832
Chem. Res. Toxicol. 2003, 16, 832-837
Antiestrogenic and DNA Damaging Effects Induced by Tamoxifen and Toremifene Metabolites Xuemei Liu,† Emily Pisha,† Debra A. Tonetti,‡ Dan Yao,† Yan Li,† Jiaqin Yao,† Joanna E. Burdette,† and Judy L. Bolton*,† Department of Medicinal Chemistry and Pharmacognosy (M/C 781), and Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illnois 60612 Received January 14, 2003
The antiestrogen, tamoxifen, has been extensively used in the treatment and prevention of breast cancer. Although tamoxifen showed benefits in the chemotherapy and chemoprevention of breast cancer, epidemiological studies in both tamoxifen-treated breast cancer patients and healthy women indicated that treatment caused an increased risk of developing endometrial cancer. These troubling side effects lead to concerns over long-term safety of the drug. Therefore, it is important to fully understand the relationship between the antiestrogenic and the genotoxic mechanisms of tamoxifen, other antiestrogens, and their metabolites. Previously, we have shown that o-quinone formation from tamoxifen and its analogues, droloxifene and 4-hydroxytoremifene, may not contribute to the cytotoxic effects of these antiestrogens; however, these o-quinones can form adducts with deoxynucleosides and this implies that the o-quinone pathway could contribute to the genotoxicity of the antiestrogens in vivo. To further investigate this potential genotoxic pathway, we were interested in the role of estrogen receptor (ER)1 R and β since work with catechol estrogens has shown that ERs seem to enhance DNA damage in breast cancer cell lines. As a result, we investigated the binding affinities of 4-hydroxy and 3,4-dihydroxy derivatives of tamoxifen and toremifene to ER R and β. The antiestrogenic activities of the metabolites using the Ishikawa cells were also investigated as well as their activity in ERR and ERβ breast cancer cells using the transient transfection reporter, estrogen response element-dependent luciferase assay. The data showed that the antiestrogenic activities of these compounds in the biological assays mimicked their activities in the ER binding assay. To determine if the compounds were toxic and if ERs played a role in this process, the cytotoxicity of these compounds in ERβ412 (ERβ), S30 (ERR), and MDA-MB-231 (ER-) cell lines was compared. The results showed that the cytotoxicity differences between the metabolites were modest. In addition, all of the metabolites showed similar toxicity patterns in both ER positive and negative cell lines, which means that the ER may not contribute to the cytotoxicity pathway. Finally, we compared the amount of DNA damage induced by these metabolites in these cell lines using the comet assay. The catechols 3,4-dihydroxytoremifene and 3,4-dihydroxytamoxifen induced a greater amount of cellular single strand DNA cleavage as compared with the phenols in all cell lines. The different amounts of DNA damage in ER positive and negative cell lines suggested that the ERs might play a role in this process. These data suggest that the formation of catechols represents a minor role in cytotoxic and antiestrogenic effects in cells as compared with their phenol analogues. However, catechols induced more DNA damage at nontoxic doses in breast cancer cells, which implies that o-quinones formed from catechols could contribute to genotoxicity in vivo, which is ERdependent.
Introduction TAM,1,2 a nonsteroidal antiestrogen, has been used since the early 1970s to treat estrogen responsive breast * To whom correspondence should be addressed. Fax: (312)9967017. E-mail:
[email protected]. † Department of Medicinal Chemistry and Pharmacognosy. ‡ Department of Biopharmaceutical Sciences. 1 Abbreviations: 3,4-Di-OHTAM, 3,4-dihydroxytamoxifen; 3,4-diOHTOR, 3,4-dihydroxytoremifene; DMSO, dimethyl sulfoxide; ED50, dose of 50% cells killed; ER, estrogen receptor; EtBr, ethidium bromide; IC50, dose of half-maximal induction; LMAgarose, low melting agarose; 4-OHTAM, 4-hydroxytamoxifen; 4-OHTOR, 4-hydroxytoremifene; P450, cytochrome P450; PSF, penicillin-streptomycin-fungizome; SERMs, selective estrogen receptor modulators; TAM, tamoxifen; TOR, toremifene.
cancer. Because of observations of decreases in contralateral breast cancer in women treated with TAM, the National Institutes of Health initiated a large cancer chemoprevention trial of TAM in women of high risk in the 1990s. The study was terminated early when TAM reduced the overall risk of developing breast cancer by 49% (1). However, a recent report has confirmed an increased incidence of endometrial cancer among TAM recipients with a relative risk up to seven as compared with nonusers and an increased mortality from the disease (2). In addition, short- and long-term treatment 2 Described in “MDA-MB-231 estrogen receptor beta stable clones exhibit distinct growth and transcriptional characteristics” (Tonetti, and et al., submitted to Int. J. Cancer).
