Identification of hepatic tamoxifenDNA adducts in mice:
-(N2-deoxyguanosinyl)tamoxifen and
-(N2-deoxyguanosinyl)tamoxifen N-oxide
Atsushi Umemoto3,
Yasumasa Monden,
Masato Suwa1,
Yoshikazu Kanno1,
Masanobu Suzuki1,
Chun-Xing Lin,
Yuji Ueyama,
Md.Abdul Momen,
Anisetti Ravindernath2,
Shinya Shibutani2 and
Kansei Komaki
Second Department of Surgery, School of Medicine, University of Tokushima, Kuramoto-cho 3-18-15, Tokushima 770-8503,
1 Pharmaceuticals Group, Nippon Kayaku Co. Ltd, Shimo 3-31-12, Kita-ku, Tokyo 115-8588, Japan and
2 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA
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Abstract
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TamoxifenDNA adducts detected in the liver of mice treated with tamoxifen have not yet been identified. In the present study a new type of tamoxifenDNA adduct, four stereoisomers of
-(N2-deoxyguanosinyl)tamoxifen N-oxide 3'-monophosphate (dG3'P-N2-TAM N-oxide) were prepared as standard DNA adducts by reacting 2'-deoxyguanosine 3'-monophosphate with trans-
-acetoxytamoxifen N-oxide in addition to four stereoisomers of
-(N2-deoxyguano- sinyl)tamoxifen 3'-monophosphate (dG3'P-N2-TAM) that was reported previously. Liquid chromatography-electrospray ionizationmass spectrometry of the reaction products gave the most abundant ion at m/z 731 ([M H]), which corresponded to dG3'P-N2-TAM N-oxide. The modified products digested by alkaline phosphatase corresponded to the isomers of dG-N2-TAM N-oxide whose structures were identified previously by mass spectrometry and nuclear magnetic resonance. Using these standard markers, we analyzed the hepatic DNA adducts of female DBA/2 mice treated with tamoxifen at a dosage of 120 mg/kg/day for 7 days by 32P-post-labeling coupled with an HPLC/radioactive detector. Mixtures of eight isomers of dG3'P-N2-TAM and dG3'P-N2-TAM N-oxide were separated into six peaks, since each of the cis epimers were not separated under the present HPLC conditions. Nine adducts were detected in all liver samples of mice. An epimer of trans-dG3'P-N2-TAM was detected as the principal DNA adduct at a level of 29.0 adducts/108 nucleotides, which accounted for 53.3% of the total tamoxifenDNA adducts. Lesser amounts of cis-dG3'P-N2-TAM (2.8%) were also observed. An epimer of the trans-dG3'P-N2-TAM N-oxide (3.9 adducts/108 nucleotides) was detected as the third biggest adduct (7.2% of the total). The cis-dG3'P-N2-TAM N-oxide (0.4 adducts/108 nucleotides) accounted for 0.7% of the total. Thus, dG3'P-N2-TAM and dG3'P-N2-TAM N-oxide were identified in tamoxifen-treated mouse liver.
Abbreviations: dG, 2'-deoxyguanosine; dG3'P, 2'-deoxyguanosine 3'-mono-phosphate; dG3'P-N2-TAM N-oxide,
-(N2-deoxyguanosinyl)tamoxifen N-oxide 3'-monophosphate; dG3'P-N2-TAM,
-(N2-deoxyguanosinyl)tamoxifen 3'-mono-phosphate; dG-N2-TAM N-oxide,
-(N2-deoxyguanosinyl)tamoxifen N-oxide; ESI-MS, electrospray ionization mass spectrometry.
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Introduction
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Since 1973, tamoxifen, (Z)-1-{4-[2-(dimethylamino)ethoxy]phenyl}-1,2-diphenyl-1-butene, has been widely adopted as a first-line endocrine therapy for breast cancer patients (1,2). This drug was recently approved by the FDA for use as a prophylactic agent in healthy women at high risk of develop- ing breast cancer (3,4). However, increased incidence of endometrial cancers were observed in tamoxifen-treated patients (5,6) as well as healthy women enrolled in chemopreventive trial (3). In general, many anti-cancer drugs including tamoxifen are shown to be mutagenic and carcinogenic due to their ability to chemically modify DNA (7,8). Among such anti-cancer drugs, the treatment period of tamoxifen is exceptionally long (35 years). Therefore, careful evaluation of safety for chronic use of tamoxifen is required.
