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[Cancer Research 61, 3925-3931, May 15, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

Mechanism of Lower Genotoxicity of Toremifene Compared with Tamoxifen1

Shinya Shibutani2, Anisetti Ravindernath, Isamu Terashima, Naomi Suzuki, Y. R. Santosh Laxmi, Yoshikazu Kanno, Masanobu Suzuki, T. Idil Apak, Jonathan J. Sheng and Michael W. Duffel

Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651 [S. S., A. R., I. T., N. S., Y. R. S. L.]; Pharmaceuticals Group, Nippon Kayaku Co. Ltd., Tokyo 115-8588, Japan [Y. K., M. S.]; and Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242 [T. I. A., J. J. S., M. W. D.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the {alpha}-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using 32P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When {alpha}-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of {alpha}-hydroxytamoxifen. {alpha}-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of {alpha}-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of {alpha}-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TAM3 (the structure in Fig. 1Citation ) is widely used as a first-line therapy for breast cancer patients (1) . A randomized clinical trial for healthy women at high risk of developing this disease, conducted by the National Surgical Adjuvant Breast and Bowel Project, showed that therapeutic doses of TAM reduced the risk of invasive breast cancer approximately 50% (2) . Therefore, this drug was approved in 1998 by the Food and Drug Administration for use as a chemopreventive agent. Unfortunately administration of TAM to breast cancer patients was associated with an increased risk of endometrial cancer (3, 4, 5, 6, 7, 8) . The increased incident of endometrial cancers was also observed at the TAM chemoprevention trial (2 , 4) . TAM is a potent hepatocarcinogen in rats (9, 10, 11) and produces a high level of TAM-DNA adducts in the liver of rats treated with this drug (12, 13, 14, 15) . TAM was listed in 1996 as a human carcinogen by the International Agency of Research on Cancer (16) .



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  		Fig. 1. Structures of TAM and TOR.

 
TAM is metabolized in the liver of rodents and humans to {alpha}-OHTAM, N-desTAM, tamoxifen N-oxide, and 4-hydroxytamoxifen (17, 18, 19) . Among these TAM metabolites, we found that {alpha}-OHTAM is sulfonated by hydroxysteroid sulfotransferases (20 , 21) and reacts with the exocyclic amino group of guanine in DNA, resulting in the formation of two trans (fr-1 and fr-2) and two cis (fr-3 and fr-4) diastereoisomers of dG-N2-TAM (Fig. 2Citation ; Refs. 13 and 22 ). A mass-spectroscopy analysis (23) and 32P-postlabeling/HPLC analysis (24 , 25) demonstrated that dG-N2-TAM and {alpha}-(N2-deoxyguanosinyl)-N-desmethyltamoxifen are major DNA adducts in the liver of rodents treated with TAM. These indicated that TAM-DNA adducts are primarily formed via sulfonation of {alpha}-hydroxylated TAM metabolites such as {alpha}-OHTAM and {alpha}-hydroxy-N-desmethyltamoxifen (Fig. 2)Citation . A high frequency of mutations was observed in the liver DNA of {lambda}/lacI transgenic rats treated with TAM (26) . dG-N2-TAM adducts display a high miscoding and mutagenic potential and generate primarily G->T transversions in mammalian cells (27 , 28) . Significant level of dG-N2-TAM adducts have been detected in the endometrium of certain women treated with TAM (29 , 30) . Such TAM adducts, if not repaired (31) , may cause mutations, leading to the development of endometrial cancers.



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  		Fig. 2. Mechanism for the formation of TAM-DNA adducts.