10.1021/tx030004s CCC: $25.00 © 2003 American Chemical Society Published on Web 05/17/2003
Activities of Tamoxifen and Toremifene Metabolites Scheme 1. Metabolism of TAM (X ) H) and TOR (X ) Cl)
Chem. Res. Toxicol., Vol. 16, No. 7, 2003 833 unless stated otherwise. 4-Hydroxy and 3,4-di-OHTAM and TOR derivatives were synthesized as described previously (15, 17, 18). The comet assay kit for the detection of DNA damage was purchased from Trevigen Inc. (Gaithersburg, MD). Human ER proteins were obtained from Panvera Corp. (Madison, WI). Cell Culture Conditions. The S30 cell line was a generous gift from V. C. Jordan’s laboratory (Northwestern University, Chicago, IL) (19). Both S30 and ERβ41 cells were maintained in minimum essential medium with 1% nonessential amino acids, 6 µg/L insulin, 1% PSF, 500 µg/mL G418, 1% glutamax (Gibco-BRL, Grand Island, NY), 5% charcoal-dextran-treated fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), and 5% CO2 at 37 °C. The MDA-MB-231 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in MEME medium supplemented with 1% PSF, 6 µg/L insulin, 1% glutamax, 5% FBS, and 5% CO2 at 37 °C. The cells (105 cells/mL) were grown for 24-48 h to maintain logarithmic growth and then treated with various concentrations of the TAM analogues or DMSO in fresh medium, with the final DMSO concentration of 0.05%.
of rats with TAM resulted in hepatocarcinomas (3-5). TAM-DNA adducts found in the endometrium and leukocytes of women undergoing TAM treatment were similar to those found in the rat hepatocarcinomas (6, 7). These disturbing side effects add to the concern of long-term safety and have led to the development of structural analogues of TAM (8, 9). One potential cytotoxic mechanism for TAM and some of its analogues involves metabolism to quinoids (10). For example, TAM and its β-cholorinated analogue, TOR, readily undergo 4-hydroxylation via P450 2D6 and P450 3A (11, 12), respectively, and further oxidation by P450s results in the formation of the electrophilic quinone methides (13). In addition, the 4-hydroxy metabolites can be further hydroxylated at the 3-position leading to the formation of the 3,4-dihydroxy catechols (14) and both catechols are further oxidized to o-quinones (Scheme 1). Previously, we showed that both the TAM-o-quinone and the TOR-o-quinone reacted with deoxynucleosides to give corresponding adducts and the catechols showed less cytoxic effects in breast cancer cells as compared to phenols (15). In the present study, we investigated the relationship of antiestrogenic effects and DNA damage induced by TAM and TOR metabolites. Relative affinities of test compounds to ERs were examined by competitive binding assay, and antiestrogenic activities were determined using Ishikawa cells and estrogen response element (ERE) luciferase assays. The data indicated that catechols had a lower binding affinity to ERs and reduced antiestrogenic effects as compared to their phenol analogues. In addition, cytotoxicity was measured in ERR, ERβ, and ER negative breast cancer cell lines to determine the relative toxic potency of these metabolites. Finally, the capability of these metabolites to induce DNA damage in these cell lines at a nontoxic dose was studied. The data showed that the formation of o-quinone may play a minor role in cytotoxic and antiestrogenic effects but could induce more DNA damage, which is ERdependent.
