Tamoxifen−DNA Adducts Formed by α-Acetoxytamoxifen N-Oxide

Oct 23, 1999 - development of endometrial cancer in breast cancer patients taking tamoxifen and ... minor adducts were detected in endometrial samples...
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Chem. Res. Toxicol. 1999, 12, 1083-1089

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Tamoxifen-DNA Adducts Formed by r-Acetoxytamoxifen N-Oxide† Atsushi Umemoto,*,‡ Yasumasa Monden,‡ Kansei Komaki,‡ Masato Suwa,§ Yoshikazu Kanno,§ Masanobu Suzuki,§ Chun-Xing Lin,‡ Yuji Ueyama,‡ Md. Abdul Momen,‡ Anisetti Ravindernath,| and Shinya Shibutani| Second Department of Surgery, School of Medicine, University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan, Pharmaceuticals Group, Nippon Kayaku Co., Ltd., 3-31-12, Shimo, Kita-ku, Tokyo 115-8588, Japan, and Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651 Received July 13, 1999

DNA adduct formation is assumed to be a major carcinogenic event, leading to the development of endometrial cancer in breast cancer patients taking tamoxifen and healthy women enrolled in a tamoxifen chemopreventive trial. To determine whether DNA adducts were formed by tamoxifen, trans- and cis-R-acetoxytamoxifen N-oxides were synthesized as model-activated forms via major tamoxifen metabolites, tamoxifen N-oxide and R-hydroxytamoxifen N-oxide. When R-acetoxytamoxifen N-oxide was reacted with human DNA, at least three DNA adducts were detected by 32P-postlabeling coupled with HPLC. The total amount of DNA adducts formed by trans-R-hydroxytamoxifen N-oxide was 1.5-fold higher than that formed by the cis form. Both trans- and cis-R-acetoxytamoxifen N-oxide reacted with 2′-deoxyguanosine, resulting in the formation of three adducts (fr-1, fr-2-1, and fr-2-2). These products were studied using mass spectroscopy and proton magnetic resonance spectroscopy. fr-1 was identified as a mixture of the epimers of trans-R-(N2-deoxyguanosinyl)tamoxifen N-oxide. fr-2-1 and fr-2-2 were determined to be epimers of cis-R-(N2-deoxyguanosinyl)tamoxifen N-oxide.

Introduction Tamoxifen, (Z)-1-{4-[2-(dimethylamino)ethoxy]phenyl}1,2-diphenyl-1-butene, is widely used as first-line endocrine therapy for patients in all stages of breast cancer (1, 2). It is also being tested as a preventive agent against the development of breast cancer for high-risk women (3, 4). Many case reports and recent large-scale randomized clinical trials have shown, however, that patients treated with tamoxifen are at increasing risk for endometrial cancer (5, 6). An increased incidence of endometrial cancer was also observed for healthy women enrolled in a tamoxifen chemopreventive trial (3). Tamoxifen is a potent carcinogen targeting the liver in rats (7, 8), where it produces a large number of different DNA adducts, the majority of which are produced via the R-hydroxylation pathway (9, 10). Sulfation of R-hydroxytamoxifen catalyzed by rat and human hydroxysteroid sulfotransferases is involved in the formation of DNA adducts, i.e., R-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM)1 (11). This is regarded as a † Preliminary reports of this work were presented before the American Association for Cancer Research, March 1999, in Philadelphia, PA. * To whom correspondence should be addressed. Phone: +81-88-633-7143. Fax: +81-88-633-7144. E-mail: umemoto@ clin.med.tokushima-u.ac.jp. ‡ University of Tokushima. § Nippon Kayaku Co., Ltd. | State University of New York at Stony Brook. 1 Abbreviations: dG, 2′-deoxyguauosine; PEI, poly(ethyleneimine); dG-N2-TAM, R-(N2-deoxyguanosinyl)tamoxifen; dG-N2-TAM N-oxide, R-(N2-deoxyguanosinyl)tamoxifen N-oxide; ODS, octadecylsilane; FAB, fast atom bombardment; DMSO, dimethyl sulfoxide.

