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Carcinogenesis Advance Access originally published online on October 25, 2006
Carcinogenesis 2007 28(3):738-748; doi:10.1093/carcin/bgl180
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system

Claudia Steiner, Wilbert H.M. Peters1, Evan P. Gallagher2, Pamela Magee3, Ian Rowland3 and Beatrice L. Pool-Zobel*

Department of Nutritional Toxicology, Institute for Nutrition Friedrich-Schiller University Jena, Dornburger Straße 25, 07743 Jena, Germany
1 Department of Gastroenterology, St Radboud University Hospital Nijmegen, The Netherlands
2 Department of Environmental and Occupational Health Sciences, University of Washington 4225 Roosevelt Way NE, Suite 100, Seattle Washington, DC, USA
3 Northern Ireland Centre for Diet and Health, University of Ulster Coleraine, Northern Ireland BT52 1SA

*To whom correspondence should be addressed. Tel: +49 0 3641 949670; Fax: +49 0 3641 949672; Email: b8pobe{at}uni-jena.de


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 REFERENCES
 
Epidemiological studies have shown that ingestion of isoflavone-rich soy products is associated with a reduced risk for the development of breast cancer. In the present study, we investigated the hypothesis that genistein modulates the expression of glutathione S-transferases (GSTs) in human breast cells, thus conferring protection towards genotoxic carcinogens which are GST substrates. Our approach was to use human mammary cell lines MCF-10A and MCF-7 as models for non-neoplastic and neoplastic epithelial breast cells, respectively. MCF-10A cells expressed hGSTA1/2, hGSTA4-4, hGSTM1-1 and hGSTP1-1 proteins, but not hGSTM2-2. In contrast, MCF-7 cells only marginally expressed hGSTA1/2, hGSTA4-4 and hGSTM1-1. Concordant to the protein expression, the hGSTA4 and hGSTP1 mRNA expression was higher in the non-neoplastic cell line. Exposure to genistein significantly increased hGSTP1 mRNA (2.3-fold), hGSTP1-1 protein levels (3.1-fold), GST catalytic activity (4.7-fold) and intracellular glutathione concentrations (1.4-fold) in MCF-10A cells, whereas no effects were observed on GST expression or glutathione concentrations in MCF-7 cells. Preincubation of MCF-10A cells with genistein decreased the extent of DNA damage by 4-hydroxy-2-nonenal (150 µM) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (50 µM), compounds readily detoxified by hGSTA4-4 and hGSTP1-1. In conclusion, genistein pretreatment protects non-neoplastic mammary cells from certain carcinogens that are detoxified by GSTs, suggesting that dietary-mediated induction of GSTs may be a mechanism contributing to prevention against genotoxic injury in the aetiology of breast cancer.

Abbreviations: AP-1, activator protein-1; ARE, antioxidant response element; BPDE, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide; BSA, bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; ER, estrogen receptor; ERE, estrogen responsive element; ERK1/2, extracellular regulated protein kinase 1/2; GSH, reduced glutathione; GST, glutathione S-transferase; 4-HNE, 4-hydroxy-2-nonenal; MAPK, mitogen-activated protein kinases; NQO1, ; NAD(P)H, quinone oxidoreductase; PAH, polycyclic aromatic hydrocarbon; ROS, reactive oxygen species.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 REFERENCES
 
Breast cancer is the most common cancer among women in countries with western style diet and the third most common cancer among women world-wide (1). Historically, the incidence breast cancer rates are substantially lower in China, Japan and other Asian countries than in the USA and Europe (2). Migrants from Asia to the USA typically acquire the breast cancer risk of their host nation by the second generation, which suggests a marked influence of environmental and life-style factors (3). The low prevalence of breast cancer in Asia is associated with the consumption of a diet abundant in soy (4). Specifically, it has been estimated that the approximate daily soy intake is 10–50 g in Asia, as compared with 1–3 g in the US and in Europe (5). Associated with this is an estimate that 20–100 mg isoflavones per day are ingested in Asia, whereas in Europe and in the USA daily intake is <1 mg (6–9).

Several observational epidemiological studies have examined the relationship between soy and breast cancer. Case–control studies examining the association between soy intake and breast cancer risk in Asian women (10,11) and Caucasian women (12) consistently suggest an inverse association for both pre- and post-menopausal breast cancer. It was also reported that the breast cancer preventive effects of dietary soy correlate with adolescent dietary exposure (11). There is substantial evidence that isoflavones contribute strongly to the chemoprotectant properties of soy products, and specifically with regards to inhibition of breast cancer development. Isoflavones constitute a group of phytoestrogens, i.e. naturally occurring non-steroidal plant compounds containing a diphenolic structure, that are able to exert estrogenic effects (13). These compounds can interact with human estrogen receptors (ER), inhibit their action, suppress cell growth and protect against the development of breast cancer (14). The isoflavone genistein has been a major focus of attention since it has a number of activities, which prevent the progression of initiated cells to tumours. These activities include induction of mammary gland differentiation in rodents (15), inhibition of cell proliferation by cell cycle modulation and induction of apoptosis (16,17), inhibition of angiogenesis (18) and tyrosine kinases (19). Interestingly, soy isoflavones such as genistein can also enhance DNA damage in different cell cultures at doses >1 µM (20–24). However, when mice or humans are exposed to dietary relevant or higher levels of genistein or isoflavones, genetic damage is not observed (22,25). Furthermore, studies in our laboratory have shown that doses of genistein up to 30 µM are not genotoxic in human mammary cell lines (C. Steiner, unpublished data). Thus, it appears that the genotoxic effects by genistein may be dose-specific, as well as dependent on the timing of exposure. Moreover, the genotoxic effects could be highly cell type-specific as well. For example, we have found that in human colonocytes, genistein induces strand breaks (100 µM exposure for 15 min) (26) whereas in human prostate cells, no increase in DNA damage was observed (80 µM exposure for 1 h (27).

Blocking agents such as phytoestrogens have promise for overall cancer risk reduction (28) by reducing exposure to genotoxic metabolites at critical cell targets through the induction of detoxification genes (29–31). Of particular importance with respect to the detoxification of carcinogenic intermediates are the GSTs, a supergene family of cytosolic, mitochondrial and microsomal enzymes (32), most of which have the ability to conjugate electrophilic chemicals with glutathione (GSH) (33). The cytosolic transferases are represented by the classes alpha, mu, pi, sigma, theta, omega and zeta. In addition to the cytosolic forms, mitochondrial GSTs comprise the kappa class GST, whereas microsomal GSTs represent a distinct gene family of membrane-associated proteins that are active in eicosanoid and glutathione metabolism (32). Several GST isoforms are also of importance in protecting against oxidative stress by conjugating reactive quinones arising from catecholestrogens, reactive {alpha}, ß-unsaturated aldehydes produced during lipid peroxidation, and also by reducing organic hydroperoxides via selenium-independent glutathione peroxidase activity (32,34,35). Although GSTs are constitutively expressed in human tissues, tissue- and cell-specific isoform expression shows marked variability, a factor, which can greatly influence susceptibility to toxicity and disease (32).

