Identification of tamoxifenDNA adducts in the endometrium of women treated with tamoxifen
Shinya Shibutani3,
Anisetti Ravindernath,
Naomi Suzuki,
Isamu Terashima,
Steven M. Sugarman1,
Arthur P. Grollman and
Michael L. Pearl2
Laboratory of Chemical Biology, Department of Pharmacological Sciences,
1 Division of Oncology and
2 Division of Obstetrics and Gynecology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794, USA
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Abstract
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The risk of developing endometrial cancer increases significantly for women treated with tamoxifen (TAM); the present study was designed to investigate the mechanism of this carcinogenic effect. Endometrial tissue was obtained from 16 women treated for varying lengths of time with TAM and from 15 untreated control subjects. DNA was analyzed with a 32P-post-labeling/HPLC on-line monitoring assay capable of detecting 2.5 adducts/1010 nucleotides. Using this sensitive and specific assay, TAMDNA adducts were detected in eight women. The major adducts found were trans and cis epimers of
-(N2-deoxyguanosinyl) tamoxifen (dG-N2-TAM); levels ranged between 0.212 and 1.68.3 adducts/108 nucleotides, respectively. There was marked inter-individual variation in the relative amounts of cis and trans adducts present. Low levels (0.741.1 adducts/108 nucleotides) of trans and cis forms of dG-N2-TAM N-oxide were detected in one patient. DNA adducts derived from 4-hydroxytamoxifen quinone methide were not observed. We conclude from this analysis that trans and cis dG-N2-TAMs accumulate in significant amounts in the endometrium of many, but not all, women treated with this drug. The level of adducts found, coupled with the previous demonstration of their mutagenicity [Cancer Res., 59, 2091, 1999], suggest that a genotoxic mechanism may be responsible for TAM-induced endometrial cancer.
Abbreviations: 4-OHTAM, 4-hydroxytamoxifen; dG, 2'-deoxyguanosine; dG3'P, 2'-deoxyguanosine 3'-monophosphate; dG-N2-TAM,
-(N2-deoxyguanosinyl)tamoxifen; TAM N-oxide, tamoxifen N-oxide; TAM, tamoxifen;
-OHTAM,
-hydroxytamoxifen.
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Introduction
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The anti-estrogen tamoxifen (TAM) is widely used in the treatment of human breast cancer; more than 500 000 women in the USA are currently being treated with this drug (1). TAM was recently approved for use as a chemopreventive agent in women at high risk of developing this disease, when a randomized clinical trial showed that therapeutic doses of this drug significantly reduced the risk of invasive breast cancer (2).
Administration of TAM to women is associated with an increased risk of endometrial cancer (26); the cellular mechanism responsible for this carcinogenic effect has not been defined (79). TAM induces hepatocellular carcinomas in rats (10,11) and TAMDNA adducts have been identified in rat liver (7,1214). However, in women treated with TAM, the risk of developing hepatocellular cancer is minimal and the failure to detect TAMDNA adducts (15,16) or to identify such lesions convincingly in human endometrial tissue (17,18) or in liver (19) supports the view held by some investigators (9,16) that estrogenic (tumor promoter) or other epigenetic effects of TAM account for the carcinogenic properties of the drug.
TAM is actively metabolized in the liver of rodents and humans to
-hydroxytamoxifen (
-OHTAM), 4-hydroxytamoxifen (4-OHTAM), N-desmethyltamoxifen (N-desmethylTAM) and tamoxifen N-oxide (TAM N-oxide) (2022). The different metabolism and activation of TAM in rats and human has been reviewed recently (23). Following O-sulfation or O-acetylation,
-OHTAM reacts preferentially with the exocyclic amino group of guanine in DNA to form two cis and two trans epimers of
-(N2-deoxyguanosyl)tamoxifen (dG-N2-TAM; Figure 1
) (13,24,25).