Materials and Methods Caution: Catechols were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (16). Materials. Chemicals were purchased from Fisher Scientific (Itasca, IL), Aldrich (Milwaukee, WI), or Sigma (St. Louis, MO)
ER Competitive Binding Assay. The procedure of Obourn et al. (20) was used with minor modifications (21). Briefly, 24 h prior to the assay, a 50% v/v hydroxylapatite slurry was prepared using 10 g of hydroxylapatite in 60 mL of TE buffer (50 mM Tris-Cl, 1 mM EDTA, pH 7.4) and stored at 4 °C. The ER binding buffer consisted of 10 mM Tris-Cl (pH 7.5), 10% glycerol, 2 mM dithiothrietol, and 1 mg/mL bovine serum albumin. The ERR wash buffer contained 40 mM Tris-Cl (pH 7.5), 100 mM KCl, and 1 mM EDTA, and the ERβ wash buffer was 40 mM Tris-Cl (pH 7.5). A “Hot Mix” of 400 nM [3H]estradiol was freshly prepared consisting of 3.2 µL of 25 µM [3H]estradiol (83 Ci/mmol), 98.4 µL of ethanol, and 98.4 µL of ER binding buffer. The reaction mixture consisted of 5 µL of test sample, 5 µL of pure human recombinant diluted ERR or ERβ (0.5 pmol), 5 µL of Hot Mix, and 85 µL of ER binding buffer. The incubations were carried out at room temperature for 2 h, and then, 100 µL of 50% hydroxylapatite slurry was added and the tubes were incubated on ice for 15 min with vortexing every 5 min. The appropriate ER wash buffer was added (1 mL), and the tubes were vortexed and centrifuged at 2000g for 5 min. The supernatant was discarded, and the wash step was repeated three times. The hydroxylapatite pellet containing the ligand-receptor complex was resuspended in 200 µL of ethanol and transferred to scintillation vials. Cytoscint (4 mL/vial) was added, and the tubes were counted using a Beckman LS 5801 (Schaumburg, IL) liquid scintillation counter. The percent inhibition of [3H]estradiol binding to each ER was determined as follows: [(dpmsample - dpmblank)/(dpmDMSO - dpmblank) - 1] × 100. The binding capability (%) of the samples was calculated in comparison to estradiol (50 nM, 100%). The data represent the average ( SD of three determinations. Inhibition of Estrogen-Induced Alkaline Phosphatase Activity in Ishikawa Cells. The procedure of Pisha et al. was used as described previously (22). Briefly, Ishikawa cells (5 × 104 cells/mL) were incubated overnight with estrogen-free media in 96 well plates. Test samples (with 1 nM 17β-estradiol) and appropriate controls were added; the 0 day control did not contain any additional estradiol. The cells, in a total volume of 200 µL/well, were incubated at 37 °C for 4 days. The cells were washed three times with PBS and lysed by freeze-thawing in the presence of 0.1 M Tris, pH 8.0. Enzyme activity was measured by reading the liberation of p-nitrophenol from 1 µM p-nitrophenolphosphate at 340 nm every 15 s for 16-20 readings with an ELISA reader (Power Wave 200 Microplate Scanning Spectrophotometer, Bio-Tek Instrument, Winooski, VT). The maximum slope of the lines generated by the kinetic readings was calculated by the Kinecalc computer program (Bio-Tek Instrument). For antiestrogenic activity, the percent induction was determined as follows: [(slopesample - slopecells)/(slopeestrogen - slopecells)] × 100. The data represent the average ( SD of three determinations.
834
Chem. Res. Toxicol., Vol. 16, No. 7, 2003
Liu et al.
Table 1. Antiestrogenic Effects of TAM and TOR Metabolites ER binding assaya IC50 (nM)
inhibition of alkaline phosphatase activityb
percent induction of luciferase activityc
compounds
ERR
ERβ
R/β
IC50 (nM)
S30
ERβ41
TAM 4-OHTAM 3,4-di-OHTAM 4-OHTOR 3,4-di-OHTOR
197 ( 18 1.0 ( 0.3 17.6 ( 6.5 1.0 ( 0.3 5.3 ( 0.8
120 ( 23 3.2 ( 0.4 5.0 ( 1.2 1.0 ( 0.2 5.6 ( 2.0
1.6 0.3 3.5 1.0 1.0
65.0 ( 8.6 2.4 ( 1.1 52.0 ( 11.0 1.6 ( 0.8 36.0 ( 6.0
8.3 ( 0.6 4.7 ( 0.1 17.6 ( 0.3 5.4 ( 0.1 10.4 ( 0.2
44.1 ( 1.0 9.0 ( 0.1 46.5 ( 0.2 6.0 ( 0.1 43.3 ( 0.3
a The reaction mixture consisted of 5 µL of test samples in DMSO, 5 µL of pure human recombinant diluted ER in 85 µL of ER binding buffer, and 5 µL of Hot Mix. Samples were incubated for 2 h. The data represent the average ( SD of three determinations. Experimental details are described in the Material and Methods. b Ishikawa cells (5 × 104 cells/well) were treated with test compounds with 17βestradiol (1 nM) for 4 days. The data represent the average ( SD of three determinations. Experimental details are described in the Material and Methods. c Cells (4 × 104 cells/well) were treated with test compounds (100 nM) with estradiol (1 nM) for 1 day. The normalized induction of 1 nM estradiol was set at 100. The results are expressed as the average ( SD of six determinations. Experimental details are described in the Material and Methods.