 
TOR (Fig. 1)Citation , a chlorinated TAM derivative, has been used for breast cancer therapy in 27 countries, including in Finland since 1988, in Sweden since 1994, in Japan since 1995, and in the United States since 1998. TOR is metabolized by human liver microsomes, resulting in the formation of N-desmethyltoremifene, 4-hydroxytoremifene, and deamino-hydroxytoremifene; the formation of N-desmethyltoremifene and deamino-hydroxytoremifene are mediated by P450 1A and 3A4 enzymes (32) . The N-desmethylated and 4-hydroxylated metabolites were detected in human blood (33) and mainly excreted in rat feces (34 , 35) . Small amounts of these metabolites are excreted in rat urine; {alpha}-OHTOR and {alpha}-hydroxy-N-desmethyltoremifene were also detected using mass spectroscopy (35) . Unlike TAM, TOR produces only a small amount of DNA adducts in rat liver (11 , 36 , 37) and does not promote hepatocarcinoma in rats (11 , 36) .

In the present study, to investigate the mechanism of the lower genotoxicity of TOR, we synthesized {alpha}-OHTOR and {alpha}-acetoxyTOR as model activated forms of TOR. We determined whether {alpha}-OHTOR can be sulfonated by hydroxysteroid sulfotransferases and whether {alpha}-acetoxyTOR can react with DNA.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
[{gamma}-32P]ATP (specific activity, 6000 Ci/mmol) was obtained from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). PEI-cellulose plates were purchased from Machery-Nagel (Dueren, Germany). Calf thymus DNA, proteinase K, potato apyrase, dG3'P, PAP, and PAPS were purchased from Sigma Chemical Co.-Aldrich (St. Louis, MO). RNase A, RNase T1, micrococcal nuclease, and spleen phosphodiesterase were obtained from Worthington Biochemical Corp. (Lakewood, NJ). T4 polynucleotide kinase was purchased from Stratagene (La Jolla, CA). hHST (SULT2A1) was purchased from PanVera Corporation (Madison, WI).

Synthesis of {alpha}-OHTOR.
(E)-1-bromo-2-{4-[2-(dimethylamino)-ethoxy]-phenyl}-1,2-diphenyl ethene was synthesized by the established protocol (38) . n-Butyl lithium (2.4 mmol; 1.5 ml of 1.6 M solution in hexane) was added to a solution of (E)-1-bromo-2-{4-[2-(dimethylamino)-ethoxy]-phenyl}-1,2-diphenyl ethene (720 mg; 1.7 mmol) in dry tetrahydrofuran (12 ml) at -78°C under N2. Distilled chloroacetaldehyde (0.39 g; 5.1 mmol) in 5 ml of tetrahydrofuran was added 5 min later. The mixture was warmed to 0°C in 20 min, quenched with water (10 ml), extracted with ether (2 x 20 ml), dried over anhydrous sodium sulfate, and then concentrated. The residue was purified by column chromatography using silica gel (eluent, 10% triethylamine in hexane:ether, 1:1, for the trans and 20% of triethylamine for the cis isomer) to yield trans-{alpha}-OHTOR (215 mg; 30%) and cis-{alpha}-OHTOR (107 mg; 20%). NMR results for trans-{alpha}-OHTOR ({delta}, ppm, CDCl3): 1H-NMR: 2.23 [s, 6H, N(CH3)2], 2.61 [t, 2H, J = 5.8 Hz, CH2-N(CH3)2], 3.40 (m, 2H, CH2-Cl), 3.89 (t, 2H, 5.8 Hz, O-CH2), 4.84 (dd, 1H, J = 6.4 and 6.2 Hz, CH -OH), 6.56 (d, 2H, J = 8.8 Hz, H 3,5 of CC6H4O), 6.82 (d, 2H, J = 8.8 Hz, H 2,6 of CC6H4O), and 7.14–7.34 (m, 10H, 2Ph); and 13C-NMR: 45.65 [N(CH3)2], 47.35 [CH2-N(CH3)2], 57.98 (CH2-Cl), 65.46 (O-CH2), 71.74 (CH-OH), and (113.32, 126.73, 127.12, 127.78, 128.19, 129.51, 130.88, 131.42, 134.15, 137.02, 127.76, 141.49, 144.03, 157.07), aromatic and olefin carbons.