-OHTAM is a substrate for rat and human hydroxysteroid sulfotransferases, suggesting a metabolic pathway by which TAM could be activated to react with DNA and thereby exert genotoxic effects in target tissues (2628).
In a previous study (29), we used a 32P-post-labelingTLC technique to detect TAMDNA adducts in endometrial tissue. This analytical method fails to resolve the two trans epimers of dG-N2-TAM and to clearly separate them from other TAMDNA adducts. The present demonstration that TAMDNA adducts are formed in the human endometrium was achieved by coupling high resolution 32P-post-labeling/HPLC (30) with partial purification of DNA adducts (29). We find that cis and trans TAMDNA adducts are present in significant amounts in endometrial tissue in eight of 16 women treated with TAM. We attribute the failure of other investigators to detect these lesions (15,16) to a relative lack of sensitivity of methods used for the analysis. TAMDNA adducts are miscoding lesions (31) and have been shown to be mutagenic in mammalian cells (32). This fact, coupled with their presence in the endometrium, suggests that a genotoxic mechanism may be responsible for TAM's carcinogenic effect on this tissue.
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Materials and methods
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Chemicals
[
-32P]ATP (sp. act. 6000 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL). Polyethylenimine (PEI)cellulose plates were purchased from Machery-Nagel (Duren, Germany), proteinase K and potato apyrase from Sigma (St Louis, MO) and nuclease P1 from Boehringer Mannheim (Indianapolis, IN). RNase A, RNase T1, micrococcal nuclease and spleen phosphodiesterase were obtained from Worthington Biochemical Co. (Freehold, NJ).
Synthesis of TAM derivations
TAM
-sulfate (24),
-acetoxyTAM (24) and 4-OHTAM quinine methide (33) were synthesized by established methods.
-AcetoxyTAM N-oxide was prepared by a minor modification of a method described in a previous report (34): 30% aqueous hydrogen peroxide (2 ml) was added to a solution containing the trans form of
-acetoxyTAM (50 mg, 113 mmol) dissolved in 3 ml of dichloromethane. The solution was stirred vigorously for 16 h at room temperature. The organic layer was separated and dried over anhydrous Na2SO4. Solvent was concentrated under reduced pressure, producing a pale yellow gummy substance.
-AcetoxyTAM N-oxide was purified by silica column chromatography using dichloromethanemethanol. The physical properties of trans
-acetoxyTAM N-oxide were consistent with those reported previously (34).
DNA extraction from endometrial tissues
Endometrial tissue was collected by biopsy utilizing a Pipelle needle or following hysterectomy and samples were stored at 80°C until the DNA was extracted. This clinical research study was approved by the Institutional Review Board of the State University of New York at Stony Brook (972578 and 992578) and informed consent was obtained from all patients. Endometrial DNA was isolated as described previously (29). The concentration of DNA was estimated as 50 µg = OD260 nm of 1.0.
Digestion of DNA samples
DNA (5 µg) was incubated at 37°C for 2 h in 30 µl of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2, 1.5 U of micrococcal nuclease and 0.15 U of spleen phosphodiesterase. Nuclease P1 (1 U) was added and the reaction was incubated for an additional 1 h. Samples were dried, dissolved in 100 µl of distilled water and extracted twice with 200 µl of butanol. The butanol fraction was back-extracted with 50 µl of distilled water, the organic layers were combined, evaporated to dryness and used for analysis of TAMDNA adducts. Approximately 95% of the TAM adducts present were recovered by this procedure.