major DNA adduct in the rat liver (12, 13) and in endometrial tissue obtained from women treated with tamoxifen (14). Since these adducts were highly mutagenic in mammalian cells (15), tamoxifen-DNA adducts may trigger endometrial cancers. In addition to major dG-N2-tamoxifen adducts, several minor adducts were detected in endometrial samples from patients taking tamoxifen (14), an indication that other pathways for DNA adduct formation remain to be clarified. In in vitro and in vivo experimental systems, R-hydroxylation occurs in several tamoxifen metabolites, N-desmethyltamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide, as well as in tamoxifen (16-18). Adducts formed via the 4-hydroxytamoxifen pathway account for only minor DNA adducts in the rat and mouse (9). Recently, an N-desmethyltamoxifen-deoxyguanosine adduct was also suggested to be formed in rats, although this conclusion was based only upon mass spectroscopy; no synthetic standards were available (19). It remains to be clarified whether N-oxide metabolites of tamoxifen are related to DNA adduct formation. Tamoxifen N-oxide and R-hydroxytamoxifen N-oxide, known metabolites in rat and human liver microsomal incubations containing tamoxifen, may be intermediates for the formation of DNA adducts (16, 18, 20). For binding to DNA via the R-position of tamoxifen, O-sulfation or O-acetylation is required for generating reactive carbocation. We prepared R-acetoxytamoxifen N-oxides (trans and cis forms) as model-activated forms, and their binding with DNA and 2′-deoxyguanosine was studied (Figure 1).

10.1021/tx990132+ CCC: $18.00 © 1999 American Chemical Society Published on Web 10/23/1999

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Table 1. Formation of DNA Adducts of r-Acetoxytamoxifen N-Oxides adduct level (adducts/106 nucleotides)

a

R-acetoxytamoxifen N-oxide

peak 1

peak 2

peak 3

total

trans form cis form

5.2 ( 0.4a (14%)b 3.1 ( 0.3 (13%)

20.5 ( 1.3 (55%) 14.1 ( 2.4 (56%)

11.5 ( 0.4 (31%) 7.8 ( 1.4 (31%)

37.2 ( 1.3 (100%) 25.0 ( 4.1 (100%)

Mean ( SD, calculated from duplicate experiments. b Percentage against total adduct level (peak 1 + peak 2 + peak 3).

Figure 1. Structures of R-hydroxytamoxifen N-oxides and R-acetoxytamoxifen N-oxides.

Experimental Procedures Caution: R-Acetoxytamoxifen and R-acetoxytamoxifen Noxide may be genotoxic agents in humans, and should be handled with care. Materials. Tamoxifen, proteinase K, and potato apyrase were purchased from Sigma Chemical Co. (St. Louis, MO). 2′Deoxyguanosine (dG) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). RNase A, RNase T1, micrococcal nuclease, and spleen phosphodiesterase were purchased from Worthington Biochemical Co. (Freehold, NJ). Nuclease P1 was obtained from Yamasa Shoyu Co. (Choshi, Japan) and T4 polynucleotide kinase obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). [γ-32P]ATP (>7000 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). The poly(ethyleneimine)cellulose sheet (Polygram Cell 300 PEI) was purchased from Machery-Nagel (Du¨ren, Germany). Preparation of r-Acetoxytamoxifen N-Oxide. R-Acetoxytamoxifen was synthesized as a precursor compound as reported previously (12) (Figure 1). N-Oxides of trans- and cis-R-acetoxytamoxifen were obtained with hydrogen peroxide oxidation; 30% hydrogen peroxide (0.22 mL) was added dropwise to transR-acetoxytamoxifen (8.3 mg) in methanol (0.5 mL), and the mixture was stirred for 24 h at room temperature. Products were purified by a Waters Sep-Pak C18 cartridge (washed with 40% aqueous methanol and eluted by methanol), yielding trans-Racetoxytamoxifen N-oxide (7.2 mg, 84%); similarly, 30% hydrogen peroxide (2.0 mL) was added to cis-R-acetoxytamoxifen (32 mg) in methanol, yielding cis-R-acetoxytamoxifen N-oxide (33 mg, 99%). FAB mass spectroscopy was conducted using a Micromass Limited Auto Spec Q (Manchester, U.K.). 1H NMR spectroscopy was carried out using a Bruker Avance 400 spectrometer. 1H NMR for the trans form (CDCl3): δ 1.27 (3H, d, J ) 6.6 Hz, CH3), 1.90 (3H, s, CH3CO), 3.27 [6H, s, (CH3)2N(O)], 3.64 (2H, t, J ) 4.4 Hz, NCH2), 4.39 (2H, t, J ) 4.4 Hz, OCH2), 5.75 (1H, q, J ) 6.6 Hz, CH), 6.53 (2H, d, J ) 8.9 Hz, H3, H5 of alkoxyphenyl), 6.81 (2H, d, J ) 8.9 Hz, H2, H6 of alkoxyphenyl), 7.1-7.5 (10H, m, phenyl). 1H NMR for the cis

