* Institute for Risk Assessment Sciences (IRAS), Universiteit Utrecht, Utrecht, The Netherlands; and St. Antonius Hospital, Department of Internal Medicine, Nieuwegein, The Netherlands
Received May 4, 2004; accepted July 5, 2004
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ABSTRACT |
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Key Words: phytochemicals; catechol estrogens; COMT; DNA damage.
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INTRODUCTION |
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Low COMT activity has been associated with increased breast cancer risk (reviewed by Yue et al., 2003). There are several ways in which COMT activity might be altered. Lachman et al. have described a low-activity form of COMT resulting from a genetic polymorphism (Lachman et al., 1996
). A single nucleotide substitution in codon 108 causes an amino acid transition (Val
Met) which results in a high- (Val/Val) or low-activity (Met/Met) form of the COMT enzyme with a three-to-four-fold difference in activity. COMT activity can also be inhibited by substrate competition for the enzyme. There are many naturally occurring substrates for COMT in the body, but some exogenous compounds have also been identified as substrates for the enzyme. For example, certain catechol metabolites of PCBs have been shown to inhibit COMT activity (Garner et al., 2000
). In addition, many dietary catechols, such as phytochemicals, can be a substrate for COMT. Phytochemicals are a diverse group of chemicals which can be found in fruits and vegetables. This group of biologically active compounds occurs in high concentrations in our diet, and the daily intake can comprise a few hundreds of milligrams per day (Hollman and Katan, 1999
). As a result, submicromolar plasma levels can be reached (Hollman and Katan, 1999
; Rein et al., 2000
; Warden et al., 2001
). Phytochemicals have been shown to possess antioxidant, anticancer, and antiviral properties. Because of these properties, they are generally regarded as safe, and many phytochemicals are sold in high concentrations as dietary supplements with recommended intake levels that exceed normal daily intake up to a 100-fold. However, in addition to the beneficial properties, phytochemicals may also affect various enzyme activities. For example, Zhu and Liehr described the effect of quercetin, a phytochemical found in many food items, on COMT activity in male Syrian hamsters (Zhu and Liehr, 1996
). In hamsters fed with quercetin, a decreased COMT activity was found that resulted in increased catechol estrogen concentrations in the kidneys and subsequent enhancement of estradiol-induced tumorigenesis.
In the present study, we investigated COMT activity in healthy mammary tissues, where COMT plays an important role in the inactivation potentially genotoxic catechol estrogens. We studied the constitutive rates of O-methylation of catechol estrogens and the effects of phytochemicals on this activity in healthy human mammary tissue cytosol. We hypothesized that phytochemicals with a catechol structure, like quercetin, catechin, and (-)-epicatechin (chemical structures in Fig. 1), make a suitable substrate for the COMT enzyme and thus potentially inhibit the formation of methoxy estrogens. We also investigated the effects of several phytochemicals without a catechol structure (genistein, chrysin, and flavone) on COMT activity. Ro 41-0960, a known selective COMT inhibitor, was used as a standard positive control. COMT inhibition results in decreased inactivation of catechol estrogens, which in turn may lead to increased DNA damage. Therefore, we studied the implications of decreased COMT activity caused by phytochemicals on catechol estrogen-induced DNA damage by performing the alkaline comet assay using the malignant human mammary tumor cell line MCF-7.
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MATERIALS AND METHODS |
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Sample preparation. Tissues from reduction mammoplasty (n = 7) were obtained from the Antonius Hospital (Nieuwegein, the Netherlands). The study was approved (number TME/Z-02.09) by the medical ethical committee of the hospital. All women gave permission for the removed tissue to be used by an informed consent. Tissues were diagnosed as histologically normal breast tissue by a pathologist. Upon arrival in our laboratory, fresh tissues were snap frozen in liquid nitrogen and stored at 70°C until use. Before preparation of cytosolic fractions, the tissues were thawed at 4°C and kept on ice. Adipose tissue was removed with a surgical knife, and the remaining parenchyma was cut into small pieces. The tissue pieces were weighed, and 3 ml cold phosphate buffer (50 mM, pH 7.6 containing 0.1 mM EDTA) was added per mg tissue. This mixture was homogenized with a Potter-Elvehjem Teflon-glass homogenizer. Cytosolic fractions were prepared through ultracentrifugation (Beckman L7-55). Homogenates were first centrifuged at 10,000 x g for 15 min at 4°C to remove the cell debris and remaining adipose tissues. Subsequently, the supernatant was centrifuged at 100,000 x g for 75 min at 4°C to separate the cytosolic (supernatant) from the microsomal (pellet) fractions. Aliquots of the cytosolic fractions were stored at 70°C until analysis. Protein contents of the fractions were determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as protein standard.