A particularly important data gap is a lack of information regarding GST isoform expression in breast tissue. The most prevalent isoenzyme in extrahepatic tissues (36) and the major GST isoenzyme in human breast tissue (37) hGSTP1-1 is inducible by flavonoids and isothiocyanates (38,39), by the gut fermentation product butyrate (40,41), as well as by antiestrogens (37). In the present study, we characterized GST isoform expression in human mammary epithelial cells and tested the hypothesis that genistein induces GST isoforms and protects against the genotoxic injury of the mutagenic compounds 4-HNE and BPDE. We used MCF-7, a mammary carcinoma cell line frequently used in studies involving genistein and related chemoprotective compounds. We also used MCF-10A cells, since they are derived from non-malignant tissue and exhibit a stable karyotype appropriate for genotoxic and gene expression studies (42) targeting mechanisms of chemoprotection that occur prior to initiation.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 REFERENCES
 
Chemicals
Cell culture materials, including Dulbecco's Modified Eagle Medium (DMEM), non-essential amino acids (MEM), penicillin/streptomycin, fetal calf serum (FCS) and trypsin were obtained from Invitrogen GmbH (Karlsruhe, Germany). Mammary Epithelial Growth Medium (MEGM) was provided by Promocell (Heidelberg, Germany), charcoal-stripped fetal calf serum (CCS) by Biological Industries (Israel) and trypsin/EDTA solution was purchased from Biochrom AG (Berlin, Germany). Genistein was supplied by TCI Tokyo Kasei (Japan). Choleratoxin, DMSO, metaphosphoric acid, 3,3'-diamino benzidine (DAB) and hydrogen peroxide were purchased from Sigma-Aldrich GmbH (Steinheim, Germany), TRIzol® reagent, desoxyribonuclease I, and SuperScriptTM First-Strand Synthesis System were purchased from Invitrogen GmbH (Karlsruhe, Germany). The QIAmp Mini Kit and Hot Star Taq DNA Polymerase were provided by Qiagen GmbH (Hilden, Germany). The RNeasy Mini Kit was provided by Qiagen Ltd. (Crawley, UK). Rabbit anti-mouse Ig conjugated with horseradish peroxidase was purchased from DAKO (Hamburg, Germany), and goat anti-chicken IgY conjugated with horseradish peroxidase from CalBiotech (Santa Cruz, USA). Enhanced chemiluminescence (ECL)TM western blotting detection reagents were provided by Amersham Biosciences Europe GmbH (Freiburg, Germany). 1-chloro-2,4-dinitrobenzene (CDNB) and GSH were obtained from Merck (Darmstadt, Germany). The Glutathione Assay-Kit and 4-hydroxy-2-nonenal (4-HNE) were purchased from Calbiochem-Novabiochem GmbH (Bad Soden, Germany). Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was provided by Biochemical Institute for Environmental Carcinogens (Grosshansdorf, Germany).

Cell culture experiments and exposures
Two cell lines, MCF-10A and MCF-7, differing in the transformation stage were chosen for the experiments. The non-tumourigenic cell line MCF-10A derived from reduction mammoplasty tissue (43) was obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in MEGM supplemented with 100 ng/ml of choleratoxin at 37°C in humidified atmosphere (95% air, 5% CO2). The mammary tumour cell line MCF-7 (44) was obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were maintained in DMEM supplemented with 10% (v/v) FCS, 1% (v/v) MEM and 1% (v/v) penicillin/streptomycin at 37°C in a humidified incubator (95% air, 5% CO2). MCF-10A cells have only a marginal ERß expression whereas MCF-7 cells show both ER{alpha} and ERß expression (45). The cells were trypsinized with 1.5 ml of trypsin/EDTA and 10x trypsin, respectively, for up to 4 min and sub-cultured at a dilution of 1:4 with culture medium. The medium was changed every 3 days, and passages 7–30 were used for the experiments. Prior to experiments, cells were transferred to phenol-red free medium containing CCS in order to eliminate potential estrogenic compounds of the serum.

In chemoprotection experiments, the MCF-7 and MCF-10A cells were plated in 25 cm2 cell culture flasks and incubated with genistein at concentrations of 1–30 µM, with a final DMSO concentration of 0.1%. These concentrations were based on sub-toxic doses (<IC50) determined previously for these cell lines (C. Steiner, C.E. Rüfer, S.E. Kulling, K. Wähälä, B.L. Pool-Zobel, manuscript in preparation). After 48 h of treatment, the cells were trypsinized and washed in cold phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4·2H2O, 1.5 mM KH2PO4 and 0.5% BSA; pH 7.4). Cell number and cell viability were determined with trypan blue dye exclusion using a haemocytometer, and analysis of GST expression, GSH concentrations and DNA damage were conducted as described below.

Comparative analysis of GST isoenzyme mRNA expression in the breast cancer cell lines by semi-quantitative RT–PCR
Total RNA was isolated using the TRIzol method. Following DNA digestion, cDNA equivalent to 5 µg total RNA was prepared by first-strand synthesis using oligo(dT) primer. Semi-quantitative reverse transcriptase PCR was then used to characterize steady-state GST mRNA expression pattern for hGSTA1, hGSTA2, hGSTA4, hGSTM1, hGSTM2, hGSTP1 and hGSTT1 in the breast cell lines (sequences of the primers and their product sizes are listed in Table I). HT29 colon cells were used, as a positive control cell line, for GST genotyping and also for GST mRNA expression by RT–PCR. The PCR conditions were as follows; 10 ng total cDNA, 1 unit of Hot Star Taq DNA Polymerase, 20 pM of primers and 1.5 mM MgCl2. The cDNA was amplified by PCR, with 38 cycles. After primary denaturation at 95°C for 15 min, the PCR cycles were run at 94°C for 30 s for denaturation, 55°C (A1, A2), 60°C (M1, M2, P1) or 63°C (A4, T1) for 30 s for annealing and 72°C for 1 min for extension.


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  		Table I Primer sequences for RT–PCR of GSTs

 
Effect of genistein on quantitative expression of hGSTA4 and hGSTP1 mRNA levels
After incubation with genistein (1–30 µM) for 8, 24 and 48 h, breast cells were trypsinized and washed twice with PBS/BSA followed by the RNA isolation using RNeasy Mini Kit according to the manufacturer's protocol. RNA quantity and quality were assessed spectrophotometrically (260/280 absorbance ratios) and by gel electrophoresis. After ensuring RNA quality, 500 ng RNA were reverse transcribed using random hexamer primers. A region of hGSTA4 and hGSTP1 mRNA was amplified using the primers GSTA4-F (5'-GGATCTGCTGGAACTGCTTATCAT-3'), GSTA4-R (5'-TGTCCGTGACCCCTTAAAATCTT-3') and GSTP1-F (5'-CTCACTCAAAGCCTCCTGCCTAT-3'), GSTP1-R (5'-CAG GATGGTATTGGACTGGTACAG-3'). Primers and probes for the endogenous reference ß-actin, as a ready-to-use kit, were supplied by Applied Biosystems (UK). TaqMan® real-time PCR was performed in 96-well plates, following the guidelines of the manufacturer (Applied Biosystems, USA). All experiments were performed in triplicate and a validation experiment was performed to confirm the specificity of the target amplification, and also the efficiency of the reference amplification. hGSTA4 and hGSTP1 mRNA expression was then normalized to the endogenous reference ß-actin using the comparative CT method ({Delta}{Delta}CT = {Delta}CT control – {Delta}CT treatment) according to the manufacturer's guidelines. The effect of genistein on GST mRNA expression change was calculated according to the efficiency method (fold change= 2–{Delta}{Delta}CT), where it is assumed that the primer efficiency is 100%.