Figure 2. 32P-Postlabeling and HPLC analysis of DNA reacted with trans- and cis-R-acetoxytamoxifen N-oxide; 5 µg of DNA was reacted with 50 µg of R-acetoxytamoxifen N-oxide at 37 °C for 1 h. The resulting DNA was labeled with 32P, and an aliquot (about 0.14 µg of DNA) was subjected to HPLC using a radioactivity detector. (A) Control DNA incubated without R-acetoxytamoxifen N-oxide. (B) DNA reacted with trans-Racetoxytamoxifen N-oxide. (C) DNA reacted with cis-R-acetoxytamoxifen N-oxide. form (CDCl3): δ 1.29 (3H, d, J ) 6.6 Hz, CH3), 1.93 (3H, s, CH3CO), 3.32 [6H, s, (CH3)2N(O)], 3.65-3.75 (2H, m, NCH2), 4.554.65 (2H, m, OCH2), 5.81 (1H, q, J ) 6.6 Hz, CH), 6.85-7.05 (4H, m, phenyl), 6.92 (2H, d, J ) 8.7 Hz, H3, H5 of alkoxyphenyl), 7.1-7.3 (6H, m, phenyl), 7.22 (2H, d, J ) 8.7 Hz, H2, H6 of alkoxyphenyl). FAB mass spectroscopy of both stereoisomers gave the same ions at m/z 446 ([M + H]+), 430 (446 - O), 386 (446 - AcOH), and 370 (386 - O). trans-R-Acetoxytamoxifen N-oxide was partly hydrolyzed to trans-R-hydroxytamoxifen N-oxides during purification by Sep-Pak C18 cartridge under aqueous conditions. Such degradation was not observed in cisR-acetoxytamoxifen N-oxide. Reaction of r-Acetoxytamoxifen N-Oxide with DNA. Human placental DNA (5 µg) was incubated with 50 µg of transor cis-R-acetoxytamoxifen N-oxide in 100 µL of 100 mM TrisHCl buffer (pH 8.0) at 37 °C for 1 h. The reaction was terminated by adding 800 µL of ethanol, and the sample was held at -80 °C for 30 min. Precipitated DNA was washed twice with 500 µL of cold ethanol and evaporated to dryness. 32P-Postlabeling of DNA Adducts. The resulting DNA was digested to deoxynucleoside 3′-monophosphates with micrococcal nuclease (3 units) and spleen phosphodiesterase (0.03 unit) at 37 °C for 3.5 h. The adduct was enriched with nuclease P1 (2.4

