Carcinogenesis

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Carcinogenesis, Vol. 21, No. 8, 1461-1467, August 2000
© 2000 Oxford University Press

Accelerated Paper

Identification of tamoxifen–DNA 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

	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, TAM–DNA 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 0.2–12 and 1.6–8.3 adducts/10⁸ nucleotides, respectively. There was marked inter-individual variation in the relative amounts of cis and trans adducts present. Low levels (0.74–1.1 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; dG_3'P, 2'-deoxyguanosine 3'-monophosphate; dG-N²-TAM, -(N²-deoxyguanosinyl)tamoxifen; TAM N-oxide, tamoxifen N-oxide; TAM, tamoxifen; -OHTAM, -hydroxytamoxifen.

	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 (2–6); the cellular mechanism responsible for this carcinogenic effect has not been defined (7–9). TAM induces hepatocellular carcinomas in rats (10,11) and TAM–DNA adducts have been identified in rat liver (7,12–14). However, in women treated with TAM, the risk of developing hepatocellular cancer is minimal and the failure to detect TAM–DNA 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) (20–22). 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 (26–28).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Formation of trans and cis forms of TAM–DNA adducts via -hydroxylation of TAM metabolites.

 
In a previous study (29), we used a ³²P-post-labeling–TLC technique to detect TAM–DNA 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 TAM–DNA adducts. The present demonstration that TAM–DNA 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 TAM–DNA 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. TAM–DNA 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.

	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 dichloromethane–methanol. 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 (97–2578 and 99–2578) 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 = OD_{260 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 TAM–DNA adducts. Approximately 95% of the TAM adducts present were recovered by this procedure.

Determination of ³²P-labeled TAM–DNA adducts by HPLC
Pooled extracts containing DNA adducts were labeled with ³²P (35), applied to 10x10 cm PEI–cellulose 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 20–45% 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 (dG_3'p)-N²-TAM (26) and dG_3'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 dN_3'P; the specific activity of [-³²P]ATP, corrected for radioactive decay, was 5.06x10⁷ d.p.m./pmol. The limit of detection of TAM–DNA adducts using this technique was ~2.5 adducts/10¹⁰ nucleotides.

Preparation of TAM–DNA adduct standards
DNA (10 µg) was mixed with 400 µg of TAM -sulfate or -acetoxyTAM N-oxide dissolved in 100 µl of 100 mM Tris–HCl 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 TAM–DNA adducts in endometrial tissue.

	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. TAM–DNA adducts were analyzed as described in Materials and methods, using the HPLC gradient system developed by Martin et al. (30).

View this table: [in this window] [in a new window]   Table I. Patient information

 
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 dG_3'p-N²-TAM, were 22.2, 26.4 and 59.4 min, respectively (Figure 2). TAM–DNA 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 TAM–DNA adducts were identical to those of standard trans and cis forms of dG_3'p-N²-TAM. When sample T1 (Figure 3B) was co-injected with authentic ³²P-labeled dG_3'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 dG_3'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. TAM–DNA 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).

View larger version (20K): [in this window] [in a new window]   Fig. 2. HPLC analysis of TAM–DNA 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 PEI–cellulose 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 20–45% 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 PEI–cellulose 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.

 

View larger version (23K): [in this window] [in a new window]   Fig. 4. Detection of minor TAM–DNA adducts in endometrial tissue. (A) The T11 sample was analyzed by HPLC as described in the legend of Figure 2. Calf thymus DNA was reacted with TAM -sulfate (B), -acetoxyTAM N-oxide (C) or a preparation of 4-OHTAM quinone methide (D), and the adducts were isolated as described in the Materials and methods.

 

View this table: [in this window] [in a new window]   Table II. Level of TAM–DNA adducts in TAM-treated endometrial samples

 

View larger version (12K): [in this window] [in a new window]   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.

 
In T11 (Figure 4A), retention times for the three major labeled products were consistent with those observed for a standard mixture of ³²P-labeled dG_3'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 0.2–12 and 1.6–8.3 adducts/10⁸ nucleotides, respectively. Total dG-N²-TAM adducts ranged from 0.2 to 18 adducts/10⁸ nucleotides.

	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 (12–14). In the present study, a sensitive, specific, and robust method was employed to identify TAM–DNA 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 TAM–DNA adducts in endometrial tissue (15–17,29). In one study, TLC was used (15), whereas in another report, the same samples were analyzed by HPLC (16). Both studies failed to detect TAM–DNA adducts in the endometrial tissue of 20 patients treated with 20 mg/day TAM for 22–65 months. Although Carmichael et al. (16) reported an ability to detect eight TAM–DNA adducts per 10¹⁰ nucleotides (16), they were unable to reproduce the results of a study by Hemminki et al. (17) which described formation of TAM–DNA adducts (2.9–8.2 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 TAM–DNA 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 TAM–DNA adducts with butanol and recovering them from a TLC plate prior to HPLC. We found it exceedingly difficult to quantify TAM–DNA 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 TAM–DNA adducts in endometrial tissue. Using this assay, TAM–DNA 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 TAM–DNA 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 GT 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 0.2–12 and 1.6–8.3 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 TAM–DNA 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-TAM–DNA adducts observed in our study is consistent with the view that -OHTAM is the important reactive intermediate in endometrial cells (26–28,39). 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 (21–22,41,42) 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 TAM–DNA 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.