Genotyping of cell lines for GSTM1, GSTT1 and GSTP1 status
Genomic DNA was extracted from 1 x 106 cells using the QIAamp DNA Mini Kit following the manufactorer's protocol. Multiplexed PCR was used to detect hGSTM1 and hGSTT1 deletion polymorphisms (46,47), whereas the sequence polymorphism of hGSTP1 at codon 104 (A->G transition) was detected using the restriction fragment length polymorphism (RFLP)–PCR (38).

Analysis of GST proteins and catalytic activity, and intracellular GSH concentrations
Prior to cytosol preparation, the cells were resuspended in cold homogenization buffer (250 mM sucrose, 20 mM Tris–HCl, 1 mM DTT and 1 mM Pefabloc; pH 7.4) and homogenized for 1 min using ultrasound (Bandelin Electronics, Berlin, Germany). Following centrifugation (16 000 g, 60 min, 4°C), the supernatant was aliquoted and stored at –80°C until use. Western blot analyses of GST proteins were performed as described previously (48). For these experiments, defined amounts of cytosolic protein were used (MCF-7: up to 120 µg for hGSTP1-1, A1/2, M1-1, M2-2, 80 µg for A4-4; MCF-10A: 25 µg for hGSTP1-1, up to 120 µg for hGSTA1/2, M1-1, M2-2 and 80 µg for A4-4) to ensure a semi-quantitative analysis. We used monoclonal antibodies against human GST class alpha, mu and pi (49). Specifically, the alpha class GST antibodies recognize human hGSTA1-1, A1-2 and A2-2 whereas the mu class GST antibodies react against human hGSTM1a-1a, M1a-1b and M1b-1b (50) and probably against hGSTM2-2. However, because of lack of the appropriate hGSTM2-2 standard protein, the detection of hGSTM2-2 was not validated in the present study. GST protein expression was detected with DAB and hydrogen peroxide after incubation with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins as secondary antibody. Detection of hGSTA4-4 was accomplished using a polyclonal chicken anti-hGSTA4 peptide antibody as previously described (51). GST protein levels were quantitated by densitometry using Fluor S Multi Imager (Bio-Rad, München, Germany) using known quantities of purified recombinant proteins wherever possible with normalization to the expression of ß-actin.

Overall cytosolic GST activity was determined spectrophotometrically according to Habig et al. (52). Total protein content was measured by the method of Bradford using BSA as standard protein (53). All values were based on the number of cells, as described previously (54). Intracellular GSH concentrations were determined following homogenization of cells in metaphosphoric acid using a colorimetric assay. The detection of the colorimetric product was performed at 400 nm using a microplate reader (Tecan, Austria). The effects of genistein treatment on cellular GSH concentrations were expressed as percentage increase in cellular GSH relative to control.

Determination of genetic damage (Comet assay)
The effects of genistein on 4-HNE- and BPDE-induced DNA damage were measured using the Comet assay. Cells were treated for 48 h with genistein (1–30 µM) or vehicle control (0.1% DMSO). Following the treatments, the cells were harvested, washed with ice cold PBS and incubated with 4-HNE or BPDE at 37°C for 30 min and at concentrations previously established in our laboratory to induce genotoxicity that are specific concentrations for the cell lines (MCF-10A: 150 and 50 µM; MCF-7: 75 and 20 µM, respectively). The aforementioned dosing regimen had been previously established and successfully used in experiments involving combinatorial effects of genotoxic agents in these cells, and also minimizing direct interaction among the chemoprotective and genotoxic agents (55,56). Cell numbers and viabilities were determined by trypan blue dye exclusion. The cells were embedded into agarose on microscopical slides, lysed and subjected to electrophoresis as described by Singh et al. (57) with modifications by Pool-Zobel et al. (58). The extent of DNA migration was used as the basis of evaluation and quantified by image analysis (Kinetic Imaging Ltd., Liverpool, UK). Fifty DNA spots were measured per slide. Mean values of tail intensity (expressed as ‘% fluorescence in comet tail’) from three slides per experiment were calculated, and the means of three independent experiments were evaluated.

Statistical analysis
All data presented reflect the means ± SD of at least three independent experiments. The effect of the treatments on GST expression, GSH concentrations and DNA damage was analysed by One-way ANOVA followed by Dunnett's post hoc test. Where appropriate, time- and treatment-related effects were analysed by Two-way ANOVA and Bonferroni post hoc test. Treatment-related differences were considered significant at P ≤ 0.05. The relationship among hGSTP1-1 protein levels and GST catalytic activities were assessed using linear regression. All statistical testing was accomplished using the GraphPad Prism software version 4.0 for Windows (San Diego, USA).


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 REFERENCES
 
Comparative expression of GST mRNA and proteins in MCF-10A and MCF-7 cells
As observed in Table II, both mammary cell lines examined expressed considerable amounts of GST isoenzymes based on mRNA and protein measurements. However, the dysplastic cell line MCF-10A lacked detectable levels of hGSTA1, hGSTA2 and hGSTM2 mRNA, and the GSTT1*0 polymorphism was also present in the cell line (Figure 1A). Both MCF-10A, as well as, MCF-7 cells exhibited the heterozygous GSTP1*A*B genotype (Figure 1B). As observed in Table II, both cell lines expressed functional hGSTA1/2, hGSTA4-4 and hGSTM1-1 proteins. In contrast, the hGSTM2-2 protein was not detectable in either mammary cell lines. Additionally, tumourigenic MCF-7 cells did not express the hGSTP1-1 protein. The higher protein expression of the breast cancer risk factor related hGSTA4-4 and hGSTP1-1 in the dysplastic cell line MCF-10A correlated with corresponding mRNA expression. MCF-10A cells expressed 2.5-fold more hGSTA4 mRNA (17.2 ± 9.4 ng in MCF-10A versus 6.8 ± 1.5 ng in MCF-7) and 66 000-fold greater hGSTP1 mRNA than MCF-7 cells (9.9 ± 1.1 ng in MCF-10A versus 0.00015 ± 0.00011 ng in MCF-7, Table III).