DNA Adducts from R-Acetoxytamoxifen N-Oxide

Figure 3. HPLC of dG-N2-TAM N-oxides formed by R-acetoxytamoxifen N-oxides; 5 mg of trans-R-acetoxytamoxifen N-oxide was reacted with 25 mg of dG and 3 mg of cis-R-acetoxytamoxifen N-oxide with 15 mg of dG for 24 h at 50 °C. The reaction mixture (5 µL) was subjected to HPLC. A linear gradient of 10 to 80% acetonitrile containing 0.05 M triethylammonium acetate (pH 7.0) was eluted over the course of 35 min at a rate of 1.0 mL/min. (A) dG-N2-TAM N-oxides from trans-R-acetoxytamoxifen N-oxide and (B) dG-N2-TAM N-oxides from cis-R-acetoxytamoxifen N-oxide.

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Figure 5. Positive ion FAB mass spectra of dG-N2-TAM N-oxides: (A) fr-1 (dG-trans-N2-TAM N-oxides) and (B) fr-2-1 (dG-cis-N2-TAM N-oxide).

Figure 6. 1H NMR spectra of dG-N2-TAM N-oxides: (A) fr-1 (mixture of dG-trans-N2-TAM N-oxide epimers) and (B and C) fr-2-1 and fr-2-2 (dG-cis-N2-TAM N-oxide epimers).

dG-N2-TAM

Figure 4. Preparative separation of N-oxides. dG (150 mg) was reacted with 33 mg of trans-R-acetoxytamoxifen N-oxide at 50 °C for 24 h. (A) an aliquot of the reaction mixture was subjected to HPLC. A linear gradient of 10 to 80% acetonitrile containing 0.05 M triethylammonium acetate (pH 7.0) was eluted over the course of 140 min at a rate of 10 mL/ min. (B) The fr-2 was rechromatographed on the same column with water/methanol (40:60) at a rate of 8 mL/min. units) at 37 °C for 1 h (21, 22). The digest was then converted to 5′-32P-labeled deoxynucleoside 3′,5′-bisphosphates by T4 polynucleotide kinase-catalyzed transfer of 32P from [γ-32P]ATP at 37 °C for 1 h. The labeled mixture was further treated with apyrase at 37 °C for 45 min. 32P-labeled nucleoside bisphosphate adducts were purified by development on PEI-cellulose sheets with 2.3 M sodium phosphate (pH 6.0) at 22 °C for 15 h. Adducts remaining at the origin were cut out and extracted with 3.5 mL of 4 M pyridinium formate (pH 4.5). The extract was filtrated with a Millipore Millex-HA filter (0.45 µm). No radioactivity was lost in filtration. HPLC of Labeled DNA Adducts. The filtrate (10 µL) was analyzed by HPLC (Hewlett-Packard 1090 liquid chromatograph) on an ODS column [Prodigy ODS(2), 5 µm, 2.0 mm × 150 mm, Phenomenex, Torrance, CA]. Solvent A was 0.2 M ammonium formate and 20 mM phosphoric acid (pH 4.2); solvent B was methanol. The elution was carried out at a rate of 0.2 mL/min using the following linear gradient: from 25 to 65% B from 0 to 40 min, from 65 to 100% B from 40 to 55 min, from 100% B from 55 to 65 min, and from 100 to 25% B from 65 to 70 min. To detect eluate radioactivity on-line, a Beckman 171 radioisotope detector with a Teflon sample loop (cell volume,