Tamoxifen is a potent carcinogen targeting the liver in rats (9,10). Carcinogenic actions of tamoxifen toward rat liver are not related to its estrogen antagonist activity but occur through DNA adduct formation (11). When liver DNA from tamoxifen-treated mice and rats were analyzed by 32P-post-labeling HPLC analysis, many tamoxifenDNA adduct peaks were observed (12). Osborne et al. (13) indicated that trans-
-(N2-deoxyguanosinyl)tamoxifen (trans-dG-N2-TAM) is a major tamoxifenDNA adduct in rat liver. Using the in vitro reaction of DNA with tamoxifen
-sulfate or
-acetoxytamoxifen, cis-dG-N2-TAM was also identified as a minor adduct by mass spectrometry and nuclear magnetic resonance (14,15). However, no information was available on whether such minor adducts are formed in tissues of tamoxifen-treated animals. In theory, reaction of
-acetoxytamoxifen or tamoxifen
-sulfate with DNA or 2'-deoxyguanosine (dG) yielded four stereoisomers of dG-N2-TAM (two epimers at
-position of the trans form and two epimers of the cis form) (14). Therefore, to identify the epimers of dG-N2-TAM adducts in tamoxifen-treated animals, authentic isomers and high-resolution HPLC conditions are required.
dG-N2-TAM is formed through
-hydroxylation by cytochrome P450, followed by O-sulfation by hydroxysteroid sulfotransferases (1618). The equivalent adducts of N- desmethyltamoxifen and N,N-didesmethyltamoxifen were also suggested to be formed in animal liver and cultured hepatocytes (1921). We recently characterized a new type of adduct,
-(N2-deoxyguanosinyl)tamoxifen N-oxide (dG-N2-TAM N-oxide) (22). dG-N2-TAM N-oxide consisted of four stereoisomers as observed for dG-N2-TAM. dG-N2-TAM N-oxide is likely to be formed more abundantly in mice than rats, since N-oxidation activity is stronger in mice than rats (23). Therefore, mouse liver is thought to be appropriate for the detection of this adduct, although this organ is not a carcinogenic target of tamoxifen. In the present study, using chemically synthesized isomers of dG3'P-N2-TAM N-oxide and dG3'P-N2-TAM as standards, we identified tamoxifenDNA adducts in the liver of tamoxifen-treated mice by 32P-post-labeling HPLC analysis (Figure 1
).
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Materials and methods
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Chemicals
Tamoxifen citrate, proteinase K, potato apyrase, alkaline phosphatase (type III), 2'-deoxyguanosine 3'-monophosphate (dG3'P) and carboxymethyl cellulose were purchased from Sigma Chemical Co. (St Louis, MO). RNase A, RNase T1, micrococcal nuclease and spleen phosphodiesterase were purchased from Worthington Biochemical Co. (Freehold, NJ). Nuclease P1 and T4 polynucleotide kinase were obtained from Yamasa Shoyu Co. (Choshi, Japan) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. [
-32P]ATP (>7000 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). Polyethyleneimine (PEI)cellulose sheet (Polygram Cell 300 PEI) was purchased from Machery-Nagel (Düren, Germany). Four isomers of dG3'P-N2-TAM were prepared as described previously (14).