Synthesis of {alpha}-AcetoxyTOR.
Acetic anhydrate (1 ml; 1 mmol) and 4-dimethylaminopyridine (10 mg) were added to a solution of the trans-{alpha}-OHTOR (110 mg; 0.26 mmol) in dry triethylamine (2.5 ml). The reaction mixture was stirred at room temperature for 12 h, and then concentrated and purified by column chromatography using silica gel (eluent, 10% triethylamine in hexane:ether, 1:1) to yield {alpha}-acetoxyTOR (79 mg; 65%). NMR results for trans-{alpha}-OHTOR: ({delta}, ppm, CDCl3): 1H-NMR: 1.92 (s, [3H], COCH3), 2.22 [s, 6H, N(CH3)2], 2.57 [t, 2H, J = 5.8 Hz, CH2-N(CH3)2], 3.45 (m, 2H, CH2-Cl), 3.85 (t, 2H, J = 5.8 Hz, O-CH2), 5.85 (dd, 1H, J = 6.4 and 6.5 Hz, CH -OAc), 6.50 (d, 2H, J = 8.8 Hz, H 3,5 of CC6H4O), 6.76 (d, 2H, J = 8.8 Hz, H 2,6 of CC6H4O), and 7.18–7.42 (m, 10H, 2Ph); and 13C-NMR: 20.78 (COCH3), 43.46 [CH2-N(CH3)2], 45.73 [N(CH3)2], 58.09 (CH2-Cl), 65.57 (O-CH2), 74.23 (CH-OAc), and (113.33, 126.88, 127.34, 127.86, 128.22, 129.23, 130.70, 131.29, 133.40, 133.79, 137.71, 141.18, 146.02, 157.29, aromatic and olefin carbons), and 169.90 (COCH3).

Preparation of STa.
A rat liver STa (99.2 units/mg protein; 39 ) and a recombinant STa (62 units/mg protein; Ref. 40 ) were prepared as described previously. Enzyme units are expressed as nanomoles of sulfuric acid ester product formed from dehydroepiandrosterone per min (39 , 40) .

Sulfonation of {alpha}-OHTAM or {alpha}-OHTOR by Hydroxysteroid Sulfotransferases.
A trans-form of {alpha}-OHTAM was synthesized by the established protocol (38) . PAPS was purified by chromatography on DEAE-cellulose according to a published procedure (41) . Both {alpha}-OHTAM and {alpha}-OHTOR were evaluated as substrates of recombinant STa using a published HPLC procedure for determination of the concentration of PAP in the reaction (42) . The assay mixtures (30 µl, total volume) contained 0.25 M potassium phosphate buffer (pH 7.0), 8.3 mM 2-mercaptoethanol, and 200 µM PAPS. Substrates were dissolved in 50% acetone/50% water and added to the assay mixtures; the final concentration of acetone in all reaction mixtures was 2.5% (v/v). All reactions were initiated by the addition of 0.5 µg STa, incubated at 37°C for 15 min, and terminated by the addition of 30 µl of methanol. HPLC was performed on an Alltech Econosphere C18 column (4.6 mm x 250 mm; 5 µm), eluted at a flow rate of 2 ml/min with 60 mM potassium phosphate (pH 5.45), and 60 mM ammonium chloride in 12% methanol (42) . The limit of detection in these assays was 1.9 nmol product/min/mg STa.

Reaction of {alpha}-OHTAM or {alpha}-OHTOR with DNA in the Presence of PAPS and Hydroxysteroid Sulfotransferase.
Calf thymus DNA (10 µg) was incubated at 37°C for 1 h with 200 µM PAPS, variable amounts of rat liver STa or hHST, and either 100 µM {alpha}-OHTAM or {alpha}-OHTOR in 30 µl of 0.25 M potassium phosphate buffer (pH 7.0) containing 8.3 mM mercaptoethanol. After the reaction, the DNA was recovered by phenol/chloroform extraction and used for determination of DNA adducts using 32P-postlabeling/HPLC analysis.