Figure 1
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  		Fig. 1 (A).Agarose gel of GSTT1 and GSTM1 deletion polymorphisms in MCF-10A and MCF-7 cell lines. The human colon tumour cell line HT29 was used as a positive control (without polymorphisms). MCF-10A showed the null genotype of GSTT1 (GSTT1*0) while a functional GSTM1 was present in both mammary cell lines. ß-globin served as an internal control for the PCR-based reactions. (B) Agarose gel showing the GSTP1 genotype in human mammary cell lines MCF-10A and MCF-7 by RFLP-PCR, using the restriction enzyme Alw 26 I. The 176 bp PCR product corresponds to the wild-type allele GST*A, whereas the 91 and 85 bp fragments indicate the presence of the mutant allele GSTP1*B. Both cell lines exhibited the heterozygous GSTP1*A*B genotype.

 


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  		Table II Detection of hGST mRNAs and proteins in human mammary cells

 


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  		Table III Quantification of basal levels of hGST mRNAs, GST proteins, GST activity, total protein and intracellular GSH in human mammary cells

 


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  		Table IV Effect of genistein on expression levels of GSTA4 protein (fold induction) human mammary cells

 
The lack of the hGSTP1-1 protein expression and the marginal expression of the alpha and mu class GSTs in MCF-7, all of which conjugate CDNB, were reflected by 100-fold lower GST activity in MCF-7 cells relative to MCF-10A cells (Table III). In contrast, overall GSH concentrations did not differ among the two cell types (Table III). In general, the MCF-10A cells expressed more overall GST protein and GST subunit levels than MCF-7 cells, consistent with the enzymatic analysis.

Modulation of hGSTP1 and hGSTA4 mRNA and protein expression, GST activity and intracellular GSH level by genistein
As observed in Figure 2A, exposure of MCF-10A cells to 30 µM of genistein elicited a significant 4.7-fold increase in overall GST-CDNB activity relative to controls. This effect was accompanied by an increase in cellular GSH concentrations (Figure 2B). The increased GST-CDNB activities in MCF-10A cells appear to be due to a significant induction of hGSTP1-1 protein (3.1-fold from 851 ± 261 to 2633 ± 390 ng/106 cells at 30 µM; Figure 3). There was a significant correlation between the hGSTP1-1 protein concentrations and the overall GST-CDNB activity at the significant modulating concentration of 30 µM. The modulation of hGSTP1-1 protein expression correlated with genistein-mediated changes in mRNA expression. Genistein marginally enhanced hGSTP1 mRNA expression after 8 h (30 µM, 1.3-fold) and 24 h (10 and 30 µM, 1.3- and 1.2-fold, respectively) of exposure. A further enhancement of hGSTP1 mRNA expression was observed after 48 h incubation (1, 10, 30 µM for 2.3-, 1.8- and 1.7-fold, respectively, Figure 4A). Although exposure to genistein (1–30 µM) did not affect hGSTA4-4 protein expression in MCF-10A cells (Table IV), it did down-regulate hGSTA4 mRNA expression (Figure 4B). In contrast, exposure to genistein did not alter GST-CDNB activities or GSH concentrations in MCF-7 cells (Figure 2A and B). While hGSTP1-1 protein was not detectable in MCF-7 cells, hGSTA4-4 protein expression was not affected by genistein (Table IV). But as shown in Figure 4B, there was a time dependent modulation of hGSTA4 mRNA expression. After a 1.3-fold increase of hGSTA4 mRNA by 30 µM of genistein following 8 h treatment, the mRNA expression was decreased by 40–60% after 24 h. Increased temporal exposure to genistein did not influence hGSTA4 mRNA expression (Figure 4B).


Figure 2
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  		Fig. 2 (A) Effect of 48 h genistein treatment on GST catalytic activity in MCF-10A and MCF-7 cells. Results are means ± SD of a minimum of three experiments. Significant effects in genistein-induced GST activities were calculated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (**P < 0.01). (B) Effect of 48 h genistein treatment on intracellular GSH levels in MCF-10A and MCF-7 cells. Results are expressed in percentage increase in cellular GSH. The corresponding GSH concentrations in control MCF-10A and MCF-7 cells were 5.2 ± 1.9 and 6.7 ± 2.8 nmol x 10–6 cells, respectively. The results represent means ± SD of seven and four experiments for MCF-10A and MCF-7 cells, respectively. Significant differences of genistein treatments were calculated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (**P < 0.01).

 


Figure 3
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  		Fig. 3 Modulation of hGSTP1-1 protein expression after 48 h exposure to genistein in MCF-10A cells. Significant differences of genistein-induced effects and control values (0 µM) were calculated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (**P < 0.01). Data represents the means ± SD of four experiments. Shown below is a representative blot of one experiment.

 


Figure 4
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  		Fig. 4 (A) Modulation of hGSTP1 mRNA expression after 8, 24 and 48 h exposure to genistein in MCF-10A cells. Data represents the means ± SD of three experiments. Significant differences of genistein induced effects and control values (0 µM) were calculated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (*P < 0.05, **P < 0.01). (B) Modulation of hGSTA4 mRNA expression after 8, 24 and 48 h exposure to genistein in MCF-10A (left) and MCF-7 cells (right). Data represents the means ± SD of three experiments. Significant differences of genistein treatments were calculated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (*P < 0.05, **P < 0.01, **P < 0.01).

 
Chemoprotection of DNA damage by genistein
Figure 5 demonstrates that the level of DNA damage in MCF-10A cells on exposure to 4-HNE and BPDE (13.5 ± 1.3% tail intensity for 150 µM 4-HNE; 49.3 ± 6.4% tail intensity for 50 µM BPDE) was suppressed by genistein. Genistein-mediated chemoprotection against 4-HNE was clearly evident at low genistein concentrations, whereas only the highest concentration of genistein (30 µM) significantly reduced the genotoxic response of BPDE. In contrast, genistein was not chemoprotective against either 4-HNE or BPDE-induced genotoxicity in the MCF-7 tumour cell line.


Figure 5
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  		Fig. 5 Effects of 48 h genistein pretreatment and subsequent challenge with (A) HNE and (B) BPDE, on DNA integrity of MCF-10A and MCF-7 cells. Results represent the means ± SD of three experiments. Significance of genistein-induced effects were calculated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test; ** differences were considered significant at P < 0.01. The HNE and BPDE concentrations in the assay were 150 and 50 µM for MCF-10A and 75 and 20 µM for MCF-7, respectively. Baseline levels of genotoxic activities (% fluorescence in tail) in MCF-10A were 14 ± 1.3 and 49 ± 6.4, for HNE and BPDE, respectively, and were significantly lower than that those observed in MCF-7 cells (61 ± 4.9 and 62 ± 5.0, respectively).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
REFERENCES
 
In the present study, we have demonstrated that genistein may act as a blocking agent in breast cancer via induction of GST. In this regard, an up-regulated detoxification system may facilitate the detoxification and/or elimination of ultimate carcinogens, thereby eliciting an inhibitory effect against the initiation of cancer and potentially reducing the risk of tumourigenesis (28). Support for this hypothesis on blocking agents in cancer chemoprevention is evidenced by several in vivo and in vitro studies (39,59–61). Our data also revealed different expression patterns of GST among the two cell lines, with higher overall GST levels in non-transformed MCF-10A cells. The extremely low expression of GST isoforms hGSTA4 and hGSTP1 in MCF-7 cancer cells is noteworthy. The lack of detectable hGSTP1-1 protein in the presence of low amounts of hGSTP1 mRNA may be a result of post-transcriptional modification of hGSTP1-1 protein (62,63). Others have shown that hGSTP1 mRNA in MCF-7 cells is highly unstable relative to ER negative breast cancer cell lines (62). We have previously shown that hGSTT1-1 protein expression may be regulated in a similar manner in human colon cells (64). Alternatively, hypermethylation of GC rich regions within the hGSTP1 promotor also contributes significantly to the maintenance of hGSTP1-1 expression in MCF-7 (65). More recently, a proteasome-dependent regulatory mechanism of hGSTP1-1 protein turnover was reported (66). Thus, a higher proteasome activity in MCF-7 cells could possibly also account for the differences in basal expression of this GST in the two cell lines.

