proliferation inhibition by genistein-8-Cglucoside The cytotoxic effect of genistein-8-C-glucoside was determined directly after 1 h of incubation with G8CG, which showed a concentration-dependent inhibition of cell growth (Fig. 2). Low-dose G8CG (2.5e20 mM) did not result in proliferation of NIH 3T3 cells when compared to control,
4. Discussion The structure of genistein resembles that of endogenous estrogens and can bind to the estrogen b receptor (ERb),
120
cell viability (
of control)
100
*
80
* 60
** ***
40
*** 20
0 control
2.5
5
7.5
10
15
20
30
50
70
9
0
110
G8CG [µM] Fig. 2. Cell viability after 1 h exposure at 37 C of NIH 3T3 cells to genistein-8-C-glucoside (G8CG). The data are expressed as percentage of the control value (value obtained for untreated cells immediately after 1 h incubation). The results were obtained from 4 individual experiments. Error bars denote SD; *P < 0.05; **P < 0.01; ***P < 0.001.
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
250
A 40
** *
* DNA in tail
30
* * *
20
*
10
0 control
2.5
5
7.5
10
15
20
30
50
70
90
110
G8CG [µM]
B 60 0 µM G8CG 50
*
2.5 µM G8CG
DNA in tail
5 µM G8CG 40
7.5 µM G8CG
*
*
10 µM G8CG
***
*
30
*
20
* 10
* *
* **
*
*
*
*
*
* *
*
*
0 control
5
10
15
30
60
90
H2O2 [µM] Fig. 3. DNA damage of NIH 3T3 cells exposed for 1 h at 37 C to: (A) genistein-8-C-glucoside (G8CG); (B) genistein-8-C-glucoside (G8CG) and hydrogen peroxide (H2O2) for 30 min. The level of DNA breaks is expressed as the percentage of DNA migrating in the tail of the comet (percentage of DNA in the tail). The number of cells in each of 4 individual treatments was 50. Error bars denote SEM.; *P < 0.05; **P < 0.01.
acting as ER agonists or antagonists. Although the biological activity of genistein has often been attributed to its competitive affinity for ER, genistein may also modify cancer risk via biochemical mechanisms such as apoptosis, cell growth modification and other signal pathways interactions that might be unrelated to its estrogenic activity. Numerous in vitro studies have reported non-glycosylated genistein to be clastogenic (chromosome breaking), DNA damaging and even mutagenic, in contrast to in vivo studies that generally have shown no genotoxicity (e.g. McClain et al., 2006). Clastogenesis or DNA mutations are thought to be carcinogenic events in cells that are damaged but not killed by exposure to toxic agents. At high concentrations, genistein induces micronucleus formation in Chinese hamster V79 cells, indicative of chromosome breakage (Snyder and Gillies, 2003). Other investigators had also reported micronucleus formation and DNA strand breaks in cultured V79 cells (Kulling and Metzler, 1997), human lymphoblastoid cells exposed to genistein (Morris et al., 1998), and human peripheral blood lymphocytes (Kulling et al., 1999) after in vitro exposure to genistein.
In contrast, other studies suggest that genistein can be beneficial in in vitro and in vivo systems (Dixon and Ferreira, 2002). Genistein possesses antioxidant properties and inhibits DNA topoisomerase II activity (Kaufmann, 1998), angiogenesis, and invasion (Shao et al., 1998; Alhasan et al., 2001), all of which can contribute to chemoprevention (Lamartiniere, 2000). Our data show that phytoestrogens G8CG have cytotoxic properties in vitro. ‘‘Physiologically relevant’’ concentrations (2.5e10 mM) of G8CG were not toxic, while higher concentrations (>20 mM) decreased proliferation of cells in a dosedependent manner (Fig. 2), which is in agreement with other in vitro results (Zava and Duwe, 1997; Hsieh et al., 1998; Chen and Donovan, 2004), which indicated that the high concentrations (>20 mM) showing that non-glycosylated genistein caused cytotoxicity and DNA ladder formation. Gupta et al. (2002) demonstrated that all flavonoids are cytotoxic at high concentrations, but more realistically the effects of flavonoids on cell damage will depend on their structure (Casagrande and Darbon, 2001). Oxidative DNA damage may play an important role in carcinogenesis (Shibutani et al., 1991). In NIH 3T3 cells,
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
251
15 µM G8CG 20 µM G8CG 60
30 µM G8CG 50 µM G8CG
DNA in tail
70 µM G8CG 90 µM G8CG
40
110 µM G8CG * *
*
*
*
*
* *
20
*
*
*
* **
*
*
** **** **
* **
*****
*
** **** **
0 0
0.5
3
6
12
18
[h] Fig. 4. DNA damage of NIH 3T3 cells exposed for 1 h at 37 C to the concentrations of genistein-8-C-glucoside (G8CG): 15, 20, 30, 50, 70, 90 and 110 mM. The level of DNA breaks is expressed as the percentage of DNA migrating in the tail of the comet (percentage of DNA in the tail). The data demonstrated the DNA damage and DNA repair after 0.5, 3, 6, 12 and 18 h after removing the G8CG. The number of cells in each of 4 independent treatments was 50. Error bars denote SEM.; *P < 0.05; **P < 0.01; ***P < 0.001.