Determination of 32P-labeled TAMDNA adducts by HPLC
Pooled extracts containing DNA adducts were labeled with 32P (35), applied to 10x10 cm PEIcellulose thin-layer plates and developed for 16 h with 1.7 M sodium phosphate buffer (pH 6.0) as eluant, using a paper wick. 32P-labeled material remaining on the TLC plate was recovered by eluting with 4 M pyrimidinium formate (pH 4.3). Recovery of 32P-labeled products was ~84%. Using a minor modification of the method reported by Martin et al. (30), 32P-labeled products were placed on a Hypersil BDS C18 analytical column (0.46x25 cm, 5 µm; Shandon) and eluted over 40 min at a flow rate of 1.0 ml/min with an isocratic solution of 2.0 M ammonium formate (pH 4.0), containing 20% acetonitrile:methanol (6:1 v/v), after which a linear gradient of 2045% was applied to the column for 25 min. Radioactivity was monitored using a radioisotope detector (Berthold LB506 C-1, ICON Scientific Inc.) connected to a Waters 990 HPLC instrument. Standards (both epimers) of 2'-deoxyguanosine 3'-monophosphate (dG3'p)-N2-TAM (26) and dG3'p-N2-TAM N-oxide (34) were prepared using published methods and labeled with 32P (35).
Adduct levels were calculated as described by Levay et al. (36) (total d.p.m. in adducts)/7.69x1011 d.p.m., assuming that 5 µg of DNA represents 1.52x104 pmol of dN3'P; the specific activity of [
-32P]ATP, corrected for radioactive decay, was 5.06x107 d.p.m./pmol. The limit of detection of TAMDNA adducts using this technique was ~2.5 adducts/1010 nucleotides.
Preparation of TAMDNA adduct standards
DNA (10 µg) was mixed with 400 µg of TAM
-sulfate or
-acetoxyTAM N-oxide dissolved in 100 µl of 100 mM TrisHCl buffer (pH 8.0) then incubated at 37°C for 1 h. Similarly, DNA was reacted with 4-OHTAM quinone methide generated by oxidation of 4-OHTAM (33). Samples were evaporated to dryness, 800 µl of ethanol was added, and the solution was centrifuged to recover precipitated DNA. Aliquots (0.4 µg) of each sample were digested enzymatically as described for analysis of endometrial DNA (29). Following digestion, reaction mixtures were dissolved in 100 µl of distilled water and extracted twice with 100 µl of butanol. The butanol fractions were dried, dissolved in a small volume of water, and used as standards for the analysis of TAMDNA adducts in endometrial tissue.
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Results
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Sixteen endometrial samples were collected from women treated with TAM (20 mg/day); 15 additional samples were obtained from women who did not receive this drug or other form of hormonal therapy. Clinical diagnoses and duration of treatment for these subjects are listed in Table I
. All but one patient had some form of malignant disease; two patients (T8 and T11) had endometrial cancer. TAMDNA adducts were analyzed as described in Materials and methods, using the HPLC gradient system developed by Martin et al. (30).
Retention times for fr-1 and fr-2, representing the two trans-form epimers, and for fr-3 and fr-4, a co-eluting mixture of the cis-form epimers of 32P-labeled dG3'p-N2-TAM, were 22.2, 26.4 and 59.4 min, respectively (Figure 2
). TAMDNA adducts were detected in eight of 16 endometrial samples obtained from patients treated with TAM (T1, T4, T7, T8 and T10 in Figure 2
; T11 in Figure 4A
; T12 and T14 in Table II
). Retention times for the major TAMDNA adducts were identical to those of standard trans and cis forms of dG3'p-N2-TAM. When sample T1 (Figure 3B
) was co-injected with authentic 32P-labeled dG3'p-N2-TAM standard (Figure 3A
), the adducts co-eluted with fr-1 and fr-2 (trans isomers) and the mixture of cis isomers (fr-3 and fr-4) (Figure 3C
). Trans and cis forms of dG3'P-N2-TAM were both present in T1, T4 and T11 (Figures 2 and 4A
). Trans forms were the major adducts found in T7 and T10 while cis forms were the major products in T8 and T14. TAMDNA adducts were not detected in eight of the 16 endometrial samples obtained from TAM-treated women (e.g. T6 in Figure 2
) nor in any of the 15 samples obtained from untreated controls (e.g. C1 in Figure 2
).