The lack of appreciable hGSTP1-1 protein and the weak expression of alpha and mu class GSTs in MCF-7 cells, all of which conjugate CDNB, is consistent with the low level of GST-CDNB activity in MCF-7 cells relative to the MCF-10A cells, although both cell lines had comparable levels of GSH. Our observations are supported by a study by Fields et al. (67) who also observed relatively low GST-CDNB activity in MCF-7 cells. As discussed, the presence of allelic and deletion variants as well as mRNA expression levels appear to be collectively crucial for the higher susceptibility of MCF-7 to cancer associated risk factors 4-HNE and BPDE (data not shown).

Our findings strongly support a general up-regulation of GSTs in non-neoplastic MCF-10A cells on exposure to genistein, with the possible exception that hGSTA4 mRNA expression was downregulated by genistein. Despite the negative effect of genistein on hGSTA4 mRNA expression, we found no modulation of hGSTA4-4 protein. An explanation for this discrepancy could be reduced mRNA stability, which we have previously reported for hGSTT1-1 in human colon cells exposed to butyrate and attributed to a shortened hGSTT1 mRNA half-life (64). The high correlation between hGSTP1-1 protein expression and GST-CDNB activity indicates that hGSTP1-1 induction is likely a major component responsible for the increase in GST-CDNB activity in MCF-10A cells. However, we could not exclude an additional induction of other GST isoenzymes which may have contributed to increased GST activity toward CDNB, or that have differing affinities toward CDNB.

The consequences of GST up-regulation in primary breast cells or cells in early stages of cell transformation could be a beneficial mechanism to mitigate the damaging impact toward carcinogenic compounds. Support for this hypothesis is provided from studies investigating nutrient related compounds (38,40,61,68). In this regard, nutritional status appears to be a key modulating factor in the generation and amelioration of toxic metabolites of relevance to breast cancer (69). Compounds of toxicological significance include the mutagenic 4-hydroxy-2-alkenal 4-HNE, which is formed during the physiological process of lipid peroxidation, leading to etheno- and propano-DNA adducts (70,71). 4-HNE is genotoxic in human colon cells (40,72) and may induce damage in the tumour suppressor gene TP53 via DNA adduct formation (42,73). Conversely, benzo(a)pyrene is a well-characterized PAH produced during food heating and a substrate for CYP 1A-enzymes which biotransform the parent compound to the mutagenic and carcinogenic BPDE (74). This compound induces DNA damage and mutations by forming DNA adducts (75). The genotoxic effects of 4-HNE and BPDE can be prevented by conjugation with GSH catalysed by GSTs (32). The GST isoenzyme hGSTA4-4 has an unusually high substrate specificity toward 4-HNE (51,76) whereas hGSTP1-1 conjugates both 4-HNE (77) and BPDE (78).

Our results of the chemoprotection experiments showed that 4-HNE genotoxicity was markedly reduced following genistein pretreatment in MCF-10A cells, whereas no modulation was observed in the tumour cell line MCF-7. 4-HNE is a high-affinity substrate for hGSTA4-4. Studies from one of our laboratories have demonstrated that hGSTA4-4 can be preferentially localized in the mitochondria relative to the cytosol (51), but can exist in phosphorylated forms in the cytoplasm of many cells and tissues (79,80). The reduction of 4-HNE genotoxicity in genistein treated MCF-10A cells, however, can not be explained by an enhanced GSTA4-4 activity, since its expression is relatively low in cells (32) and moreover, since it was not significantly modulated by genistein. In contrast, hGSTP1-1, which also may detoxify 4-HNE (77), was highly expressed in MCF-10A cells, and was induced by genistein. Collectively, these data support the hypothesis that hGSTP1-1 is a major enzyme involved in detoxification of 4-HNE in these non-neoplastic cells. This, despite the fact that we observed a discrepancy in the dose–response profiles, was associated with the effects of genistein on DNA damage and GST expression. Specifically, at relatively low and biologically relevant doses (1 µM) genistein inhibited DNA damage, whereas higher doses of 10 or 30 µM were needed to significantly modulate GST mRNA and protein levels, respectively. As discussed above, these observations could be due to the contribution of other HNE-detoxification pathways (e.g. aldehyde reductases, alcohol dehydrogenases) that were potentially modulated at lower doses of 1 µM genistein.

Increased GSTP1-1 activity may also be responsible for the reduction of BPDE-mediated DNA damage by genistein. The direct conjugation and detoxification of BPDE by hGSTP1-1, however, may only partially explain the protective role of genistein, since BPDE also has indirect modes of genotoxicity. Thus, in vitro studies have shown that comet DNA tailing effects induced by benzo(a)pyrene or other bulky adduct-forming mutagens arise mainly from transient DNA single strand breaks formed during excision repair (81). This suggests that increased DNA excision repair by genistein could possibly contribute to the effect of GST-mediated metabolic deactivation. Previous cDNA macroarray experiments from our group confirmed a genistein-mediated up-regulation of XRCC repair genes (results not shown). Our conclusion is that the relatively high basal expression of hGSTP1-1 and associated high GST activity protected against 4-HNE and BPDE-induced DNA damage. This is consistent with a study by Fields et al. (82) where no protection against genotoxicity was seen at low GST expression and activity levels as shown in MCF-7 cells. Collectively, these studies indicate that a threshold level of GST activity may be required to achieve adequate protection against cellular damage by carcinogens.

Little is known about the exact underlying mechanisms of the modulation of hGSTP1 and hGSTA4 mRNA expression by phytoprotective compounds such as genistein. In fact, the regulation of GSTs is governed by a complex set of exogenous and endogenous parameters. In addition to dietary phytochemicals, other environmental factors such as exposure to carcinogens and endogenous metabolites such as ROS and lipid peroxidation products may modulate cellular detoxification capacities. The aforementioned compounds appear to confer resistance against carcinogenesis through their ability to generate a redox signal that stimulates increased expression of protective detoxification enzymes such as GSTs (32,83–86). However, it must be pointed out that the complex in vivo environment may not be reflected using the in vitro approaches with cell lines. Nevertheless, our studies do imply that the dietary phytoestrogens, especially genistein, can additionally improve the selected detoxification systems and thus may contribute to the prevention of human mammary carcinogenesis.