Preparation of dG3'P-tamoxifen N-oxide adducts
Trans-
-acetoxytamoxifen N-oxide was prepared according to a previously described method (22). For the preparation of
-(N2-deoxyguanosinyl) tamoxifen N-oxide 3'-monophosphate (dG3'P-N2-TAM N-oxide), 3.0 mg of dG3'P in 0.2 ml of distilled water was added to 7.0 mg of
-acetoxytamoxifen N-oxide in 0.3 ml of acetonitrile, and then 0.3 ml of 0.l M TrisHCl buffer (pH 8.0) was added and stirred for 24 h at 40°C. The reaction products were separated by two HPLC (Hewlett Packard 1090) systems equipped with a photodiode array detector, using a µBondapak C18 column (7.8x300 mm). Eluant A was 50 mM triethylammonium acetate (pH 7.0); eluant B was acetonitrile. The elution was carried out at a flow rate of 2.0 ml/min by the following linear gradient: 1035% B in 020 min; 3580% B in 2045 min; 80% B in 4555 min. Chromatograms were scanned for UV absorbance at 260 nm. Another HPLC system was used to purify the modified nucleotides by the same column with a linear gradient of distilled water to acetonitrile (10% in 05 min; 1035% in 525 min; 3580% in 2555 min) at a flow rate of 2.0 ml/min.
Characterization of dG3'P-tamoxifen N-oxide adducts
For on-line liquid chromatography-electrospray ionizationmass spectrometry (LCESIMS) analysis, each product was separated by a Gulliver HPLC system (JASCO Co., Tokyo, Japan) on a µBondapak C18 column (3.9x150 mm) with a linear gradient of water to acetonitrile (10% in 05 min; 1035% in 525 min; 3580% in 2555 min) at a flow rate of 0.5 ml/min. The main flow of HPLC eluate to the photodiode array detector was split into a Micromass Limited Quattro II mass spectrometer (Manchester, UK) at a flow rate of ~5 µl/min, operating in the negative ion mode. Capillary and cone voltages were 3.04 kV and 30 V, respectively. The source temperature was 100°C.
To examine the correspondence of these dG3'P products and the authentic dG-N2-TAM N-oxides, whose structures were identified previously (22), the HPLC-purified products were incubated at 37°C for 1 h with alkaline phosphatase (3 U) in 100 µl of 100 mM TrisHCl buffer (pH 7.0). Each reaction mixture was extracted twice with 100 µl of butanol. The extract was evaporated to dryness under reduced pressure, subjected to a µBondapak C18 column (3.9x150 mm) and eluted over 30 min at a flow rate of 1.0 ml/min with a linear gradient of 1050% acetonitrile containing 100 mM triethylammonium acetate (pH 7.0). The eluate was monitored using the photodiode array detector.
Animal treatment and DNA isolation
Female DBA/2 mice (5 weeks old) were supplied by NSLC Co. (Hamamatsu, Japan). The animals were housed in polycarbonate cages (three animals per cage) in the pathogen-free room of the animal facilities, and beds were changed twice per week. They were kept under conditions of controlled temperature (22 ± 2°C) and humidity (55 ± 5%) with a 13 h light/11 h dark cycle. The animals were provided a standard laboratory chow MFTM (Oriental Yeast Co. Ltd, Tokyo, Japan) and tap water ad libitum. Tamoxifen citrate was administered daily to female DBA/2 mice (6 weeks old, n = 5) by intragastric instillation at doses of 120 mg/kg for 7 days. The vehicle was 0.5% (w/v) carboxymethyl cellulose sodium salt in water. For the control mice, only the vehicle was administered (n = 3). The animals were asphyxiated with CO2 24 h after the final administration of tamoxifen, and the livers were collected and stored at 80°C until use. DNA was isolated as reported previously (24). The concentration of the DNA was adjusted to 1 mg/ml by estimating spectrophotometrically (OD260 1.0 = 50 µg/ml).
32P-post-labeling of liver DNA adducts and synthetic tamoxifen-dG3'P
Liver DNA (10 µg) was digested to deoxynucleoside 3'-monophosphate with micrococcal nuclease and spleen phosphodiesterase at 37°C for 3.5 h. The adduct was enriched with nuclease P1 at 37°C for 1 h (25). The digest was then converted to 5'-32P-labeled deoxynucleoside 3',5'-diphosphate by T4 polynucleotide kinase and [
-32P]ATP at 37°C for 1 h. For the synthetic tamoxifen-dG3'P adducts, 32P-post-labeling reactions were started directly from treatment with T4 polynucleotide kinase. After labeling, the mixture was further treated with apyrase at 37°C for 45 min. 32P-labeled nucleoside diphosphate was partially purified by development on PEI-cellulose sheets with 1.7 M sodium phosphate (pH 6.0) at 22°C for 15 h. The adducts that remained around the origin on the TLC plate were cut out and extracted with 4 M pyridinium formate (pH 4.5) twice (2.0 and 1.5 ml). The extract was filtered by a Millipore Millex-HA filter (0.45 µm) and evaporated to dryness.