Reactivity of {alpha}-AcetoxyTAM or {alpha}-AcetoxyTOR with DNA.
{alpha}-AcetoxyTAM was synthesized by the established protocol (22) . Calf thymus DNA (10 µg) was reacted at 37°C for 1 h with variable amounts of {alpha}-acetoxyTAM or {alpha}-acetoxyTOR in 200 µl of 100 mM Tris-HCl buffer (pH 7.5). The DNA was recovered by phenol/chloroform extraction and used for 32P-postlabeling/HPLC analysis.

Enzymatic Digestion of DNA Samples.
DNA sample (0.1–3.0 µg) was enzymatically digested at 37°C for 2 h in 30 µl of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2, using 15 units of micrococcal nuclease and 0.1 units of spleen phosphodiesterase, and incubated for an additional 1 h with 1 unit of nuclease P1, as described previously (20) . After the reaction, 100 µl of distilled water was added to the digested sample and extracted twice with 100 µl of butanol. The butanol fractions were back-extracted with 50 µl of distilled water. The butanol fractions were evaporated to dryness and used for analysis of DNA adducts.

Detection of DNA Adducts by 32P-postlabeling/HPLC Analysis.
The pooled extracts of digests were incubated at 37°C for 40 min with 30 µCi of [{gamma}-32P]ATP and 3 µl of T4 polynucleotide kinase (10 units/µl; Ref. 43 ) and subsequently incubated for 1 h with potato apyrase (8 x 10-2 units). A part of the 32P-labeled sample was spotted on a PEI-cellulose TLC plate (10 x 10 cm) and developed for 16 h using 1.7 M sodium phosphate buffer (pH 6.0) with a paper wick (20) . The position of adducts was established by a ß-phosphorimaging (Molecular Dynamic, Inc.) or autoradiography using Kodak Xomat XAR film. DNA adducts that remained around the original spot were recovered using 4 M pyridinium formate (pH 4.3). 32P-labeled products were placed on a Hypersil BDS C18 analytical column (0.46 x 25 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 Berthold LB506 C-1 radioisotope detector (ICON Scientific, Inc.) connected to a Waters 990 HPLC instrument. Standards of dG3'p-N2-TAM (22) were prepared using published methods and labeled with 32P (43) . The relative adduct levels were calculated according to Levay et al. (44) , using dpm instead of cpm: (total dpm in adducts)/1. 36 x 10 11 dpm, assuming that 3 µg of DNA was 9.09 x 103 pmol of dN3'P and the specific activity of the [{gamma}-32P]ATP was 1. 50 x 107 dpm/pmol. The specific activity of the [{gamma}-32P]ATP was corrected by calculating the extent of decay. A peak counting >200 dpm by radioisotope detector was judged to be significant. The limit of detection of DNA adducts using this technique was ~1.5 adducts/109 nucleotides.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Formation of DNA Adducts via Sulfonation of {alpha}-OHTAM or {alpha}-OHTOR.
To explore the formation of DNA adducts through O-sulfonation of {alpha}-OHTAM or {alpha}-OHTOR, these compounds were incubated with calf thymus DNA, PAPS, and with or without rat STa. When STa was incubated with {alpha}-OHTAM, three TAM-DNA adducts were detected at 21.7 min (fr-1), 25.7 min (fr-2), and 59.6 min (fr 3 & 4), respectively (Fig. 3A)Citation . The tRs of fr-1 and fr-2 were consistent with those of the two diastereoisomers of trans-dG-N2-TAM and the tR of fr 3 & 4 was consistent with that of a mixture of two diastereoisomers of cis-dG-N2-TAM (Fig. 3C)Citation . The level of fr-2 (16 adducts/106 nucleotides) was higher than the other adducts (Table 1)Citation . The total amounts of dG-N2-TAM adducts were 21 adducts/106 nucleotides. With {alpha}-OHTAM alone, a small amount (total, 0.27 adducts/106 nucleotides) of TAM adducts were detected. No DNA adducts were detected when DNA was incubated without {alpha}-OHTAM and STa, (data not shown).