G8CG induced detectable damage (comets) only at high, cytotoxic concentrations (Fig. 3A). Studies conducted in vitro of non-glycosylated genistein suggest it is mutagenic. The positive findings for genotoxicity have only been observed in in vitro systems, generally being negative in vivo. The mechanism of genistein genotoxicity may involve inhibition of topoisomerase II, an enzyme involved in DNA replication. Genistein binds to and stabilizes the topoisomeraseeDNA complex inhibiting re-ligation, resulting in DNA strand breaks (Markovits et al., 1989; Kaufmann, 1998). Treatment with H2O2 caused significant DNA damage in our NIH fibroblasts. In contrast, cells previously supplemented with G8CG (2.5e 10 mM) were from oxidative injury to their DNA (Fig. 3B). The antioxidant activity of genistein with respect to DNA damage has been reported indicating that it inhibits carcinogenesis induced by H2O2, as well as protecting DNA against UV light and Fenton reaction induced damage (Wei et al., 1996). Our data are also consistent with that of Foti et al. (2005), who showed that the DNA protective effect of genistein is lost at above 10 mM. Genistein is an effective scavenger of free radicals as well as a strong inhibitor of hydrogen peroxide formation, not only in vivo but also in vitro (Wei et al., 1993; Record et al., 1995). Ruiz-Larrea et al. (1997) have shown that genistein is the most potent antioxidant among isoflavones in both the aqueous and lipophilic phases of cells, suggesting the importance of the specific molecular structure of genistein, not only of the 40 hydroxyl group but also the 50 -hydroxyl group. In vitro lipoprotein oxidation studies suggest that some oxidative metabolites of genistein, particularly 3-OH-genistein, may be more potent than genistein itself (Rufer and Kulling, 2006). The possibility of DNA to repair the damage caused by G8CG has been shown in Fig. 4. Twelve hours was the shortest time at which effective repair could be seen. In previous studies (Rucinska et al., 2007), we reported that genistein-8-Cglucoside induces oxidative DNA damage and acts as an antioxidant to potentially prevent such damage. However,
repair process of DNA damage in the present study occurred considerably faster and more efficiently. In conclusion, our study reports a hypothesis that, not only genistein, but also genistein-8-C-glucoside demonstrates a double nature of action in vitro. G8CG at ‘‘physiological concentrations’’ increases cell protection against oxidative stress, while higher concentrations are cytotoxic and genotoxic to NIH 3T3 fibroblasts. Acknowledgements We would like to thank Dr Sergej Kirko (National Academy of Sciences, Belarus) for the isolation of genistein8-C-glucoside. References Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, et al. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987;262:5592e5. Alhasan SA, Aranha O, Sarkar FH. Genistein elicits pleiotropic molecular effects on head and neck cancer cells. Clin Cancer Res 2001;7:4174e81. Allred CD, Ju YH, Allred KF, Chang J, Helferich WG. Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein. Carcinogenesis 2001;22:1667e73. Andlauer W, Kolb J, Furst P. Absorption and metabolism of genistin in the isolated rat small intestine. FEBS Lett 2000;475(2):127e30. B1asiak J, Kowalik J. A comparison of the in vitro genotoxicity of tri- and hexavalent chromium. Mutat Res 2000;469:135e45. Boos G, Stopper H. Genotoxicity of several clinically used topoisomerase II inhibitors. Toxicol Lett 2000;116:7e16. Casagrande F, Darbon JM. Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: regulation of cyclin-dependent kinases CDK2 and CDK1. Biochem Pharm 2001;61:1205e15. Chen AC, Donovan SM. Genistein at a concentration present in soy infant formula inhibits Caco-2BBe cell proliferation by causing G2/M cell cycle arrest. J Nutr 2004;134:1303e8. Choi EJ, Kim T, Lee M. Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells. Life Sci 2007;80: 1403e8.