COMT activity. In order to study the O-methylation activity, cytosolic protein (300 µg) was incubated with 50 mM phosphate buffer (pH 7.6), 5 mM MgCl2, 150 µM SAM, 1 mM DTT, and various concentrations of a phytochemical or the solvent vehicle (0.1% v/v MeOH) to a final volume of 492.5 µl. Reaction mixtures were incubated at 37°C for 5 min before the reaction was started by adding 2-OHE2 and 4-OHE2 (3.75 µM each). After 30 min, the reaction was stopped by putting the reaction tubes on ice. The metabolite extraction procedure was adapted from Spink et al. and performed as described previously (Spink et al., 1990; van Duursen et al., 2003
). Briefly, the internal standard (20 µl equilin, 10 µM) was added, and 2- and 4-MeOE2 were extracted with dichloromethane. Trimethylsilyl derivatives of the estrogens were prepared and analyzed by GC/MS. Peak areas were determined at m/z 446 and 340 for 2- and 4-MeOE2 and equilin, respectively. Peak identification and quantification was performed with the corresponding standards.
Cell lines and cell culture. MCF-7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco's Modified Eagle's Medium supplemented with 0.01 mg/ml insulin, 5% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.
Cell viability. The cell viability was determined by measuring the capacity of the cells to reduce MTT (3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the blue-colored formazan (Denizot and Lang, 1986). Cell cultures of MCF-7 were exposed to the catechol estrogens (7.5 µM 2- and 4-OHE2), Ro 41-0960 (10 µM), or quercetin (30 µM) for 6 h. Then, serum-free medium containing 1 mg MTT/ml was added for 1 h. The medium containing MTT was then removed, and the cells were washed twice with warm PBS. Formazan was extracted by adding 1 ml isopropanol at room temperature. Formazan formation was measured spectrophotometrically at an absorbance wavelength of 560 nm, and cell viability was calculated using solvent vehicle-treated cells (ethanol, methanol, and DMSO, total of 0.17% v/v) as 100% viable control cells.
Alkaline single-cell gel electrophoresis (comet) assay. The effect of COMT inhibition on DNA damage caused by catechol estrogens was determined using the single-cell gel electrophoresis (comet) assay as described by Singh et al. with some modifications (Singh et al., 1988). For this assay, 5 x 105 MCF-7 cells were plated onto 12-well plates and placed in a humidified atmosphere with 5% CO2 at 37°C. The next day, cells were exposed for 5 h to serum-free medium containing the solvent vehicles (ethanol, methanol, and DMSO, total of 0.17% v/v) and catechol estrogens (7.5 µM 2-OHE2 and 4-OHE2), Ro 41-0960 (10 µM), and quercetin (10 µM or 30 µM), alone or in combination. Then, media were removed and analyzed for methoxy estradiol concentrations as described above. The cells were washed with PBS, and 100 µl trypsin was added. As soon as the cells detached, 1 ml of warm medium containing 5% FBS was added, and the cells were suspended and transferred to a 1.5-ml Eppendorf cup. The cell suspension was briefly centrifuged, and 1000 µl of the supernatant was removed. The remaining cells were gently resuspended, and a 10 µl aliquot was added to 90 µl warm 0.5% low-melting agarose. This mixture was spread onto a frosted slide covered with 1.5% normal melting agarose and placed on a ice-cold glass plate to solidify. Then, the slides were placed in freshly prepared cold lysis solution (2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Tris containing 1% Triton-X, and 10% DMSO, pH 10) for 1 h at 4°C. After this, the slides were kept in the dark to prevent DNA damage by exposure to direct light. Subsequently, the slides were placed in a horizontal slide holder in an electrophoresis unit containing cold electrophoresis solution (0.3 M NaOH, 0.001 M EDTA) for 25 min and then electrophoresed for 25 min at 25 V, 290310 mA. Then, the slides were washed three times with a sterile neutralization buffer (0.4 M Tris/HCl, pH 7.5) and dehydrated for 10 min in 100% ethanol. The slides were kept in a dark box at 4°C until analysis. Prior to analysis, the slides were stained with ethidium bromide (20 µg/ml). Analysis was performed under a fluorescence microscope using a 20x objective and a filter of 450490 nm equipped with a digital camera. Of each treatment 175200 cells (four slides per treatment, 4050 cells per slide) were analyzed and the tail moment (comet tail length x % tail DNA) was determined using the PC image-analysis program Casp described by Konca et al. (2003)
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Data analysis. Enzyme kinetic parameters (Vmax and Km values in pmol/min/mg protein and µM, respectively) were calculated with Prism 3.0 (GraphPad Software, San Diego, CA). Statistical significance of difference of the mean was determined by the Student's t-test. Variance and differences among the means were determined by a one-way ANOVA with a Tukey-Kramer Multiple Comparisons test using GaphPad InStat 3.06 (GraphPad Software Inc., San Diego, CA).