Determination of Inhibition of Estrogen-Induced Luciferase Activity. S30 and ERβ41 cells (2 × 104 cells/mL) were plated onto 12 well plates and transiently transfected with ERE luciferase and β-galactosidase using Fugene 6 (Roche Biochemicals, Germany). ERE luciferase (1 µg) and β-galactosidase plasmids (1 µg) (both gifts from Ah-Ng Tony Kong of Rutgers University, NJ) were mixed with 3 µL of Fugene in clear, nonsupplemented MEME for a final volume of 100 µL and added to each well. The cells were transfected overnight and then incubated with fresh media containing 100 nM compounds with and without 1 nM 17β-estradiol and controls (DMSO and 1 nM 17β-estradiol) for 24 h at which time the cells were harvested for luciferase and β-galactosidase activity in 50 µL of passive lysis buffer (Promega, Madison, WI). Luciferase activity was determined with 10 µL aliquots of cell lysates. The lysates were mixed with 100 µL of the Luciferase Assay kit (Promega) and read on a Lumistar galaxy luminometer (BNG Labtechnologies, Durham, NC) every 0.5 s for 25 s in a 96 well plate format. The β-galactosidase activity was determined with 10 µL aliquots of the lysates added to 400 µL of Z buffer (0.06 M Na2HPO4, 0.04 M NaH2PO4, 0.01 M KCl, 0.001 M MgSO4, and 0.05 M β-mercaptoethanol) and 100 µL of 4 mg/mL o-nitophenyl-β-D-galactopyranosidase in Z buffer. The reactions were incubated overnight at room temperature and stopped by the addition of 500 µL of 1 M Na2PO3. The resulting yellow color was read at 420 nm on a microplate spectrophotometer. The luciferase activity was normalized to the β-galactosidase activity. Percent estrogen induction was calculated as follows: [(Luciferasesample - LuciferaseDMSO)/(Luciferaseestradial - LuciferaseDMSO)] × 100. The results are expressed as the average ( SD of six determinations. Evaluation of the Cytotoxic Potential in Cancer Cell Lines. Cell viability was determined by trypan blue exclusion. Briefly, the cells (105 cells/mL) were incubated with various concentrations of TAM derivatives or DMSO for 18 h. After they were treated, floating cells were collected by centrifugation at 3000 rpm for 5 min and attached cells were first trypsinized and then harvested by centrifugation. Floating cells and attached cells were combined, washed with PBS, and stained with 0.4% trypan blue. A drop of cell suspension was placed on a hemocytometer, and the cell number was determined using a light microscope. The LC50 values were obtained by linear regression analysis, and the data represent the average ( SD of four determinations. DNA Damage in Breast Cancer Cell Lines Using the Alkaline Single Cell Gel Electrophoresis Assay (Comet Assay). The Comet assay was carried out as recommended by the manufacturer (Trevigen Inc.). Briefly, after they were incubated with the test compounds for 90 min, the attached cells were trypsinized and combined with the suspended cells in the medium by centrifugation. The cells were washed with PBS and resuspended in PBS at a concentration of 2 × 105 cells/mL. Cells (30 µL) were combined with LMAgarose (300 µL, 42 °C), and 50 µL of the mixture was immediately placed onto a CometSlide. The slides were incubated at 4 °C in the dark for 10 min followed
by immersion in prechilled Lysis Solution at 4 °C for 30 min and then incubated in freshly prepared alkali electrophoresis solution at room temperature in the dark for 45 min. Following electrophoresis in alkali solution at 320 mA for 30 min, the slides were immersed in ethanol for 5 min and air-dried. The slides were then stained with SYBr Green and viewed under a fluorescence microscope. The DNA was scored from 0 (intact DNA) to 4 (completely damaged DNA with tail only). Scores were calculated using the following formula in which NA (intact DNA), NB, NC, ND, and NE (completely damaged DNA) were the number of different kinds of comets: scores (S) ) (NA × 0 + NB × 1 + NC × 2 + ND × 3 + NE × 4)/(NA + NB + NC + ND + NE) × 100. Duplicated samples were prepared for each treatment, and at least 100 cells were scored per sample.