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Fig. 2. HPLC analysis of TAMDNA adducts in endometrial tissues. A standard of the trans (fr-1 and fr-2) or cis forms (a mixture of fr-3 and fr-4) of 32P-labeled dG3'P-N2-TAM recovered from a PEIcellulose TLC plate, as described in Materials and methods, was subjected to a Hypersil BDS C18 analytical column (0.46x25 cm, 5 µm), eluted at a flow rate of 1.0 ml/min with an isocratic condition of 2.0 M ammonium formate (pH 4.0), containing 20% acetonitrile:methanol (6:1 v/v), for 40 min followed by a linear gradient of 2045% for 25 min, using a minor modification of method established by Martin et al. (30). The radioactivity was measured by a radioisotope monitor (Berthold LB506 C-1) linked to a Waters 990 HPLC instrument. Five micrograms of endometrial DNA obtained from TAM-treated patients (T1, T4, T7, T8, T10 and T6) or from untreated patients (C1) were digested enzymatically and extracted by butanol, as described in Materials and methods. The butanol fraction was evaporated to dryness then labeled with 32P. 32P-labeled samples were developed on a PEIcellulose TLC plate using 1.7 M sodium phosphate buffer (pH 6.0), with a paper wick. 32P-labeled products remained on the TLC were recovered using 4 M pyrimidinium formate (pH 4.3) and loaded on the Hypersil BDS C18 analytical column.
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Fig. 3. Co-elution of adducts in T1 and a standard sample of 32P-labeled dG3'P-N2-TAM. Standard 32P-labeled dG3'P-N2-TAM (A) was mixed with T1 sample (B) and used for the experiment in (C). HPLC chromatography was performed as described in the legend of Figure 2 .
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In T11 (Figure 4A
), retention times for the three major labeled products were consistent with those observed for a standard mixture of 32P-labeled dG3'p-N2-TAM adducts produced by reacting calf thymus DNA with TAM
-sulfate (Figure 4B
). Several minor products also were observed. Retention times for peaks a (32.0 min), b (38.2 min) and c (61.8 min) were consistent with those of adducts produced when DNA reacts with
-acetoxyTAM N-oxide (Figure 4C
). Therefore, peaks a and b were identified as epimers of the trans form of
-(N2-deoxyguanosinyl)tamoxifen N-oxide; peak c as a mixture of epimers of the cis form. Adduct levels were 0.90, 1.1 and 0.74 adducts/108 nucleotides, respectively.
Three DNA adducts are formed by reacting DNA with 4-OHTAM quinone methide (Figure 4D
). Peaks corresponding to the major products (retention times, 14.6 and 23.0 min) were not observed in any endometrial samples obtained from women treated with TAM; the retention time for the third peak (60.3 min) is longer than that of peak c in T11.
The level of dG-N2-TAM adducts found in the eight positive endometrial samples are summarized in Table II
. The total and relative amounts of trans and cis forms of dG-N2-TAM recovered varied significantly. Levels of trans- and cis-form adducts ranged from 0.212 and 1.68.3 adducts/108 nucleotides, respectively. Total dG-N2-TAM adducts ranged from 0.2 to 18 adducts/108 nucleotides.
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Discussion
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TAM exhibits an unusual pattern of carcinogenicity in that it induces hepatocellular adenomas and carcinomas in rats, but not in mice or hamsters, whereas women treated with TAM develop endometrial but not hepatocellular cancer. Human endometrial cancer has been attributed variously to the weak estrogenic (partial agonist) effects of TAM on the human uterus (9,16) and/or to the genotoxic effects of the drug (1214). In the present study, a sensitive, specific, and robust method was employed to identify TAMDNA adducts, providing direct evidence that this drug can exert a genotoxic effect on endometrial tissue.