	Notes

 
³ To whom correspondence should be addressed Email: shinya{at}pharm.sunysb.edu

	Acknowledgments

 
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.

	References

Top Abstract Introduction Materials and methods Results Discussion References

 

1. Osborne,C.K. (1998) Tamoxifen in the treatment of breast cancer. New Eng. J. Med., 339, 1609–1617.[Free Full Text]
2. 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, 1371–1388.[Abstract/Free Full Text]
3. 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, 527–537.[Abstract/Free Full Text]
4. 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, 1451–1467.[Web of Science][Medline]
5. 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, 1654–1662.[Abstract/Free Full Text]
6. Killackey,M., Hakes,T.B. and Pierce,V.K. (1985) Endometrial adenocarcinoma in breast cancer patients receiving antiestrogens. Cancer Treat. Rep., 69, 237–238.[Web of Science][Medline]
7. Tannenbaum,S.R. (1997) Comparative metabolism of tamoxifen and DNA adduct formation and in vitro studies on genotoxicity. Semin. Oncol., 24, 81–86.
8. Wogan,G.N. (1997) Review of the toxicology of tamoxifen. Semin. Oncol., 24 (Suppl. 1), 87–97.
9. Stearns,V. and Gelman,E.P. (1998) Does tamoxifen cause cancer in human? J. Clin. Oncol., 16, 779–792.[Abstract]
10. 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, 315–317.[Abstract/Free Full Text]
11. 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.[Abstract/Free Full Text]
12. Han,X. and Liehr,J.G. (1992) Induction of covalent DNA adducts in rodents by tamoxifen. Cancer Res., 52, 1360–1363.[Abstract/Free Full Text]
13. Osborne,M.R., Hewer,A., Hardcastle,I.R., Carmichael,P.L. and Phillips,D.H. (1996) Identification of the major tamoxifen–deoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res., 56, 66–71.[Abstract/Free Full Text]
14. Divi,R.L., Osborne,M.R., Hewer,A., Phillips,D.H. and Poirier,M.C. (1999) Tamoxifen–DNA adduct formation in rat liver determined by immunoassay and ³²P-post-labeling. Cancer Res., 59, 4829–4833.[Abstract/Free Full Text]
15. 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, 1475–1479.[Abstract/Free Full Text]
16. 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 tamoxifen–DNA adducts in the human endometrium. Carcinogenesis, 20, 339–342.[Abstract/Free Full Text]
17. Hemminki,K., Rajaniemi,H., Lindahl,B. and Moberger,B. (1996) Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res., 56, 4374–4377.[Abstract/Free Full Text]
18. 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.[Free Full Text]
19. 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, 1651–1654.[Abstract/Free Full Text]
20. 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, 1487–1492.[Abstract/Free Full Text]
21. 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, 683–688.[Abstract/Free Full Text]
22. 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, 377–382.[Abstract]
23. White,I.N. (1999) The tamoxifen dilemma. Carcinogenesis, 20, 1153–1160.[Abstract/Free Full Text]
24. Dasaradhi,L. and Shibutani,S. (1997) Identification of tamoxifen–DNA adducts formed by -sulfate tamoxifen and -acetoxytamoxifen. Chem. Res. Toxicol., 10, 189–196.[Web of Science][Medline]
25. Osborne,M.R., Hardcastle,I.R. and Phillips,D.H. (1997) Minor products of reaction of DNA with -acetoxytamoxifen. Carcinogenesis, 18, 539–543.[Abstract/Free Full Text]
26. 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, 647–653.[Abstract/Free Full Text]
27. 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, 2007–2011.[Abstract/Free Full Text]
28. 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, 861–866.[Abstract/Free Full Text]
29. Shibutani,S., Suzuki,N., Terashima,I., Sugarman,S.M., Grollman,A.P. and Pearl,M.L. (1999) Tamoxifen–DNA adducts in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol., 12, 646–653.[Web of Science][Medline]
30. 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, 1061–1069.[Abstract/Free Full Text]
31. Shibutani,S. and Dasaradhi,L. (1997) Miscoding potential of tamoxifen-derived DNA adducts: -(N²-deoxyguanosinyl)tamoxifen. Biochemistry, 36, 13010–13017.[Medline]
32. 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, 2091–2095.[Abstract/Free Full Text]
33. Marques,M.M. and Beland,F.A. (1997) Identification of tamoxifen–DNA adducts formed by 4-hydroxytamoxifen quinone methide. Carcinogenesis, 18, 1949–1954.[Abstract/Free Full Text]
34. 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) Tamoxifen–DNA adducts formed by -acetoxytamoxifen N-oxide. Chem. Res. Toxicol., 12, 1083–1089.[Web of Science][Medline]
35. 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.
36. 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, 1181–1186.[Abstract/Free Full Text]
37. Ottender,M. and Lutz,S.K. (1999) Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts. Mutat. Res., 424, 237–247.[Web of Science][Medline]
38. 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, 13513–13514.
39. Potter,G.A., McCague,R. and Jarman,M. (1994) A mechanism hypothesis for DNA adduct formation by tamoxifen hepatic oxidative metabolism. Carcinogenesis, 15, 439–442.[Abstract/Free Full Text]
40. 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, 471–477.[Abstract/Free Full Text]
41. 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, 827–834.[Web of Science][Medline]
42. 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, 589–593.[Abstract/Free Full Text]

Received March 21, 2000; revised May 22, 2000; accepted May 25, 2000.

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