252
A. Rucinska, T. Gabryelak / Cell Biology International 33 (2009) 247e252
Dixon RA, Ferreira D. Genistein. Phytochemistry 2002;60:205e11. Duthie SJ, Ma O, Ross MA, Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res 1996;56:1291e5. Fioravanti L, Cappelletti V, Miodini P, Ronchi E, Brivio M, Di Fronzo G. Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro. Cancer Lett 1998;130:143e52. Foti P, Erba D, Riso P, Spadafranca A, Criscuoli F, Testolin G. Comparison between daidzein and genistein antioxidant activity in primary and cancer lymphocytes. Arch Biochem Biophys 2005;433:421e7. Gupta S, Saha B, Giri AK. Comparative antimutagenic and anticlastogenic effects of green tea and black tea: a review. Mutat Res 2002;512:37e65. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 1998;58:3833e8. Kato K, Takahashi S, Cui L, Toda T, Suzuki S, Futakuchi M, et al. Suppressive effects of dietary genistin and daidzin on rat prostate carcinogenesis. Japan J Cancer Res 2000;91:786e91. Kaufmann SH. Cell death induced by topoisomerase targeted drugs: more questions than answers. Biochim Biophys Acta 1998;1400:195e211. Kulling SE, Metzler M. Induction of micronuclei, DNA strand breaks and HRRT mutations in cultured Chinese hamster V79 cells by the phytoestrogen coumoestrol. Food Chem Toxicol 1997;35:605e13. Kulling SE, Rosenberg B, Jacobs E, Metzler M. The phytoestrogens coumoestrol and genistein induce structural chromosomal aberrations in cultured human peripheral blood lymphocytes. Arch Toxicol 1999;73: 50e4. Laman NA, Volynets AP. Flavonoids in ontogenesis of Lupinus luteus L. Plant Physiol (Rus) 1974;21:737e45. Lamartiniere CA. Protection against breast cancer with genistein: a component of soy. Am J Clin Nutr 2000;71:1705Se7S. Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and genistein contents of vegetables. J Nutr 2000;84:717e25. Markovits J, Linassier C, Fosse P, Couprie J, Pierre J, Jacquemin-Sablon A, et al. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 1989;49:5111e7. McClain RM, Wolz E, Davidovich A, Bausch J. Genetic toxicity studies with genistein. Food Chem Toxicol 2006;44:42e55.
Morris SM, Chen JJ, Domon OE, McGarrity LJ, Bishop ME, Manjanatha MG, et al. p53, mutations, and apoptosis in genistein-exposed human lymphoblastoid cells. Mutat Res 1998;405:41e56. Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity. J Immunol Methods 1983;65: 55e63. Po LS, Wang TT, Chen ZY, Leung LK. Genistein-induced apoptosis in MCF-7 cells involves changes in Bak and Bcl-x without evidence of anti-oestrogenic effects. J Nutr 2002;88:463e9. Record IR, Dreosti IE, McInerney JK. The antioxidant activity of genistein in vitro. J Nutr Biochem 1995;6:481e5. Rucinska A, Kirko S, Gabryelak T. Effect of the phytoestrogen, genistein-8-Cglycoside on Chinese hajster ovary cells in vitro. Cell Biol Int 2007;31: 1371e8. Rufer CE, Kulling SE. Antioxidant activity of isoflavones and their major metabolites using different in vitro assays. J Agric Food Chem 2006;54: 2926e31. Ruiz-Larrea MB, Mohan AR, Paganda G, Miller NJ, Bolwell GP, RiceEvans CA. Antioxidant activity of phytoestrogenic isoflavones. Free Rad Res 1997;26:63e70. Shao ZM, Wu J, Shen ZZ, Barsky SH. Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 1998;58:4851e7. Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 1991; 349:431e4. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184e91. Snyder RD, Gillies PJ. Reduction of genistein clastogenicity in Chinese hamster V79 cells by daidzeiny and other flavonoids. Food Chem Toxicol 2003;41:1291e8. Wei H, Cai Q, Rahn RQ. Carcinogenesis 1996;17:73e7. Wei H, Wie L, Frenkel K, Bowen R, Barnes S. Inhibition of tumor promoterinduced hydrogen peroxide formation in vitro and in vivo by genistein. Nutr Cancer 1993;20:1e12. Zava D, Duwe G. Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer 1997; 27:31e40.