Several investigators have combined 32P-labeling with chromatography to detect TAMDNA adducts in endometrial tissue (1517,29). In one study, TLC was used (15), whereas in another report, the same samples were analyzed by HPLC (16). Both studies failed to detect TAMDNA adducts in the endometrial tissue of 20 patients treated with 20 mg/day TAM for 2265 months. Although Carmichael et al. (16) reported an ability to detect eight TAMDNA adducts per 1010 nucleotides (16), they were unable to reproduce the results of a study by Hemminki et al. (17) which described formation of TAMDNA adducts (2.98.2 adducts/109 bases) in four of six women treated with 20 or 40 mg of this drug. Chemically defined standards were not employed by Hemminki et al. and an alternative interpretation of their experimental data has been offered (18). Common to all three studies is the presence of large amounts of free 32P and 32P-labeled non-adducted nucleotides at the point of the analysis when TAMDNA adducts are subjected to TLC or HPLC. This factor produces a high background level of radioactivity that interferes significantly with adduct detection. We avoided this problem by extracting TAMDNA adducts with butanol and recovering them from a TLC plate prior to HPLC. We found it exceedingly difficult to quantify TAMDNA adducts in endometrial tissue when these steps were omitted (29). By removing free 32P and 32P-labeled nucleotides from the sample, a low background (<200 d.p.m.) in the HPLC chromatogram is readily achieved (Figure 2
). Coupled with the use of chemically defined standards, the increased sensitivity of our procedure permits the unequivocal demonstration of cis and trans TAMDNA adducts in endometrial tissue. Using this assay, TAMDNA adducts can be detected at a level of 2.5 adducts/1010 nucleotides, 4-fold lower than the detection limit reported by Carmichael et al. (16).
DNA adduct levels in target organs have been correlated with tumor incidence in experimental animals (37); comparable data do not exist for human subjects. The level of TAMDNA adducts in patients T1 and T11 was 5.0- and 4.4-fold lower, respectively, than the eight adducts/107 nucleotides found in the liver of rats treated with a dose of TAM normalized to the amount resulting in 50% tumor incidence in a 2 year bioassay (37). dG-N2-TAM adducts are miscoding lesions (31) and generate targeted mutations, primarily G
T transversions, in mammalian cells (32). We conclude that therapeutic doses of TAM in susceptible subjects could initiate endometrial cancer. As discussed below, the level of endometrial TAM adducts varied significantly among individual subjects and may reflect the relative degree of risk of developing drug-induced endometrial cancer.
The major DNA adducts detected in endometrial tissue were identified as epimers of the trans and cis forms of dG-N2-TAM with adduct levels ranging between 0.212 and 1.68.3 adducts/108 nucleotides, respectively. These values are slightly higher than those estimated by 32P-post-labeling-TLC analysis (29). Epimers are generated when the sulfuric acid ester of trans-
-OH-TAM reacts with the exocyclic amino group of guanine in DNA (24). Trans and cis forms of dG-N2-TAM interconvert via a short-lived carbocation intermediate (38). This explains why, although the trans form of TAM is administered to all patients, cis adducts were found in endometrial tissue of some women in this study.
There was marked inter-individual variation in the level of TAMDNA adducts detected in endometrial tissue. One subject (T1) developed a high level of adducts after treatment with 20 mg of TAM daily for 4 months; in contrast, adducts were not detected in eight of 16 patients. These include several subjects (e.g. T14 and T16) who had been treated with TAM for 5 years. This variability may represent differences in the activity of enzymes involved in the oxidative metabolism of TAM and/or cellular sulfotransferases that convert
-OHTAM to an activated form that reacts with DNA. Variable adduct levels also could reflect differences in nucleotide excision repair.
Several metabolites of TAM have been identified, some of which may be activated enzymatically and react with DNA (Figure 1
). The pattern of trans and cis dG-TAMDNA adducts observed in our study is consistent with the view that
-OHTAM is the important reactive intermediate in endometrial cells (2628,39). 4-OHTAM quinone methide, produced by oxidation of 4-OHTAM, reacts with dG in vitro to form trans and cis dG-N2-4-OH-TAM (33). These adducts are not found in the liver of rats treated with TAM or
-OHTAM (30,40) and were not detected in the present study of endometrial tissue.