A potential mechanism to induce GST expression is thereby the activation of signalling pathways. Previous work in our laboratory indicated that the fermentation product butyrate activated MAP kinase cascade pathways by enhancing ERK1/2 phosphorylation in human colon cells (40). The activation of MAP kinase signalling pathways was also discussed for known phase II gene inducers such as phenolic antioxidants, isothiocyanates and phenolic flavonoids (87–89). Binding of AP-1 like transcription factors such as Jun, Fos and Nrf2 to the antioxidant responsive element-like sequences within the hGSTP1 promoter is a downstream consequence of ERK phosphorylation events (90–93).

In breast cells, there exist other ER-dependent and ER-independent signalling pathways for estrogens and phytoestrogens. In ER positive cells, the formed activated hormone receptor complex can bind directly to an ERE binding site within the promoter sequences of AP-1 forming transcriptions factors (94), or can bind directly to members of the AP-1 complex such as Jun, Fos, Nrf2 (37,89,95). ER{alpha} and ERß exert differential actions on the AP-1 complex. If estrogens bind to ERß the transcription via AP-1 will be inhibited, while the antiestrogen-ERß complex activates the transcription via AP-1 (95). The high affinity of phytoestrogens for ERß could be a possible activation pathway of GSTs by genistein. Another ER-mediated inductive mechanism is the indirect activation of MAP kinase cascades like ERK1/2 and JNK (96). Consequently, the resulting AP-1 complex activates hGSTP1 transcription. The ER-independent gene regulation is mediated by G protein-coupled receptors (GPR). Estradiol and genistein induce ERK1/2 phosphorylation via GPR30 (97) leading to activation of AP-1. Thus, hormones and phytohormones do not require ER expression and the MAPK pathway emerges as crucial.

It must be noted that the concentrations of genistein (1–30 µM) used in the present study were above isoflavone concentrations reported in plasma after dietary consumption of isoflavones (≤1 µM) (98). However, in the design of the present study, we took into account that short-term (24–72 h) exposure durations using the cell lines were of different duration than those in vivo situations in which more chronic dietary exposure of soy isoflavones results in low micromolar concentrations in serum. Moreover, continuous soy consumption throughout the life span may potentially result in accumulation of isoflavones in target tissues at levels exceeding those in serum. Several reports involving either single dose or short-term isoflavone treatments (including genistein) have shown increased plasma and tissue concentrations well above basal levels in respective control groups (99–104). Furthermore, infants exposed to dietary levels of 4.5–8.0 mg total isoflavones per kg body weight in soy-based formulas displayed plasma genistein and daidzein concentrations of 2.5 µM (105).

In conclusion, the results of the present study indicate that genistein mediates an enhancement of GST activity through induction of hGSTP1 mRNA and protein expression in human mammary non-neoplastic MCF-10A cells. Furthermore, modulation of GSTs on exposure to genistein leads to reduced genotoxicity on exposure to relevant proximate carcinogens, 4-HNE and BPDE. Our data are supportive of the hypothesis that genistein may have chemopreventive actions related to the early events of breast cancer. Confirmation for this hypothesis we got previously showing an up-regulation of a further phase II-enzyme, NAD(P)H: quinone oxidoreductase, in non-malignant mammary cells by genistein resulting in enhanced cellular protection against carcinogens (manuscript in preparation). Of future interest would be to determine the effects of high soy consumption on GST expression in breast cells in vivo. Ultimately, a more detailed understanding of modulation of human GST isoforms in vivo will enable clarification regarding their roles in the etiology of human breast cancer.


   Acknowledgments
 
This study was carried out with financial support from the Commission of the European Communities specific RTD programme ‘Quality of Life and Management of Living Resources’ (project no QLK-2000-00266). It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area. Additional support was provided by a grant from the National Institutes of Health (R01-ES09427).

Conflict of Interest Statement: None declared.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 REFERENCES
 