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RESULTS |
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The constitutive rates of methylation of catechol estrogens in the various tissue samples are shown in Table 1. The mean 2-MeOE2 formation of seven tissues was 8.12 ± 1.32 pmol/min/mg protein with a range of activity of 2.1419.03 pmol/min/mg protein. For 4-MeOE2, the mean metabolite formation and range of activity were 1.83 ± 0.29 and 0.373.81 pmol/min/mg protein, respectively. ANOVA analysis showed significant differences in 2- and 4-MeOE2 formation between the tissue samples (p = 0.0004 and p = 0.0044, respectively). Further analysis showed that cytosol from tissue sample 3 had substantially higher rates of methoxy estradiol formation compared with other tissue samples. However, despite the variation in rates of methylation, the ratio of 4-MeOE2/2-MeOE2 formation at 7.5 µM 2- and 4-OHE2 was not significantly different statistically between the tissues (ANOVA analysis, p = 0.125). The average 4-/2-MeOE2 ratio in seven tissue samples was 0.26 ± 0.02.
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Catechol Estrogen-Induced DNA Damage in MCF-7 Cells
To investigate the implications of possible COMT inhibition by quercetin in whole cells, the effects of quercetin on catechol estrogen-induced DNA damage were studied. MCF-7 cells were exposed to catechol estrogens with or without Ro 41-0960 and quercetin. Then the amount of DNA damage was determined using the comet assay, and methoxy estrogen levels were determined in the culture medium by GC/MS analysis.
An equimolar concentration of catechol estrogens (7.5 µM), catechol estrogens together with quercetin (30 µM) or Ro 41-0960 (10 µM), or a combination of the two COMT inhibitors, did not cause cytotoxicity in MCF-7 cells, as determined by the MTT test (data not shown). Incubation of MCF-7 cells with catechol estrogens, Ro 41-0960, or quercetin alone did not cause a significant increase of DNA damage compared with the solvent vehicle-treated cells (Fig. 4). The extent of background DNA damage was significantly increased (by about 200%) in catechol estrogen-exposed cells when COMT was inhibited by 10 µM Ro 41-0960 (p < 0.05). When quercetin was added to the cells together with Ro 41-0960, catechol estrogen-induced DNA damage increased even further, in an apparent concentration-dependent manner, compared with Ro 41-0960 treated cells. A concentration-dependent increase of catechol estrogen-induced DNA damage was also seen after incubation with catechol estrogens and quercetin alone. Catechol estrogen-induced DNA damage levels increased 76 and 160% when cells were co-incubated with 10 and 30 µM quercetin, respectively, compared with vehicle-treated control cells.
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DISCUSSION |
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Effect of Phytochemicals on COMT Activity
In this study we showed that phytochemicals with a catechol structure are capable of inhibiting COMT activity in cytosolic fractions of healthy human mammary tissues. Zhu and Liehr reported that quercetin acted as inhibitor of COMT activity in hamster kidney cytosol, with IC50 values of 8 µM and 2 µM at substrate concentrations of 10 µM 2-OHE2 and 4-OHE2, respectively (Zhu and Liehr, 1996). We found in this study lower IC50 values for COMT inhibition with quercetin (0.5 µM for both 2-OHE2 and 4-OHE2). This is probably due to differences in study design; we added both 2- and 4-OHE2 together to the cytosol, and we used human COMT. Zhu and Liehr concluded that quercetin acted as a noncompetitive inhibitor of COMT activity by competing for SAM. Quercetin and other phytochemicals containing a catechol structure have shown to be a substrate for COMT and, thus, compete for cofactors necessary for O-methylation of the substrate, such as SAM (Zhu et al., 2000
). Upon O-methylation, a methyl group from SAM is transferred to the catechol substrate resulting in S-adenosyl-L-homocysteine (SAH). An increasing concentration of SAH was shown to (noncompetitively) inhibit the association of the methyl donor SAM with COMT. This might also explain why phytochemicals reduced COMT activity to 60% of the control activity, while Ro 41-0960 fully inhibited methylation. Ro 41-0960 is a poor substrate for COMT, but it binds tightly to the catalytic site of the enzyme, thus inhibiting methylation of other substrates without depletion of cofactors (Backstop et al., 1989
; Ding et al., 1996
).