-OHTAM N-oxide and desmethyl TAM are major TAM metabolites (2122,41,42) which, like the parent drug, could be activated and form trans and cis adducts in DNA. The retention times of several minor adducts detected in sample T11 are consistent with those derived from dG-N2-TAM-N-oxide (34). Since a defined standard of desmethyl TAM was not available, we cannot exclude its presence in endometrial DNA.
In conclusion, we have established the presence of TAMDNA adducts in the endometrial tissue of some women treated with TAM. The level of adducts found, coupled with the previous demonstration of their mutagenic potential, suggests that a genotoxic mechanism may be responsible for the endometrial carcinogenicity associated with the use of this drug.
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Notes
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3 To whom correspondence should be addressed Email: shinya{at}pharm.sunysb.edu 
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Acknowledgments
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A preliminary report of this study was presented at the 35th Annual Meeting of the American Society of Clinical Oncology, May 1999, in Atlanta, GA. This research was supported by the National Institute of Environmental Health Sciences grant ES09418 and by a generous gift from the Norman H. Morris Foundation.
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References
|
---|
-
Osborne,C.K. (1998) Tamoxifen in the treatment of breast cancer. New Eng. J. Med., 339, 16091617.[Free Full Text]
-
Fischer,B., Costantino,J.P., Wickerham,L., Redmond,C.K., Kavanah,M., Cronin,W.M., Botel,V., Robidoux,A., Dimitrov,N., Atkins,J. et al. (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, 13711388.[Abstract/Free Full Text]
-
Fischer,B., Costantino,J.P., Redmond,C.K., Fisher,E.R., Wickerham,D.L. and Cronin,W.M. (1994) Endometrial cancer in tamoxifen-treated breast cancer patients: findings from the National Surgical Adjuvant Breast and Bowel Project (NSABP, B-14). J. Natl Cancer Inst., 86, 527537.[Abstract]
-
Clarke,M., Cillins,R., Davies,C., Godwin,J., Gray,R. and Peto,R. (1998) Tamoxifen for early breast cancer: an overview of the randomized trials. Lancet, 351, 14511467.[ISI][Medline]
-
Bernstein,L., Deapen,D., Cerhan,J.R., Schwartz,S.M., Liff,J., McGann-Maloney,E., Perlman,J.A. and Ford,L. (1999) Tamoxifen therapy for breast cancer and endometrial cancer risk. J. Natl Cancer Inst., 91, 16541662.[Abstract/Free Full Text]
-
Killackey,M., Hakes,T.B. and Pierce,V.K. (1985) Endometrial adenocarcinoma in breast cancer patients receiving antiestrogens. Cancer Treat. Rep., 69, 237238.[ISI][Medline]
-
Tannenbaum,S.R. (1997) Comparative metabolism of tamoxifen and DNA adduct formation and in vitro studies on genotoxicity. Semin. Oncol., 24, 8186.
-
Wogan,G.N. (1997) Review of the toxicology of tamoxifen. Semin. Oncol., 24 (Suppl. 1), 8797.