Results ER Competitive Binding Assay. Table 1 lists the IC50 values of the compounds needed to displace [3H]estradiol from the binding sites of ERR and ERβ. The relative affinities for ERR were 4-OHTAM ≈ 4-OHTOR > 3,4-di-OHTOR > 3,4-di-OHTAM. The affinities for ERβ gave a similar trend, and the most striking difference observed between the two receptors was the decreased affinity of ERβ for 4-OHTAM (R/β ) 0.3) and greater affinity of ERβ for 3,4-di-OHTAM (R/β ) 3.5). The other compounds appeared to have similar affinity rankings for both receptors as demonstrated by the R/β ratios. TAM was the least effective compound of the series to displace 17β-estradiol in both ERs. The introduction of a hydroxyl group at the 4-position increased the affinity of the compounds for the receptors as observed with both 4-OHTAM and 4-OHTOR. The addition of the second hydroxyl group at the 3-position decreased the affinity for ERR as seen with 3,4-di-OHTAM and 3,4-di-OHTOR. These results are consistent with previous papers, which demonstrated that TAM and TOR had similar binding affinities for the ERR (23), while 4-OHTAM displayed a binding affinity of several orders of magnitude higher (24). In addition, the values that we observed with the ERR for TAM and 4-OHTAM were similar to those reported previously using rat uterine ERs, 272 and 5.4 nM, respectively (25). Inhibition of 17β-Estradiol-Induced Alkaline Phosphatase Activity in Ishikawa Cells. Ishikawa cells are a ERR positive cell line, which is a stable human endometrial cancer cell line that displays estrogen inducible alkaline phosphatase activity (22). This cell line responds to estrogens and antiestrogens at concentrations approximating physiological levels (26). We used this assay to determine if the metabolites demonstrated
Activities of Tamoxifen and Toremifene Metabolites Table 2. Cytotoxic Potential in Breast Cancer Cellsa LC50 (µM) compounds
MDA-MB-231
S30b
ERβ41
TAM 4-OHTAM 3,4-di-OHTAM 4-OHTOR 3,4-di-OHTOR
34 ( 3 29 ( 3 37 ( 1 28 ( 3 35 ( 3
23 ( 1 19 ( 1 40 ( 2 33 ( 3 38 ( 3
22 ( 2 23 ( 3 30 ( 3 29 ( 2 38 ( 4
a
Cells (105 cells/mL) were incubated with various concentrations of sample or vehicle for 18 h. Values are expressed as the average ( SD of four determinations. Experimental details are described in the Material and Methods. b Data from ref 15.
antiestrogenic effects and if the trends were consistent with those observed in the ER binding study. Table 1 lists the IC50 values of the compounds required to inhibit alkaline phosphatase activity induced by 1 nM 17βestradiol. The compounds in general inhibited the estrogen-dependent alkaline phosphatase activity in the same nanomolar range and similar rank as demonstrated with the ERR binding results, except a decreased activity potency of 3,4-di-OHTOR was observed. On the other hand, our data showed that incubation of Ishikawa cells with 4-hydroxy metabolites at high (µM) concentrations caused a maximal 33% increase in alkaline phosphatase activity as compared with E2 (1 nM) (data not shown), which is because both TAM and TOR are SERMs and they have both estrogenic and antiestrogenic properties. This is also consistent with the previous report (27) of slight stimulatory effects of estrogen responsive activity at high (µM) concentrations of these metabolites. Inhibition of 17β-Estradiol-Induced Luciferase Activity. While the Ishikawa system is appropriate for determining ERR activity, a system to determine ERR and ERβ responsiveness within similar biological systems was required. The inhibition of luciferase activity, under the control of the ERE, in the two MDA-MB 231 stable transfectant cell lines [S30 (ERR) and ERβ41 (ERβ)] was measured (Table 1). The activities of the compounds in the biological assays mimicked their activities with the ERs. The normalized induction of 1 nM 17β-estradiol was set at 100%, and the normalized inductions of the other compounds were determined as a fraction of the 17βestradiol control. The inhibition observed with the ER positive cells for most of the compounds parallels that observed with the ER binding data. However, 3,4-diOHTAM and 3,4-di-OHTOR displayed less luciferase inhibition in ERβ41 cells. Evaluation of the Cytotoxic Potential in Breast Cancer Cell Lines. Table 2 records the LC50 values of the compounds in MDA-MB 231, S30, and ERβ41 cell lines. The data showed that the difference in cytotoxic potency between the metabolites is quite modest. In addition, all of the metabolites exhibited similar toxicity patterns in both ER positive and negative cells indicating a lack of ER-induced sensitivity for the compounds. DNA Damage in Breast Cancer Cells Using the Alkaline Single Cell Gel Electrophoresis Assay (Comet Assay). The results of the comet assay are presented in Figure 1A-C. Unlike the previous assays, the induction of single strand DNA damage does appear to be ER-dependent with the ERβ cell line (ERβ41), the most sensitive. The most striking outcomes were the increases in DNA damage with 3,4-di-OHTAM and 3,4di-OHTOR, which is not consistent with the previous data from the cytotoxicity and ER binding data. However,
Chem. Res. Toxicol., Vol. 16, No. 7, 2003 835
our previous reports indicated that 3,4-di-OHTAM and 3,4-di-OHTOR demonstrated a greater affinity for nucleosides due to their metabolism to o-quinones, which are more reactive electrophiles than quinone methides (15, 17, 18).