  1. World Cancer Research Fund/American Institute for Cancer Research. (1997) Food, Nutrition, and the Prevention of Cancer: A Global Perspective., Washington DAICR (Ed.).
  2. Parkin D.M., Bray F., Ferlay J., Pisani P. (2001) Estimating the world cancer burden: Globocan 2000. Int. J. Cancer 94:153–156.[CrossRef][ISI][Medline]
  3. Ziegler R.G., Hoover R.N., Pike M.C., et al. (1993) Migration patterns and breast cancer risk in Asian-American women. J. Natl Cancer Inst. 85:1819–1827.[Abstract/Free Full Text]
  4. Dai Q., Shu X.O., Jin F., Potter J.D., Kushi L.H., Teas J., Gao Y.T., Zheng W. (2001) Population-based case–control study of soyfood intake and breast cancer risk in Shanghai. Br. J. Cancer 85:372–378.[CrossRef][ISI][Medline]
  5. Limer J.L. and Speirs V. (2004) Phyto-oestrogens and breast cancer chemoprevention. Breast Cancer Res. 6:119–127.[CrossRef][ISI][Medline]
  6. Chen Z., Zheng W., Custer L.J., Dai Q., Shu X.O., Jin F., Franke A.A. (1999) Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in Shanghai. Nutr. Cancer 33:82–87.[ISI][Medline]
  7. Arai Y., Uehara M., Sato Y., Kimira M., Eboshida A., Adlercreutz H., Watanabe S. (2000) Comparison of isoflavones among dietary intake, plasma concentration and urinary excretion for accurate estimation of phytoestrogen intake. J. Epidemiol. 10:127–135.[Medline]
  8. de Kleijn M.J., van der Schouw Y.T., Wilson P.W., Adlercreutz H., Mazur W., Grobbee D.E., Jacques P.F. (2001) Intake of dietary phytoestrogens is low in postmenopausal women in the United States: the Framingham study(1-4). J. Nutr. 131:1826–1832.[Abstract/Free Full Text]
  9. Keinan-Boker L., Peeters P.H., Mulligan A.A., et al. (2002) Soy product consumption in 10 European countries: the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Public Health Nutr. 5:1217–1226.[CrossRef][ISI][Medline]
  10. Yamamoto S., Sobue T., Kobayashi M., Sasaki S., Tsugane S. (2003) Soy, isoflavones, and breast cancer risk in Japan. J. Natl Cancer Inst. 95:906–913.[Abstract/Free Full Text]
  11. Wu A.H., Wan P., Hankin J., Tseng C.C., Yu M.C., Pike M.C. (2002) Adolescent and adult soy intake and risk of breast cancer in Asian-Americans. Carcinogenesis 23:1491–1496.[Abstract/Free Full Text]
  12. Linseisen J., Piller R., Hermann S., Chang-Claude J. (2004) Dietary phytoestrogen intake and premenopausal breast cancer risk in a German case–control study. Int. J. Cancer 110:284–290.[CrossRef][ISI][Medline]
  13. Kurzer M.S. and Xu X. (1997) Dietary phytoestrogens. Annu. Rev. Nutr. 17:353–381.[CrossRef][ISI][Medline]
  14. Messina M.J., Persky V., Setchell K.D., Barnes S. (1994) Soy intake and cancer risk: a review of the in vitro and in vivo data. Nutr. Cancer 21:113–131.[ISI][Medline]
  15. Cotroneo M.S., Wang J., Fritz W.A., Eltoum I.E., Lamartiniere C.A. (2002) Genistein action in the prepubertal mammary gland in a chemoprevention model. Carcinogenesis 23:1467–1474.[Abstract/Free Full Text]
  16. Chang W.H., Liu J.J., Chen C.H., Huang T.S., Lu F.J. (2002) Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by fermented soy milk. Nutr. Cancer 43:214–226.[CrossRef][ISI][Medline]
  17. Shao Z.M., Alpaugh M.L., Fontana J.A., Barsky S.H. (1998) Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by P21WAF1/CIP1 induction, G2/M arrest, and apoptosis. J. Cell Biochem. 69:44–54.[CrossRef][ISI][Medline]
  18. Fotsis T., Pepper M.S., Montesano R., Aktas E., Breit S., Schweigerer L., Rasku S., Wahala K., Adlercreutz H. (1998) Phytoestrogens and inhibition of angiogenesis. Baillieres Clin. Endocrinol. Metab. 12:649–666.[CrossRef][ISI][Medline]
  19. Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y. (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262:5592–5595.[Abstract/Free Full Text]
  20. Di Virgilio A.L., Iwami K., Watjen W., Kahl R., Degen G.H. (2004) Genotoxicity of the isoflavones genistein, daidzein and equol in V79 cells. Toxicol. Lett. 151:151–162.[CrossRef][ISI][Medline]
  21. Kulling S.E., Rosenberg B., Jacobs E., Metzler M. (1999) The phytoestrogens coumoestrol and genistein induce structural chromosomal aberrations in cultured human peripheral blood lymphocytes. Arch. Toxicol. 73:50–54.[CrossRef][ISI][Medline]
  22. Record I.R., Jannes M., Dreosti I.E., King R.A. (1995) Induction of micronucleus formation in mouse splenocytes by the soy isoflavone genistein in vitro but not in vivo. Food Chem. Toxicol. 33:919–922.[CrossRef][ISI][Medline]
  23. Snyder R.D. and Gillies P.J. (2003) Reduction of genistein clastogenicity in Chinese hamster V79 cells by daidzein and other flavonoids. Food Chem. Toxicol. 41:1291–1298.[CrossRef][ISI][Medline]
  24. Anderson D., Dobrzynska M.M., Basaran N. (1997) Effect of various genotoxins and reproductive toxins in human lymphocytes and sperm in the Comet assay. Teratog. Carcinog. Mutagen. 17:29–43.[CrossRef][ISI][Medline]
  25. Miltyk W., Craciunescu C.N., Fischer L., et al. (2003) Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein, and glycitein) in 20 patients with prostate cancer. Am. J. Clin. Nutr. 77:875–882.[Abstract/Free Full Text]
  26. Pool-Zobel B.L., Adlercreutz H., Glei M., Liegibel U.M., Sittlingon J., Rowland I., Wahala K., Rechkemmer G. (2000) Isoflavonoids and lignans have different potentials to modulate oxidative genetic damage in human colon cells. Carcinogenesis 21:1247–1252.[Abstract/Free Full Text]
  27. Raschke M., Wahala K., Pool-Zobel B.L. (2006) Reduced isoflavone metabolites formed by the human gut microflora suppress growth but do not affect DNA integrity of human prostate cancer cells. Br. J. Nutr. 96:426–434.[ISI][Medline]
  28. Wattenberg L.W. (1992) Inhibition of carcinogenesis by minor dietary constituents. Cancer Res. 52:2085s–2091s.[Medline]
  29. Johnson I.T., Williamson G., Musk S.R.R. (1994) Anticarcinogenic factors in plant foods: a new class of nutrients? Nutr. Res. Rev. 7:175–204.
  30. Talalay P., Fahey J.W., Holtzclaw W.D., Prestera T., Zhang Y. (1995) Chemoprotection against cancer by phase 2 enzyme induction. Toxicol. Lett. 82-83:173–179.
  31. Birt D.F., Hendrich S., Wang W. (2001) Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol. Ther. 90:157–177.[CrossRef][ISI][Medline]
  32. Hayes J.D., Flanagan J.U., Jowsey I.R. (2005) Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45:51–88.
  33. Keppler D. (1999) Export pumps for glutathione S-conjugates. Free Radic. Biol. Med. 27:985–991.[CrossRef][ISI][Medline]
  34. Hachey D.L., Dawling S., Roodi N., Parl F.F. (2003) Sequential action of phase I and II enzymes cytochrome p450 1B1 and glutathione S-transferase P1 in mammary estrogen metabolism. Cancer Res. 63:8492–8499.[Abstract/Free Full Text]
  35. Wang L., Groves M.J., Hepburn M.D., Bowen D.T. (2000) Glutathione S-transferase enzyme expression in hematopoietic cell lines implies a differential protective role for T1 and A1 isoenzymes in erythroid and for M1 in lymphoid lineages. Haematologica 85:573–579.[Abstract/Free Full Text]
  36. Hayes J.D. and Pulford D.J. (1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev. Biochem. Mol. Biol. 30:445–600.[ISI][Medline]
  37. Montano M.M., Deng H., Liu M., Sun X., Singal R. (2004) Transcriptional regulation by the estrogen receptor of antioxidative stress enzymes and its functional implications. Oncogene 23:2442–2453.[CrossRef][ISI][Medline]
  38. Pool-Zobel B.L., Bub A., Liegibel U.M., Treptow-van Lishaut S., Rechkemmer G. (1998) Mechanisms by which vegetable consumption reduces genetic damage in humans. Cancer Epidemiol. Biomarkers Prev. 7:891–899.[Abstract]