HPLC analysis of labeled DNA adducts
HPLC analysis of DNA adducts was conducted using a slight modification of the method of Martin et al. (12). The dried extract was dissolved in an appropriate amount of 4 M pyridinium formate. An aliquot was analyzed by HPLC (Hewlett Packard 1090) on a Prodigy octadecyl silane (2) column (5 µm, 2.0x150 mm) from Phenomenex (Macclesfield, UK). Eluant A was 2 M ammonium formate (pH 4.0); eluant B was acetonitrile/methanol (6:1 v/v). The elution was carried out at a flow rate of 0.2 ml/min by the following linear gradient: 1520% B in 040 min; 2030% B in 4080 min. For the on-line radioactive detector of the eluate, the Beckman 171 radioisotope detector with a Teflon sample loop (cell volume 200 µl) was used with a mixing scintillation cocktail, Atomlight (Packard), at a flow rate of 0.1 ml/min. The detection limit of tamoxifenDNA adducts was 2.0 adducts/109 normal nucleotides.
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Results
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Characterization of dG3'P-tamoxifen N-oxide adducts
When dG3'P was reacted with
-acetoxytamoxifen N-oxide, two major products, OX-1 and OX-2, were fractionated at retention times (tR) of 30.2 and 31.1 min, respectively (Figure 2A
). The third product, OX-3, was fractionated at a tR of 37.039.0 min, although no obvious peak was visible by UV absorbance at 260 nm. In further HPLC purification, the products OX-1 and OX-2 showed tR at 20.7 and 26.3 min, respectively (Figure 2B and C
), whereas product OX-3 showed a broad peak with a shoulder at 29.7 min (Figure 2D
). UV spectra of OX-1 and OX-2 in water/acetonitrile were indistinguishable; the UV maximum of both OX-1 and OX-2 was 250 nm, whereas that of OX-3 shifted to a 4 nm shorter wavelength. ESIMS of OX-1, OX-2 and OX-3 gave the most abundant ions at m/z 731 ([M H]), as expected (Figure 3
).

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Fig. 3. Negative ion ESIMS of the products of dG3'P reacted with trans- -acetoxytamoxifen N-oxide: (A) OX-1; (B) OX-2; (C) OX-3.
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Further confirmation was performed by comparing the alkaline phosphatase-treated dG3'P products with the authentic dG-N2-TAM N-oxides. The tR of OX-1 treated with alkaline phosphatase shifted from 18.8 to 22.5 min (Figure 4
, asterisk) which is consistent with that of one of the epimers of trans-dG-N2-TAM N-oxides. In a similar manner, the tR of OX-2 (19.8 min) shifted to a tR identical to another epimer of trans-dG-N2-TAM N-oxides (22.6 min). The tR of OX-3 (24.6 min) shifted to a tR similar to epimers of cis-dG-N2-TAM N-oxides (26.3 min). The UV spectra of each product and that of the corresponding dephosphorylated products were indistinguishable from each other. Furthermore, the UV spectra of the dephosphorylated products and the UV spectra of the corresponding authentic dG-N2-TAM N-oxides were identical.

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Fig. 4. HPLC comparison of the reaction products of -acetoxytamoxifen N-oxide with dG3'P and dG. (A) OX-1 and (B) OX-1 treated with alkaline phosphatase, and (C) the mixture of epimers of trans-dG-N2-TAM N-oxide.