Discussion Although the carcinogenic effects of antiestrogens have mainly been attributed to hormonal properties (28), antiestrogens can also be metabolized to reactive intermediates, which bind to cellular macromolecules (10, 15). At least three different electrophilic bioactivation mechanisms are considered to be responsible for the carcinogenicity of antiestrogens. TAM and TOR can form 4-hydroxylated metabolites, which can be further oxidized to quinone methides capable of alkylating DNA in vivo (13). The second bioactivation pathway for antiestrogens involves o-quinone formation, which can react with deoxynucleosides in vitro (17). The third pathway, which is not emphasized in this work, involves R-hydroxylation, conjugation with sulfate, and loss of the sulfate group forming highly reactive carbocations, which react with DNA (6, 29). In the present study, we investigated the antiestrogenic effects of TAM and TOR metabolites and their ability to induce DNA damage. We found that 3,4-diOHTAM and 3,4-di-OHTOR showed a low affinity to both ERR and ERβ as compared with the 4-hydroxy metabolites, 4-OHTAM and 4-OHTOR. These results correlated with the Ishikawa and transient transfection luciferase assays, which indicated the relative antiestrogenic activity in cells. Both 3,4-di-OHTAM and 3,4-di-OHTOR showed less antiestrogenic effects than the 4-hydroxy metabolites. However, the R/β ratios from the ER binding assay were not correlated with the cell-based luciferase assay, which is probably due to the different expression levels of ERs in the two transient transfected cell lines. Previously, we showed that ERR positive cell lines are considerably more sensitive to the toxic effects of equine catechol estrogens (8), and as a result, it was of interest to determine if similar effects were observed with the antiestrogens and their metabolites. In this study, all of the metabolites showed similar cytotoxicity between MDA-MB-231, S30, and ERβ41 cells indicating a lack of ER-induced sensitivity for the compounds. Because the catechols showed less cytotoxic effects in breast cancer cells as compared to the phenols, the catechol pathway may only play a minor role in cell death and the ERs may not be involved in the cytotoxicity pathway. DNA single strand breaks detected by the comet assay showed that in all cell lines, 3,4-di-OHTOR induced more DNA single strand breaks than 4-OHTOR. The possible reason is that the catechols can be oxidized to quinoids by any oxidative enzymes or metal ions and cause a variety of damage to DNA. In contrast, quinone methides may require P450 for formation, which is not present in these cells (30). However, 3,4-di-OHTAM induced a greater amount of single strand DNA breaks only at high concentrations in these cell lines as compared with 4-OHTAM. These results are consistent with the kinetic data, which showed that the TOR-o-quinone was considerably more reactive than the TAM-o-quinone (15) and consistent with previous observations that the TOR-oquinone was much more effective at forming deoxynucleoside adducts than TAM-o-quinone (15, 17, 18). The
836
Chem. Res. Toxicol., Vol. 16, No. 7, 2003
Liu et al.
phenol analogues. As a result, the formation of oquinones from the SERMs may represent a minor role in cytotoxic and antiestrogenic pathways. Because no difference in toxicity was observed between ERβ and ER negative cell lines, ERβ may not contribute to cytotoxicity pathway for these metabolites. However, catechols induced more DNA damage in cells as compared to the phenols, which suggests that the o-quinones formed from catechols could contribute to genotoxicity in vivo and the process seems to be ER-dependent.