  39. Ambrosone C.B., McCann S.E., Freudenheim J.L., Marshall J.R., Zhang Y., Shields P.G. (2004) Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocyanates, but is not modified by GST genotype. J. Nutr. 134:1134–1138.[Abstract/Free Full Text]
  40. Ebert M.N., Beyer-Sehlmeyer G., Liegibel U.M., Kautenburger T., Becker T.W., Pool-Zobel B.L. (2001) Butyrate induces glutathione S-transferase in human colon cells and protects from genetic damage by 4-hydroxy-2-nonenal. Nutr. Cancer 41:156–164.[CrossRef][ISI][Medline]
  41. Pool-Zobel B.L., Selvaraju V., Sauer J., Kautenburger T., Kiefer J., Richter K.K., Soom M., Wolfl S. (2005) Butyrate may enhance toxicological defence in primary, adenoma and tumor human colon cells by favourably modulating expression of glutathione S-transferases genes, an approach in nutrigenomics. Carcinogenesis 26:1064–1076.[Abstract/Free Full Text]
  42. Schaeferhenrich A., Beyer-Sehlmeyer G., Festag G., et al. (2003) Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal. Mutat. Res. 526:19–32.[ISI][Medline]
  43. Soule H.D., Maloney T.M., Wolman S.R., Peterson W.D. Jr, Brenz R., McGrath C.M., Russo J., Pauley R.J., Jones R.F., Brooks S.C. (1990) Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50:6075–6086.[Abstract/Free Full Text]
  44. Soule H.D., Vazguez J., Long A., Albert S., Brennan M. (1973) A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl Cancer Inst. 51:1409–1416.[ISI][Medline]
  45. Liu S. and Lin Y.C. (2004) Transformation of MCF-10A human breast epithelial cells by zeranol and estradiol-17beta. Breast J. 10:514–521.[CrossRef][Medline]
  46. Bell D.A., Taylor J.A., Paulson D.F., Robertson C.N., Mohler J.L., Lucier G.W. (1993) Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J. Natl Cancer Inst. 85:1159–1164.[Abstract/Free Full Text]
  47. Pemble S., Schroeder K.R., Spencer S.R., Meyer D.J., Hallier E., Bolt H.M., Ketterer B., Taylor J.B. (1994) Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism. Biochem. J. 300:271–276.
  48. Ebert M.N., Klinder A., Peters W.H., Schaferhenrich A., Sendt W., Scheele J., Pool-Zobel B.L. (2003) Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate. Carcinogenesis 24:1637–1644.[Abstract/Free Full Text]
  49. Peters W.H., Boon C.E., Roelofs H.M., Wobbes T., Nagengast F.M., Kremers P.G. (1992) Expression of drug-metabolizing enzymes and P-170 glycoprotein in colorectal carcinoma and normal mucosa. Gastroenterology 103:448–455.[ISI][Medline]
  50. Grubben M.J., Van Den Braak C.C., Broekhuizen R., De Jong R., Van Rijt L., De Ruijter E., Peters W.H., Katan M.B., Nagengast F.M. (2000) The effect of unfiltered coffee on potential biomarkers for colonic cancer risk in healthy volunteers: a randomized trial. Aliment. Pharmacol. Ther. 14:1181–1190.[CrossRef][ISI][Medline]
  51. Gardner J.L. and Gallagher E.P. (2001) Development of a peptide antibody specific to human glutathione S-transferase alpha 4-4 (hGSTA4-4) reveals preferential localization in human liver mitochondria. Arch. Biochem. Biophys. 390:19–27.[CrossRef][ISI][Medline]
  52. Habig W.H., Pabst M.J., Jakoby W.B. (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249:7130–7139.[Abstract/Free Full Text]
  53. Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.[CrossRef][ISI][Medline]
  54. Treptow-van Lishaut S., Rechkemmer G., Rowland I., Dolara P., Pool-Zobel B.L. (1999) The carbohydrate crystalean and colonic microflora modulate expression of glutathione S-transferase subunits in colon of rats. Eur. J. Nutr. 38:76–83.[CrossRef][ISI][Medline]
  55. Glei M., Liegibel U.M., Ebert M.N., Bohm V., Pool-Zobel B.L. (2002) beta-Carotene reduces bleomycin-induced genetic damage in human lymphocytes. Toxicol. Appl. Pharmacol. 179:65–73.[CrossRef][ISI][Medline]
  56. Glei M. and Pool-Zobel B.L. (2006) The main catechin of green tea, (-)-epigallocatechin-3-gallate (EGCG), reduces bleomycin-induced DNA damage in human leucocytes. Toxicol. In Vitro 20:295–300.[CrossRef][ISI][Medline]
  57. Singh N.P., McCoy M.T., Tice R.R., Schneider E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175:184–191.[CrossRef][ISI][Medline]
  58. Pool-Zobel B.L., Bub A., Muller H., Wollowski I., Rechkemmer G. (1997) Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis 18:1847–1850.[Abstract/Free Full Text]
  59. Breinholt V., Lauridsen S.T., Dragsted L.O. (1999) Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat. Xenobiotica 29:1227–1240.[CrossRef][ISI][Medline]
  60. Steinkellner H., Rabot S., Freywald C., Nobis E., Scharf G., Chabicovsky M., Knasmuller S., Kassie F. (2001) Effects of cruciferous vegetables and their constituents on drug metabolizing enzymes involved in the bioactivation of DNA-reactive dietary carcinogens. Mutat. Res. 480–481:285–297.
  61. Steinkellner H., Hoelzl C., Uhl M., Cavin C., Haidinger G., Gsur A., Schmid R., Kundi M., Bichler J., Knasmuller S. (2005) Coffee consumption induces GSTP in plasma and protects lymphocytes against (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide induced DNA damage: results of controlled human intervention trials. Mutat. Res. 591:264–275.[ISI][Medline]
  62. Morrow C.S., Chiu J., Cowan K.H. (1992) Posttranscriptional control of glutathione S-transferase pi gene expression in human breast cancer cells. J. Biol. Chem. 267:10544–10550.[Abstract/Free Full Text]
  63. Jhaveri M.S., Stephens T.E., Morrow C.S. (1997) Role of posttranscriptional processes in the regulation of glutathione S-transferase P1 gene expression in human breast cancer cells. Biochem. Biophys. Res. Commun. 237:729–734.[CrossRef][ISI][Medline]
  64. Kautenburger T., Beyer-Sehlmeyer G., Festag G., et al. (2005) The gut fermentation product butyrate, a chemopreventive agent, suppresses glutathione S-transferase theta (hGSTT1) and cell growth more in human colon adenoma (LT97) than tumor (HT29) cells. J. Cancer Res. Clin. Oncol. 131:692–700.[CrossRef][ISI][Medline]
  65. Jhaveri M.S. and Morrow C.S. (1998) Methylation-mediated regulation of the glutathione S-transferase P1 gene in human breast cancer cells. Gene 210:1–7.[CrossRef][ISI][Medline]
  66. Usami H., Kusano Y., Kumagai T., Osada S., Itoh K., Kobayashi A., Yamamoto M., Uchida K. (2005) Selective induction of the tumor marker glutathione S-transferase P1 by proteasome inhibitors. J. Biol. Chem. 280:25267–25276.[Abstract/Free Full Text]
  67. Fields W.R., Li Y., Townsend A.J. (1994) Protection by transfected glutathione S-transferase isozymes against carcinogen-induced alkylation of cellular macromolecules in human MCF-7 cells. Carcinogenesis 15:1155–1160.[Abstract/Free Full Text]
  68. Kirlin W.G., Cai J., DeLong M.J., Patten E.J., Jones D.P. (1999) Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells. J. Nutr. 129:1827–1835.[Abstract/Free Full Text]
  69. Key T.J., Allen N.E., Spencer E.A., Travis R.C. (2003) Nutrition and breast cancer. Breast 12:412–416.[CrossRef][ISI][Medline]
  70. Esterbauer H., Schaur R.J., Zollner H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11:81–128.[CrossRef][ISI][Medline]
  71. Chung F.L., Chen H.J., Nath R.G. (1996) Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis 17:2105–2111.[Abstract/Free Full Text]
  72. Knoll N., Ruhe C., Veeriah S., Sauer J., Glei M., Gallagher E.P., Pool-Zobel B.L. (2005) Genotoxicity of 4-hydroxy-2-nonenal in human colon tumor cells is associated with cellular levels of glutath