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  		Fig. 3. 32P-Postlabeling/HPLC analysis of DNA incubated with {alpha}-OHTAM or {alpha}-OHTOR in the presence of PAPS and STa. Calf thymus DNA (10 µg) was incubated with 100 µM {alpha}-OHTAM (A) or 100 µM {alpha}-OHTOR (B) in 50 µl of 0.25 M potassium phosphate buffer (pH 7.0) containing 2.4 µg of STa, 200 µM PAPS, and 8.3 mM 2-mercaptoethanol. The recovered DNA (3 µg) was digested enzymatically, extracted with butanol, and labeled with 32P, as described in "Materials and Methods." Half of the 32P-labeled sample was applied to PEI-cellulose TLC chromatography and developed using 1.7 M sodium phosphate buffer (pH 6.0). The 32P-labeled products were recovered from the TLC plate and subjected to HPLC on-lined radioisotope detection, as described in "Materials and Methods." C, a mixture of two diastereoisomers (fr-1 and fr-2) of trans-form and two diastereoisomers (fr-3&4) of cis-form of 32P-labeled dG3'P-N2-TAM. D, dG3'P (0.5 mg) was reacted at 37°C for 4 h with {alpha}-acetoxyTOR (0.5 mg) in 200 µl of 100 mM Tris-HCl buffer (pH 7.5), extracted with butanol, and then labeled with 32P.

 

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  		Table 1 Formation of DNA adducts via STa-catalyzed sulfonation of {alpha}-OHTAM or {alpha}-OHTOR

 
In contrast, when {alpha}-OHTOR was incubated with STa, only a trace of DNA adduct (0. 1 adduct/106 nucleotides) was detected at 11.0 min (Table 1Citation and Fig. 3BCitation ): The tR was consistent with that of fr-b, observed when dG3'P was reacted with large amounts of {alpha}-acetoxyTOR (Fig. 3D)Citation . Without STa, no DNA adduct was detected (Table 1)Citation . The level of DNA adducts formed by sulfonation of {alpha}-OHTOR was 210 times lower than that for {alpha}-OHTAM.

Using hHST, {alpha}-OHTAM or {alpha}-OHTOR was also incubated under reaction conditions containing DNA and PAPS. With {alpha}-OHTAM, the formation of dG-N2-TAM adducts was increased, depending on the amounts of hHST used (Table 2)Citation . However, with {alpha}-OHTOR, a trace of DNA adduct can be detected only when 0.08 unit of hHST was used (Table 2)Citation . The level of TOR adduct was 50 times lower than that of TAM adducts. Like STa, hHST catalyzes {alpha}-OHTAM, but not {alpha}-OHTOR.


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  		Table 2 Formation of DNA adducts induced by hHST-catalyzed sulfonation of {alpha}-OHTAM and {alpha}-OHTOR

 
Sulfonation of {alpha}-OHTAM or {alpha}-OHTOR Catalyzed by STa.
{alpha}-OHTAM or {alpha}-OHTOR was incubated with a recombinant STa in the presence of PAPS to monitor the substrate-dependent formation of PAP in the reaction. When {alpha}-OHTAM was used, the increased formation of PAP from PAPS was observed, as compared with the control. The rate of {alpha}-OHTAM-dependent PAP formation was 15 ± 1 nmol/min/mg STa. In contrast, with {alpha}-OHTOR, no significant PAP formation was detected. PAP was not formed even with 5-fold higher amounts of {alpha}-OHTOR (500 µM). Apparently unlike {alpha}-OHTAM, {alpha}-OHTOR cannot serve as a substrate for STa.

Reactivity of {alpha}-AcetoxyTOR with dN3'p.
dG3'P (0.5 mg) was incubated at 37°C for 4 h with {alpha}-acetoxyTOR (0.5 mg) in 200 µl of 100 mM Tris-HCl buffer (pH 7.5), resulting in the formation of five adducts (a: tR = 4.3 min; b: tR = 11.0 min; c: tR = 24. 9 min; d: tR = 2 7.8 min; and e: tR = 59.8 min; Fig. 3DCitation ). The level of adducts a, b, c, d, and e were 2.0, 9.0, 0.88, 11.6, and 0.46 adducts/108 nucleotides, respectively. No adducts were observed with other deoxynucleoside 3'-monophosphates (data not shown).