200 µL) was used with a mixed scintillation cocktail, Atomlight (Packard) at a rate of 0.1 mL/min. Reaction of r-Acetoxytamoxifen N-Oxides with dG. dG (25 mg) was added to 5 mg of trans-R-acetoxytamoxifen N-oxide in 1 mL of acetonitrile, 2.5 mL of 0.1 M Tris-HCl buffer (pH 8.0) added, and the mixture stirred for 24 h at 50 °C. dG (15 mg) was added to 3 mg of cis-R-acetoxytamoxifen N-oxide in 0.6 mL of acetonitrile, 1.5 mL of 0.1 M Tris-HCl buffer (pH 8.0) added, and the mixture stirred for 24 h at 50 °C. The reaction mixture (5 µL) was subjected to HPLC (Shimadzu LC-6A, Kyoto, Japan) on an ODS column (Diachroma ODS M-15, 5 µm, 4.6 mm × 250 mm; Mitsubishi Kakoki Kaisha Ltd., Kawasaki, Japan) at 35 °C. For the preparative experiment, 150 mg of dG was added to 33 mg of trans-R-acetoxytamoxifen N-oxide in 6 mL of acetonitrile, 15 mL of 0.1 M Tris-HCl buffer (pH 8.0) added, and the mixture stirred for 24 h at 50 °C. Each of the reaction mixtures (5 mL) was subjected to HPLC (Shimadzu LC-10A, Kyoto, Japan) on an Inertsil ODS column (30 mm × 250 mm; GL Sciences Inc., Tokyo, Japan), and the adduct fractions were collected.

Results Detection of Tamoxifen-DNA Adducts. At least three adducts (peaks 1-3) were detected at retention times of 32.4, 33.6, and 42.3 min, respectively, when DNA was reacted with trans- or cis-R-acetoxytamoxifen Noxide (Figure 2B,C). The total amount of DNA adduct formation of the trans form was about 1.5-fold more than that of the cis form. The ratio of these adducts (peaks 1-3) produced by trans-R-acetoxytamoxifen N-oxide was

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Table 2. 1H NMR Spectrum of dG-N2-Tamoxifen N-Oxide Adducts

δ (ppm)a,b fr-1 tamoxifen fragment hινδ;1gβ N(O)Me2 hινδ;1gβ′ hινδ;1gR′ hινδ;1gR H3 and H5 H2 and H6 phenyl guanine fragment N2H H8 N1 sugar fragment 2′ 5′ 4′ 3′ 1′

fr-2-1

fr-2-2

1.27, d, J ) 7.0 Hz 3.00, s ndc 4.27-4.35, m 4.85-4.93, m 4.78-4.85, m (minor epimer) 6.60, d, J ) 8.8 Hz 6.81, d, J ) 8.8 Hz 7.0-7.6, m

1.26, d, J ) 6.9 Hz 3.1-3.2, m ndc 4.2-4.25, m 5.1-5.2, m

1.26, d, J ) 6.9 Hz 3.1-3.2, m ndc 4.25-4.3, m 5.18-5.24, m

7.16, d, J ) 8.7 Hz 7.32, d, J ) 8.7 Hz 6.85-7.35, m

7.14, d, J ) 8.7 Hz 7.30, d, J ) 8.7 Hz 6.85-7.35, m

6.03, d, J ) 7.7 Hz 5.99, d, J ∼ 8.0 Hz (minor epimer) 7.99, s 8.00, s (minor epimer) 10.0-10.3, br

5.90, d, J ) 9.0 Hz

5.94, d, J ) 8.8 Hz

7.95, s

7.97, s

10.3-10.5, br

10.3-10.5, br

ndd ndc 3.85-3.95, m 4.35-4.4, m 6.20, dd, J ) 6.8 and 7.4 Hz

ndd ndc 3.9-3.95, m 4.4-4.5, m 6.11, dd, J ) 5.9 and 8.1 Hz

ndd ndc 3.8-3.9, m 4.4-4.5, m 6.05, dd, J ) 6.6 and 7.6 Hz

a Recorded in DMSO-d . Chemical shifts were referenced to tetramethylsilane (TMS). b Abbreviations: s, singlet; d, doublet; dd, doublet 6 of doublets; m, multiplet; br, broad. c nd, not detected, obscured by water resonance. d nd, not detected, obscured by DMSO resonance.