Acknowledgment. This research was supported by NIH Grant CA73638 (J.L.B.). We are grateful to Dr. V. C. Jordan (Northwestern University) for the gift of the S30 cell line and Dr. A.-N. T. Kong (Rutgers University) for the ERE-luciferase and β-galactosidase plasmids.
References
Figure 1. Induction of DNA single strand breaks by TAM and TOR metabolites in (A) MDA-MB-231, (B) S30, and (C) ERβ41 cells using the alkaline single cell gel electrophoresis assay. Cells (3 × 105 cells/mL) were treated with compounds or vehicle for 90 min. Closed circles, 3,4-di-OHTOR; closed squares, 3,4-diOHTAM; open circles, 4-OHTOR; open squares, 4-OHTAM.
different patterns of DNA damage in ER positive (S30 and ERβ41) and negative cell lines (MDA-MB-231) showed that the ERs might be involved in this process. In summary, we have found that catechols showed lower binding affinity to ERs, reduced antiestrogenic effects, and decreased cytotoxicity as compared to their
(1) Fisher, B., Costantino, J. P., Wickerham, D. L., Redmond, C. K., Kavanah, M., Cronin, W. M., Vogel, V., Robidoux, A., Dimitrov, N., Atkins, J., Daly, M., Wieand, S., Tan-Chiu, E., Ford, L., and Wolmark, N. (1998) Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl. Cancer Inst. 90, 1371-1388. (2) Bergman, L., Beelen, M. L., Gallee, M. P., Hollema, H., Benraadt, J., and van Leeuwen, F. E. (2000) Risk and prognosis of endometrial cancer after tamoxifen for breast cancer. Comprehensive Cancer Centres’ ALERT Group. Assessment of Liver and Endometrial cancer Risk following Tamoxifen. Lancet 356, 881-887. (3) Carthew, P., Martin, E. A., White, I. N., De Matteis, F., Edwards, R. E., Dorman, B. M., Heydon, R. T., and Smith, L. L. (1995) Tamoxifen induces short-term cumulative DNA damage and liver tumors in rats: promotion by phenobarbital. Cancer Res. 55, 544547. (4) Greaves, P., Goonetilleke, R., Nunn, G., Topham, J., and Orton, T. (1993) Two-year carcinogenicity study of tamoxifen in Alderley Park Wistar-derived rats. Cancer Res. 53, 3919-3924. (5) Han, X. L., and Liehr, J. G. (1992) Induction of covalent DNA adducts in rodents by tamoxifen. Cancer Res. 52, 1360-1363. (6) Shibutani, S., Suzuki, N., Terashima, I., Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (1999) Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol. 12, 646-653. (7) Hemminki, K., Rajaniemi, H., Koskinen, M., and Hansson, J. (1997) Tamoxifen-induced DNA adducts in leucocytes of breast cancer patients. Carcinogenesis 18, 9-13. (8) Liu, X., Yao, J., Pisha, E., Yang, Y., Hua, Y., van Breemen, R. B., and Bolton, J. L. (2002) Oxidative DNA damage induced by equine estrogen metabolites: role of estrogen receptor alpha. Chem. Res. Toxicol. 15, 512-519. (9) McDonnell, D. P. (2000) Selective estrogen receptor modulators (SERMs): A first step in the development of perfect hormone replacement therapy regimen. J. Soc. Gynecol. Invest. 7, S10S15. (10) Bolton, J. L. (2002) Quinoids, quinoid radicals, and phenoxyl radicals formed from estrogens and antiestrogens. Toxicology 177, 55-65. (11) Dehal, S. S., and Kupfer, D. (1997) CYP2D6 catalyzes tamoxifen 4-hydroxylation in human liver. Cancer Res. 57, 3402-3406. (12) Dehal, S. S., and Kupfer, D. (1999) Cytochrome P-450 3A and 2D6 catalyze ortho hydroxylation of 4-hydroxytamoxifen and 3-hydroxytamoxifen (droloxifene) yielding tamoxifen catechol: involvement of catechols in covalent binding to hepatic proteins. Drug Metab. Dispos. 27, 681-688. (13) Fan, P. W., Zhang, F., and Bolton, J. L. (2000) 4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides. Chem. Res. Toxicol. 13, 45-52. (14) Jordan, V. C. (1982) Metabolites of tamoxifen in animals and man: identification, pharmacology, and significance. Breast Cancer Res. Treat. 2, 123-138. (15) Yao, D., Zhang, F., Yu, L., Yang, Y., van Breemen, R. B., and Bolton, J. L. (2001) Synthesis and reactivity of potential toxic metabolites of tamoxifen analogues: droloxifene and toremifene o-quinones. Chem. Res. Toxicol. 14, 1643-1653. (16) NIH Guidelines for the Laboratory Use of Chemical Carcinogens (1981) U.S. Government Printing Office, Washington, DC.