Reactivity of {alpha}-AcetoxyTAM or {alpha}-AcetoxyTOR with DNA.
To compare the reactivity of {alpha}-acetoxyTAM to DNA with that of {alpha}-acetoxyTOR, calf thymus DNA was reacted with variable amounts of {alpha}-acetoxyTAM or {alpha}-acetoxyTOR, and the formation of DNA adducts was determined by 32P-postlabeling/HPLC analysis (Table 3)Citation . When 250 pmol of {alpha}-acetoxyTAM was used, the trans-diastereoisomers (fr-1 and fr-2) of dG-N2-TAM and the cis-form (a mixture of fr-3 and fr-4) were formed (Fig. 4B)Citation ; the total amounts of TAM adducts were 99 adducts/106 nucleotides (Table 3)Citation . Using the equivalent amount of {alpha}-acetoxyTOR, a small amount of two adducts were detected (Fig. 4CCitation and Table 3Citation ). The tRs of the two adducts were consistent with that of fr-a and fr-b observed when dG3'P was reacted with {alpha}-acetoxyTOR. The total amount of TOR adducts (0.8 adducts/106 nucleotides) was 125 times lower than those induced by {alpha}-acetoxyTAM (Table 3)Citation . No adducts were observed with control DNA (Fig. 4A)Citation . Because the reactivity of {alpha}-acetoxyTOR with dG3'P was very poor, the structures of {alpha}-acetoxyTOR-modified dG3'P could not be identified by LC/MS/MS even when excess amounts of {alpha}-acetoxyTOR were reacted.


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  		Table 3 Reactivity of {alpha}-acetoxytamoxifen and {alpha}-acetoxytoremifene to DNA

 


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  		Fig. 4. Formation of DNA adducts by reacting DNA with either {alpha}-acetoxyTAM or {alpha}-acetoxyTOR. Calf thymus DNA (10 µg) was reacted at 37°C for 1 h with either 0.1 µg (250 pmol) of {alpha}-acetoxyTAM (B) or {alpha}-acetoxyTOR (C) in 200 µl of 100 mM Tris-HCl buffer (pH 7.5). A control incubation (A) with all components except for the {alpha}-acetoxy derivatives was also carried out. The DNA was recovered by phenol/chloroform extraction and then analyzed by 32P-postlabeling/HPLC method, as described in "Materials and Methods."

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TAM-DNA adducts are primarily formed from {alpha}-hydroxylated TAM metabolites including {alpha}-OHTAM via sulfonation catalyzed by hydroxysteroid sulfotransferases (20 , 21) . Because {alpha}-hydroxylated TOR metabolites such as {alpha}-OHTOR and {alpha}-hydroxy-N-desmethylTOR were detected in rat urine (35) , {alpha}-hydroxylated TOR metabolites may be precursors forming TOR-DNA adducts. However, unlike {alpha}-OHTAM, {alpha}-OHTOR produced only a small amount of DNA adducts under reaction conditions containing PAPS, DNA, and either STa or hHST. This result may be based on the fact that {alpha}-OHTOR, a poor substrate for hydroxysteroid sulfotransferases, is not readily converted to the sulfuric acid ester when compared with {alpha}-OHTAM. This may be one of reasons why TOR is less genotoxic for rats than TAM (11 , 36 , 45) .

Because the reactivity of {alpha}-acetoxyTAM with either dG or DNA was similar to that of TAM {alpha}-sulfate (22) , {alpha}-acetoxyTOR can be used as a model activated form of TOR. However, the level of DNA adducts formed by {alpha}-acetoxyTOR was much lower than that of {alpha}-acetoxyTAM. Our results were consistent with the facts that TOR produced two-orders of magnitude lower levels of DNA adducts in rat liver compared with TAM (11 , 36 , 45) and no DNA adducts in the mouse liver (46) . The lower genotoxicity of TOR may be attributable to the limited formation of DNA adducts induced by TOR.