Scheme 1. Proposed Activation Pathway for Formation of Tamoxifen-DNA Adducts

13:56:31, and that produced by cis-R-acetoxytamoxifen N-oxide exhibited a similar ratio of 14:55:31 (Table 1). In contrast, control DNA incubated in the buffer exhibited no peak in radioactivity detection (Figure 2A). These labeled adducts were stable in 4 M pyridinium formate

(pH 4.5) even after storage for 7 days at room temperature. Separation of dG-Tamoxifen Adducts. When transor cis-R-acetoxytamoxifen N-oxide was reacted with dG, two products, fr-1 (tR ) 17.3 min) and fr-2 (tR ) 19.6 min),

DNA Adducts from R-Acetoxytamoxifen N-Oxide

were obtained at a ratio of ca. 2:1 estimated by UV detection at 254 nm (Figure 3A,B). fr-1 and fr-2 were later shown to be trans and cis dG-tamoxifen N-oxide adducts. Two minor peaks were observed at 18.3 and 20.5 min, trans and cis forms of the adducts of R-acetoxytamoxifen N-oxide and tris(hydroxymethyl)aminomethane (Tris) suggested by MS and NMR. In preparative HPLC separation of trans-R-acetoxytamoxifen N-oxide-dG reaction products, fr-1 (tR ) 78 min) and fr-2 (tR ) 87 min) were obtained in accordance with the analytical HPLC (Figure 4A). Two Tris adduct peaks (trans at 81 min and cis at 89 min) and one unknown peak (at 90 min) were also detected. When each fraction was rechromatographed by the same column but using isocratic elution, only fr-2 could be separated into two peaks: tR ) 42 min for fr-2-1 and tR ) 43 min for fr-2-2 (Figure 4B). Since the separation of fr-2-1 and fr-2-2 was insufficient, only the anterior part of the fr-2-1 peak and the posterior part of the fr-2-2 peak were collected. These products (fr-1, fr-2-1, and fr-2-2) were applied to structural analysis by mass spectroscopy and nuclear magnetic resonance spectroscopy. Characterization of dG-N2-TAM N-Oxide. Positive ion FAB mass spectroscopy of all reaction products of R-acetoxytamoxifen N-oxide and dG (fr-1, fr-2-1, and fr2-2) yielded the same expected ions at m/z 653, 637, 537, and 370 (Figure 5A,B). The molecular ion at m/z 653 showed a protonated R-(N2-deoxyguanosinyl)tamoxifen N-oxide (dG-N2-TAM N-oxide). This molecular ion yielded daughter ions at m/z 637 (653 - O), 537 (653 - deoxyribosyl), and 370 (tamoxifen - 1). In the separate experiment, exact FAB mass spectroscopy showed that the molecular weights of fr-1, fr-2-1, and fr-2-2 were 653.3088, 653.3089, and 653.3087, respectively; the calculated molecular weight for C36H41N6O6 (M + H+) was 653.3088. The 1H NMR spectrum was recorded in DMSO-d6. Signals were assigned on the basis of a comparison with 1H NMR data of deoxyguanosine and trans- and cis-Racetoxytamoxifen N-oxides. Chemical shifts for fr-1, fr2-1, and fr-2-2 are listed in Table 2. In fr-1, the two doublets at δ 6.60 and 6.81 corresponding to protons H3 and H5, and H2 and H6, of alkoxyphenyl indicated trans isomer (Figure 6A). Epimers were also present in fr-1 for two H8 singlets, two N2H doublets, and two R multiplets. In contrast, two doublets of alkoxyphenyl in fr-2-1 and fr-2-2 shifted downfield (Figure 6B,C), confirming that compounds have a cis form. The doublet of N2H at δ 5.99 and 6.03 for fr-1, δ 5.90 for fr-2-1, and δ 5.94 for fr-2-2 was coupled to the R-position at δ 4.78-4.85 and 4.854.93, δ 5.1-5.2, and δ 5.18-5.24, respectively, and the shift in signals for proton H8 of guanine indicates that the tamoxifen moiety is linked to the N2 position of guanine. Thus, fr-1 was identified as the mixture of epimers of dG-trans-N2-TAM N-oxide. fr-2-1 and fr-2-2 were identified as epimers of dG-cis-N2-TAM N-oxide. Interestingly, the signal of N-methyl protons of dG-cisN2-TAM N-oxide was observed as a multiplet, due to the influence of the interaction between the tamoxifen moiety side chain and the sugar moiety.