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Identification of tamoxifenDNA adducts in mouse liver DNA
Since each cis epimer could not be separated under the present HPLC conditions, a mixture of eight trans and cis isomers of dG-N2-TAM and dG3'P-N2-TAM N-oxide were separated into six peaks (Figure 5A
). Peaks a and b represented epimers of trans-dG3'P-N2-TAM and peaks c and d represented epimers of trans-dG3'P-N2-TAM N-oxide. Peaks e and f represented a mixture of epimers of cis-dG3'P-N2-TAM and a mixture of epimers of cis-dG3'P-N2-TAM N-oxide, respectively. When 2.0 µg liver DNA samples of mice treated with tamoxifen were analyzed, nine DNA adducts were detected in all cases (Figure 5C and D
). These peaks were not detected in non-treated controls (Figure 5B
). To identify adducts, the sample was co-chromatographed with the trans-dG3'P-N2-TAM N-oxide standard (Figure 5E
). An epimer (peak b) of trans-dG3'P-N2-TAM was detected as the principal DNA adduct (peak 5) at levels of 29.0 ± 21.9 adducts/108 nucleotides (mean ± SD) that accounted for 53.3% of the total tamoxifen adduct (Table I
). The second biggest peak (peak 4) eluted 2.8 min earlier than the principal adduct; the level was 14.1 ± 10.1 adducts/108 nucleotides (25.8% of the total). The tR of this peak (35.6 min) was very close to another epimer (peak a) of trans-dG3'P-N2-TAM that eluted only 0.3 min earlier (tR 35.3 min). This indicated that small amounts of another epimer of trans-dG3'P-N2-TAM might be hidden behind this second biggest peak even if it was present. This product may be an N-desmethyltamoxifen adduct, although an authentic standard was not available (19). cis-dG3'P-N2-TAM (peak e) was also detected (peak 8), even though the levels were low (2.8% of the total adduct levels in mouse liver).

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Fig. 5. HPLC separation of 32P-labeled tamoxifenDNA adducts. (A) The 5'-32P-labeled authentic dG3'P-N2-TAM and dG3'P-N2-TAM N-oxide: a and b, epimers of trans-dG3'P-N2-TAM; c and d, epimers of trans-dG3'P-N2-TAM N-oxide; e, mixture of epimers of cis-dG3'P-N2-TAM; f, mixture of epimers of cis-dG3'P-N2-TAM N-oxide. (B) Liver DNA adduct from an untreated female DBA/2 mouse (from 8.0 µg DNA). (C and D) Liver DNA adducts from mice nos. 5 and 4 treated with tamoxifen, respectively (2.0 µg DNA). (E) Co-chromatography of mouse no. 4 mouse liver DNA adducts with trans-dG3'P-N2-TAM N-oxide (peak d). All peak numbers correspond to those listed in Table I .
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A new type of tamoxifenDNA adduct, an epimer (peak d) of the trans-dG3'P-N2-TAM N-oxide, was detected in all five mice as the third biggest peak (peak 6); the adduct level was 3.9 ± 3.5 adducts/108 nucleotides, which accounted for 7.2% of the total (Figure 5B
). cis-dG3'P-N2-TAM N-oxide (peak f) was also detected (peak 9); the adduct level was 0.4 ± 0.2 adducts/108 nucleotides, which accounted for 0.7% of the total.
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Discussion
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The identification of tamoxifenDNA adducts formed in the tissues of animals is important to investigate tamoxifen carcinogenesis. Previously, it was suggested that trans-dG-N2-TAM was a major adduct among tamoxifen-induced DNA adducts in rats (13). The mutagenic potential of all four isomers of dG-N2-TAM has already been elucidated using replication systems of site-specific modified oligodeoxynucleotides in vitro (26) and in mammalian cells (27). However, all details of DNA adducts induced by tamoxifen including distinction of epimers and other types of adducts in animals have not been entirely clarified. To elucidate the tamoxifen-induced DNA adducts in animals, we synthesized authentic standard markers, four isomers of dG3'P-N2-TAM and four isomers of dG3'P-N2-TAM N-oxide. We found that one epimer (peak b) of trans-dG-N2-TAM was the principal hepatic DNA adduct in mice that accounted for >50% of the total adducts. This major adduct is the same adduct detected in endometrial tissues from tamoxifen-treated patients (28). Presence of cis-dG-N2-TAM was also established, although its amount was less than the trans form (2.8% the total).