Activities of Tamoxifen and Toremifene Metabolites (17) Zhang, F., Fan, P. W., Liu, X., Shen, L., van Breeman, R. B., and Bolton, J. L. (2000) Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4-dihydroxytamoxifeno-quinone. Chem. Res. Toxicol. 13, 53-62. (18) Fan, P. W., Zhang, F., and Bolton, J. L. (2000) 4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides. Chem. Res. Toxicol. 13, 45-52. (19) Jeng, M. H., Jiang, S. Y., and Jordan, V. C. (1994) Paradoxical regulation of estrogen-dependent growth factor gene expression in estrogen receptor (ER)-negative human breast cancer cells stably expressing ER. Cancer Lett. 82, 123-128. (20) Obourn, J. D., Koszewski, N. J., and Notides, A. C. (1993) Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor. Biochemistry 32, 6229-6236. (21) Liu, J., Burdette, J. E., Xu, H., Gu, C., van Breemen, R. B., Bhat, K. P., Booth, N., Constantinou, A. I., Pezzuto, J. M., Fong, H. H., Farnsworth, N. R., and Bolton, J. L. (2001) Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms. J. Agric. Food Chem. 49, 2472-2479. (22) Pisha, E., and Pezzuto, J. M. (1997) Cell-based assay for the determination of estrogenic and anti-estrogenic activities. Methods Cell. Sci. 19, 37-43. (23) Roos, W., Oeze, L., Loser, R., and Eppenberger, U. (1983) Anitestrogenic action of 3-hydroxytamoxifen in the human breast cancer cell line MCF-7. J. Natl. Cancer Inst. 71, 55-59. (24) Coezy, E., Borgna, J. L., and Rochefort, H. (1982) Tamoxifen and metabolites in MCF7 cells: Correlation between binding to
Chem. Res. Toxicol., Vol. 16, No. 7, 2003 837
(25)
(26)
(27)
(28) (29)
(30)
estrogen receptor and inhibition of cell growth. Cancer Res. 42, 317-323. Martel, C., Provencher, L., Li, X., St. Pierre, A., Leblanc, G., Gauthier, S., Merand, Y., and Labrie, F. (1998) Binding characteristics of novel nonsteroidal antiestrogens to the rat uterine estrogen receptors. J. Steroid Biochem. Mol. Biol. 64, 199-205. Albert, J. L., Sundstrom, S. A., and Lyttle, C. R. (1990) Estrogen regulation of placental alkaline phosphatase gene expression in a human endometrial adenocarcinoma cell line. Cancer Res. 50, 3306-3310. Simard, J., Sanchez, R., Poirier, D., Gauthier, S., Singh, S. M., Merand, Y., Belanger, A., Labrie, C., and Labrie, F. (1997) Blockade of the stimulatory effect of estrogens, OH-tamoxifen, OH-toremifene, droloxifene, and raloxifene on alkaline phosphatase activity by the antiestrogen EM-800 in human endometrial adenocarcinoma Ishikawa cells. Cancer Res. 57, 3494-3497. Henderson, B. E., Ross, R., and Bernstein, L. (1988) Estrogens as a cause of human cancer: The Richard and Hinda Rosenthal Foundation Award Lecture. Cancer Res. 48, 246-253. Shibutani, S., Ravindernath, A., Suzuki, N., Terashima, I., Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (2000) Identification of tamoxifen-DNA adducts in the endometrium of women treated with tamoxifen. Carcinogenesis 21, 1461-1467. Bolton, J. L., and Thompson, J. A. (1991) Oxidation of butylated hydroxytoluene to toxic metabolites. Factors influencing hydroxylation and quinone methide formation by hepatic and pulmonary microsomes. Drug Metab. Dispos. 19, 467-472.
TX030004S