The tested phytochemicals with a catechol structure, quercetin, catechin, and (-)-epicatechin, all reduced COMT activity, but large differences in inhibitory potency were found. This might be a result of structural differences of these phytochemicals, but interindividual variations among the tissue samples in COMT activity and responsiveness toward inhibition might also play a role. Interindividual variation between the tissue samples was especially apparent with flavone, which showed COMT inhibition in sample 3, but not in sample 5. In an attempt to correct for interindividual variations in sensitivity toward COMT inhibition, we calculated the potency of a phytochemical relative to the potency of Ro 41-0960 to inhibit COMT activity in the same tissue sample. The relative potencies (RPs) varied less than a 100-fold with the RPs of the three phytochemicals with a catechol structure (e.g. quercetin, catechin, and epicatechin) being higher than the RP of flavone, the phytochemical without a catechol structure. However, we did not study the potencies of all phytochemicals in all the tissue samples. As a result, it is not clear whether the RPs represent the differences between individuals or differences between the potencies of the phytochemicals. Therefore, the calculated inhibition potencies of the phytochemicals, both absolute and relative to Ro 41-0960, should be considered with care.
Catechol Estrogen-Induced DNA Damage in MCF-7 Cells
Although COMT activity was inhibited by the phytochemicals in cytosol from healthy mammary tissues, the question was raised if this inhibition is relevant in a more complex system such as whole cells. We showed that incubation with Ro 41-0960 or quercetin caused a significant increase in DNA damage by catechol estrogens compared with catechol estrogens alone. Chen et al. also showed by the comet assay a low potency of another catechol estrogen, 4-hydroxyestrone (4-OHE1), to induce DNA damage in MCF-7 cells (Chen et al., 2000). They mainly attributed this low potency to the fact that 4-OHE1 does not auto-oxidize and requires oxidative enzymes to generate the highly reactive quinone. However, our study suggests that the inactivation of catechol estrogens plays an important role in the potential of these compounds to cause DNA damage. We showed that a decrease in inactivation of the potentially genotoxic catechol estrogens by COMT inhibition caused a significant increase in catechol estrogen-induced DNA damage. Our data concur with the results described by Lavigne et al. (2001)
. They found a clear association between catechol estrogen levels and 8-oxo-dG levels in MCF-7 cells after treatment with estradiol and the COMT inhibitor Ro 41-0960. These data show that catechol estrogens have the potential to induce DNA damage, but that this is strongly dependent on the cellular capacity for inactivation by COMT.
Implications for Breast Cancer Development
Phytochemicals are often studied in relation with hormone-dependent cancers such as breast cancer. The low breast cancer incidence in Asian countries is often attributed to the soy-rich diet, which contains high concentrations of isoflavones like genistein. On the other hand, some studies describe a deleterious effect of certain phytochemicals in women with breast cancer (Lesperance et al., 2002; Tagliaferri, 2001
). Our study shows that phytochemicals with a catechol structure have the potential to reduce COMT activity in cytosol of healthy mammary tissues at concentrations which are well within the range of plasma levels that are reached by regular daily intake. We found for quercetin an IC50 value of 0.5 µM for COMT inhibition. Hollman and Katan found plasma levels up to 0.74 µM quercetin after consumption of a meal rich in plant products (Hollman and Katan, 1999a). It is not unlikely that higher levels can be reached, since quercetin plasma levels return to basal levels after about 20 h, so repeated consumption of high levels of quercetin can result in accumulation in the blood. Furthermore, the COMT-inhibiting properties of quercetin also resulted in decreased inactivation of potentially genotoxic catechol estrogens and an increase in catechol estrogen-induced DNA damage. Yet, it is difficult to predict the effects of excessive phytochemical intake in individuals, as large variations in COMT activity and responses to COMT inhibition between the various breast tissue samples were found. Nevertheless, this study shows that adverse effects of high levels of certain phytochemicals are not unlikely. Therefore, high intake of phytochemicals, for example through dietary supplements, should be considered with care.
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NOTES |
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1 To whom correspondence should be addressed at Institute for Risk Assessment Sciences, Yalelaan 2, P.O. Box 80176, 3508 TD UTRECHT, The Netherlands. Fax: +31 30 253 5077. E-mail: M.vanDuursen{at}iras.uu.nl.
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