dG-N2-TAM is formed through
-hydroxylation (2931) followed by sulfation (1618). However, the presence of many DNA adducts implies that several metabolic pathways are involved in the formation of tamoxifen adducts. To clarify the in vivo metabolic activation of tamoxifen requires analysis of the metabolites in plasma. Various tamoxifen metabolites including
-hydroxy, N-desmethyl, 4-hydroxy, N,N-didesmethyl and N-oxide forms were detected in plasma of both patients and rats (32,33). DNA adduct formation via
-hydroxylation is possible in all the above tamoxifen metabolites as well as tamoxifen itself (Figure 6
).
-Hydroxylated metabolites of N-desmethyltamoxifen, 4- hydroxytamoxifen and tamoxifen N-oxide as well as tamoxifen were detected in vitro and in vivo (3133). An N-desmethyltamoxifen dG adduct was also suggested to be formed in rats, although this conclusion was based upon MS alone; no synthetic standards were available (19). However, oxidation of 4-hydroxytamoxifen promoted only minor DNA adducts in rat and mouse livers (3436).
The N-oxide form of the tamoxifenDNA adduct was expected to be formed in vivo, since it has been demonstrated that tamoxifen N-oxide,
-hydroxytamoxifen N-oxide and 4-hydroxytamoxifen N-oxide were detected in an incubation mixture of tamoxifen with liver microsomes from species including humans or in blood and tissues from tamoxifen-treated patients, rats and mice (23,29,31,32,3739). N-oxidation of tamoxifen is catalyzed by flavin-containing monooxygenase in liver microsomes (37,38); N-oxidizing activity in mice is especially strong compared with rats and humans (23,40). In the present study, we first observed that dG-N2-TAM N-oxide was formed in the mouse liver as expected due to the strong N-oxidizing activity in mice. Since the adduct level of dG-N2-TAM N-oxide should not be very high, a comparatively high dose of tamoxifen (120 mg/kg) which was reported in mice previously (12) was administered. Recently, it was demonstrated that when
-hydroxytamoxifen N-oxide was administered to F344 rats by gavage or primary cultures of rat hepatocytes, the DNA adduct patterns were very similar to those formed by tamoxifen and
-hydroxytamoxifen (21). Therefore, it was concluded that N-oxygen was lost prior to DNA binding. But, our previous study (22) and present study demonstrated that the in vitro reaction of DNA, dG and dG3'P with
-acetoxytamoxifen N-oxide did not yield dG-N2-TAM, and the dG-N2-TAM N-oxides synthesized and these in the mouse DNA were sufficiently stable during the detection procedures of 32P-post-labeling HPLC. The present mouse data suggested that tamoxifen is able to form N-oxide adducts without losing N-oxygen at least in part.
When a trans-form of tamoxifen was administered to mice, cis-forms of dG-N2-TAM and dG-N2-TAM N-oxide adducts were detected in the liver. This indicates that tamoxifen metabolites such as tamoxifen
-sulfate and tamoxifen N-oxide
-sulfate generate an allylic carbocation that allows rapid transcis conversion (41). Since the level of the cis forms was one-tenth of the trans forms, the transcis conversion in mouse liver may be much less than that observed in in vitro experiments using trans or cis forms of
-acetoxytamoxifen (14) and
-acetoxytamoxifen N-oxide (22).
In conclusion, dG-N2-TAM and dG-N2-TAM N-oxide DNA adducts were detected in mouse liver. This finding suggests that tamoxifenDNA adducts are formed via
-hydroxylation of tamoxifen and its metabolites.
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Notes
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3 To whom correspondence should be addressed Email: umemoto{at}clin.med.tokushima-u.ac.jp 
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Acknowledgments
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This study was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan (no. 10671119) and the National Institute of Environmental Health Sciences grant ES09418.
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Received March 8, 2000;
revised June 12, 2000;
accepted June 16, 2000.