One potential explanation for both the inability of hydroxysteroid sulfotransferase to catalyze sulfonation of the {alpha}-hydroxyl moiety of TOR and the low reactivity of {alpha}-acetoxyTOR with DNA may be attributable to the steric hindrance caused by the presence of a bulky chlorine atom positioned at the ethyl moiety of TOR. In addition, unlike TAM carbocation (Fig. 5Citation ; Ref. 47 ), field effect caused by the electron-withdrawing chlorine atom may diminish the effective elimination of the {alpha}-acetoxyl group from the {alpha}-carbon and therefore inhibit the formation of the carbocation intermediate that reacts with the N2 position of dG residues in DNA (48) .



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  		Fig. 5. Formation of TAM or TOR carbocation intermediates.

 
Both TAM and TOR have similar estrogenic activity in endometrial cancer cells (49) and in rat uterus (50) and produce similar increase in the endometrial thickness of postmenopausal breast cancer patients (51) . However, thus far there is no clinical report that TOR increases the incidence of endometrial cancer, although TOR has been used for breast cancer therapy since 1988 in Finland, since 1994 in Sweden, and since 1995 in Japan. This may indicate that DNA damage induced by TAM is primarily involved in the development of endometrial cancer. Because clinical efficacy of TOR for breast cancer patients are similar to that of TAM (52) , the use of TOR, instead of TAM, may minimize incidences of endometrial cancer.

RAL, a selective estrogen response modifier, was approved in 1998 by the Food and Drug Administration to use for osteoporosis, but thus far has not been approved for breast cancer therapy. RAL is unlikely to react with DNA because of the absence of the ethyl moiety, and it does not demonstrate proliferative effects on the uterus of postmenopausal women (53) . On the basis of a recent RAL chemoprevention trial, RAL also reduced the incidence of breast cancer in women at high risk of developing of breast cancer (54) . Unlike TAM, no increased incidence of endometrial cancer was observed in this trial (54) . RAL may be another alternative to diminish the risk of development of endometrial cancer.

Because TAM-DNA adducts detected in the endometrium of women treated with TAM (29 , 30) have a strong miscoding and mutagenic potential (27 , 28) , TAM adducts, if not repaired (31) , may pose a potential risk of development of endometrial cancer in women treated with TAM. TOR and RAL are likely to have less genotoxicity to the human endometrium than TAM. To avoid the secondary cancers caused by TAM, such less genotoxic antiestrogens should be used for breast cancer therapy and chemoprevention for breast cancer.


   ACKNOWLEDGMENTS
 
We thank R. Rieger for mass spectroscopy measurements.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant ES09418 from the National Institute of Environmental Health Sciences (to S. S.) and Grant CA38683 (to M. W. D.) from the National Cancer Institute, Department of Health and Human Services. Back

2 To whom requests for reprints should be addressed, at Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651. Phone: (631) 444-8018; Fax: (631) 444-3218. Back

3 The abbreviations used are: TAM, tamoxifen; dG, 2'-deoxyguanosine; dG3'P, 2'-deoxyguanosine 3'-monophosphate; HPLC, high performance liquid chromatography; TOR, toremifene; RAL, raloxifene; {alpha}-OHTAM, {alpha}-hydroxytamoxifen; {alpha}-acetoxyTAM, {alpha}-acetoxytamoxifen; dG-N2-TAM, {alpha}-(N2-deoxyguanosinyl)tamoxifen; N-desTAM, N-desmethyltamoxifen; PEI, polyethyleneimine; {alpha}-OHTOR, {alpha}-hydroxytoremifene; {alpha}-acetoxyTOR, {alpha}-acetoxytoremifene; PAP, adenosine 3',5'-diphosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; hHST, human hydroxysteroid sulfotransferase (SULT2A1); STa, rat hydroxysteroid (alcohol) sulfotransferase a; tR, retention time; NMR, nuclear magnetic resonance; s, singlet; d, doublet; t, triplet; m, multipet; dd, doublet of doublets. Back

Received 1/ 2/01. Accepted 3/19/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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