-
Stearns,V. and Gelman,E.P. (1998) Does tamoxifen cause cancer in human? J. Clin. Oncol., 16, 779792.[Abstract]
-
Williams,G.M., Iatropoulos,M.J., Djordjevic,M.V. and Kaltenberg,O.P. (1993) The triphenylethylene drug tamoxifen is a strong liver carcinogen in the rat. Carcinogenesis, 14, 315317.[Abstract]
-
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, 39193924.[Abstract]
-
Han,X. and Liehr,J.G. (1992) Induction of covalent DNA adducts in rodents by tamoxifen. Cancer Res., 52, 13601363.[Abstract]
-
Osborne,M.R., Hewer,A., Hardcastle,I.R., Carmichael,P.L. and Phillips,D.H. (1996) Identification of the major tamoxifendeoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res., 56, 6671.[Abstract]
-
Divi,R.L., Osborne,M.R., Hewer,A., Phillips,D.H. and Poirier,M.C. (1999) TamoxifenDNA adduct formation in rat liver determined by immunoassay and 32P-post-labeling. Cancer Res., 59, 48294833.[Abstract/Free Full Text]
-
Carmichael,P.L., Ugwumadu,A.H., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Lack of genotoxicity of tamoxifen in human endometrium. Cancer Res., 56, 14751479.[Abstract]
-
Carmichael,P.L., Sardar,S., Crooks,N., Neven,P., Van Hoof,I., Ugwumadu,A., Bourne,T., Tomas,E., Hellberg,P., Hewer,A. and Phillips,D.H. (1999) Lack of evidence from HPLC 32P-post-labeling from tamoxifenDNA adducts in the human endometrium. Carcinogenesis, 20, 339342.[Abstract/Free Full Text]
-
Hemminki,K., Rajaniemi,H., Lindahl,B. and Moberger,B. (1996) Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res., 56, 43744377.[Abstract]
-
Orton,T.C., Topham,J.C. and Park,A. (1997) Correspondence re: K. Hemminki et al. tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res., 57, 4148.[ISI][Medline]
-
Martin,E.A., Rich,K.J., White,I.N., Woods,K.L., Powles,T.J. and Smith,L.L. (1995) 32P-postlabelled DNA adducts in liver obtained from women treated with tamoxifen. Carcinogenesis, 16, 16511654.[Abstract]
-
Phillips,D.H., Potter,G.A., Horton,M.N., Hewer,A., Crofton-Sleigh,C., Jarman,M. and Venitt,S. (1994) Reduced genotoxicity of [D5-ethyl]-tamoxoifen implicates
-hydroxylation of the ethyl group as a major pathway of tamoxifen activation to a liver carcinogen. Carcinogenesis, 15, 14871492.[Abstract]
-
Jarman,M., Poon,G.K., Rowlands,M.G., Grimshaw,R.M., Horton,M.N., Potter,G.A. and McCague,R. (1995) The deuterium isotope effect for the
-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D5-ethyl]tamoxifen. Carcinogenesis, 16, 683688.[Abstract]
-
Poon,G.K., Walter,B., Lonning,P.E., Horton,M.N. and McCague,R. (1995) Identification of tamoxifen metabolites in human Hep G2 cell line, human liver homogenate and patients on long-term therapy for breast cancer. Drug Metab. Dispos., 23, 377382.[Abstract]
-
White,I.N. (1999) The tamoxifen dilemma. Carcinogenesis, 20, 11531160.[Abstract/Free Full Text]
-
Dasaradhi,L. and Shibutani,S. (1997) Identification of tamoxifenDNA adducts formed by
-sulfate tamoxifen and
-acetoxytamoxifen. Chem. Res. Toxicol., 10, 189196.[ISI][Medline]
-
Osborne,M.R., Hardcastle,I.R. and Phillips,D.H. (1997) Minor products of reaction of DNA with
-acetoxytamoxifen. Carcinogenesis, 18, 539543.[Abstract]
-
Shibutani,S., Dasaradhi,L., Terashimi,I., Banoglu,E. and Duffel,M.W. (1998)
-Hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts. Cancer Res., 58, 647653.[Abstract]
-
Shibutani,S., Shaw,P., Suzuki,N., Dasaradhi,L., Duffel,M.W. and Terashima,I. (1998) Sulfation of
-hydroxytamoxifen catalyzed by human hydroxysteroid sulfotransferase results in tamoxifen DNA adducts. Carcinogenesis, 19, 20072011.[Abstract]
-
Davis,W., Venitt,S. and Phillips,D.H. (1998) The metabolic activation of tamoxifen and alpha-hydroxytamoxifen to DNA-binding species in rat hepatocytes proceeds via sulphation. Carcinogenesis, 19, 861866.[Abstract]
-
Shibutani,S., Suzuki,N., Terashima,I., Sugarman,S.M., Grollman,A.P. and Pearl,M.L. (1999) TamoxifenDNA adducts in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol., 12, 646653.[ISI][Medline]
-
Martin,E.A., Heydon,R.T., Brown,K., Brown,J.E.B., Lim,C.K., White,I.N.H. and Smith,L.L. (1998) Evaluation of tamoxifen and
-hydroxytamoxifen 32P-post-labelled DNA adducts by the development of a novel automated on-line solid-phase extraction HPLC method. Carcinogenesis, 19, 10611069.[Abstract]
-
Shibutani,S. and Dasaradhi,L. (1997) Miscoding potential of tamoxifen-derived DNA adducts:
-(N2-deoxyguanosinyl)tamoxifen. Biochemistry, 36, 1301013017.[ISI][Medline]
-
Terashimi,I., Suzuki,N. and Shibutani,S. (1999) Mutagenic potential of
-(N2-deoxyguanosinyl)tamoxifen lesions, the major DNA adducts detected in endometrial tissues of patients treated with tamoxifen. Cancer Res., 59, 20912095.[Abstract/Free Full Text]
-
Marques,M.M. and Beland,F.A. (1997) Identification of tamoxifenDNA adducts formed by 4-hydroxytamoxifen quinone methide. Carcinogenesis, 18, 19491954.[Abstract]
-
Umemoto,A., Monden,Y., Komaki,K., Suwa,M., Kanno,Y., Suzuki,M., Lin,C., Ueyama,Y., Momen,M.A., Ravindernath,A. and Shibutani,S. (1999) TamoxifenDNA adducts formed by
-acetoxytamoxifen N-oxide. Chem. Res. Toxicol., 12, 10831089.[ISI][Medline]
-
Maniatis,T., Frisch,E.F. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, NY.
-
Levay,G., Pongcracz,K. and Bodell,W.J. (1991) Detection of DNA adducts in HL-60 cells treated with hydroquinone and p-benzoquinone by 32P-post-labeling. Carcinogenesis, 12, 11811186.[Abstract]
-
Ottender,M. and Lutz,S.K. (1999) Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts. Mutat. Res., 424, 237247.[ISI][Medline]
-
Sanchez,C., Shibutani,S., Dasaradhi,L., Bolton,J.L., Fan,P.F. and McClelland,R.A. (1998) Lifetime and reactivity of the ultimate tamoxifen carcinogen: the tamoxifen carbocation. J. Am. Chem. Soc., 120, 1351313514.[ISI]
-
Potter,G.A., McCague,R. and Jarman,M. (1994) A mechanism hypothesis for DNA adduct formation by tamoxifen hepatic oxidative metabolism. Carcinogenesis, 15, 439442.[Abstract]
-
Beland,F.A., McDaniel,L.P. and Marques,M.M. (1999) Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo. Carcinogenesis, 20, 471477.[Abstract/Free Full Text]
-
McCague,R. and Seago,A. (1986) Aspects of metabolism of tamoxifen by rat liver microsomes. Identification of a new metabolite: E-1-[4-(2-dimethylaminorethoxy)1-phenyl]-1,2-diphenyl-1-buten-3-ol-N-oxide. Biochem. Pharmacol., 35, 827834.[ISI][Medline]
-
Lim,C.K., Yuan,Z.X., Lamb,J.H., White,I.N., DeMatteis,F. and Smith,L.L. (1994) A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes. Carcinogenesis, 15, 589593.[Abstract]
Received March 21, 2000;
revised May 22, 2000;
accepted May 25, 2000.