Discussion Tamoxifen is metabolically activated by a microsomal fraction and binds to DNA in cultured cells and tissues of animals. N-Oxide metabolites of tamoxifen, tamoxifen N-oxide, R-hydroxytamoxifen N-oxide, and 4-hydroxy-

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tamoxifen N-oxide, were detected in an incubation mixture of tamoxifen with liver microsomes from species including humans and in blood and tissue from patients and rats treated with tamoxifen (16, 18, 20, 23-26). Formation of the N-oxides of tamoxifen is catalyzed by flavin-containing monooxygenase (23, 24). Since N-oxide products are major tamoxifen metabolites, the tamoxifen N-oxide-DNA adduct is assumed to be formed in vivo. In our preliminary study, tamoxifen N-oxide R-sulfate was synthesized as a model-activated form by N-oxidation of R-hydroxytamoxifen, followed by O-sulfation. The resulting derivative was too unstable, however, to be hydrolyzed to R-hydroxytamoxifen N-oxide. Another modelactivated form, R-acetoxytamoxifen N-oxide, was synthesized by O-acetylation of R-hydroxytamoxifen, followed by N-oxidation. R-Acetoxytamoxifen N-oxide readily reacted with DNA or dG. The DNA reactivity of R-acetoxytamoxifen N-oxide was similar to that of R-acetoxytamoxifen or tamoxifen R-sulfate (data not shown). It is not known whether tamoxifen N-oxidation precedes R-hydroxylation or vice versa. Comparing the deuterium isotope effects of R-hydroxytamoxifen and R-hydroxytamoxifen N-oxide (18) indicated that tamoxifen N-oxide rather than R-hydroxytamoxifen is an immediate precursor of R-hydroxytamoxifen N-oxide (Scheme 1). For further bioactivation of R-hydroxytamoxifen Noxide, O-acetylation is not an ordinary pathway for carcinogen-DNA binding, while O-sulfation of R-hydroxytamoxifen catalyzed by rat and/or human hydroxysteroid sulfotransferase is involved in the formation of DNA adducts (27-29). O-Sulfation rather than O-acetylation of R-hydroxytamoxifen N-oxide is thus likely to play an important role in DNA binding.

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Interestingly, when DNA reacted with trans and cis forms of R-acetoxytamoxifen N-oxide, three peaks having the same retention time were detected; the ratios of the peaks were similar. In a later study, we confirmed that peaks 1 and 2 correspond to epimers of trans-R-(N2deoxyguanosinyl)tamoxifen N-oxide 3′-monophosphate (dG3′P-trans-N2-TAM N-oxide), and peak 3 corresponds to a mixture of epimers of dG3′P-cis-N2-TAM N-oxide.2 As reported previously for tamoxifen R-sulfate (30), transor cis-R-acetoxytamoxifen N-oxide generates allylic carbocation that reacts with DNA. DNA adducts were formed via the trans-cis convertible intermediate (Scheme 2). Conversion to the trans form appears to be preferable to conversion to the cis form in equilibrium between the trans and cis forms, as observed for tamoxifen R-sulfate (11). We also found that R-acetoxytamoxifen N-oxides were reactive to dG, and formed two trans and two cis isomers of dG-N2-TAM N-oxides. These isomers were sufficiently stable to allow structural analysis by MS and NMR. We reported that dG-N2-TAM DNA adducts are detected using 32P-postlabeling and HPLC analysis in leucocytes and endometrial tissues of patients taking tamoxifen (14, 31, 32). The retention time of other adducts was consistent with that of DNA adducts observed when DNA reacted with R-acetoxytamoxifen Noxide. dG-N2-TAM N-oxide DNA adducts thus also exist in human tissue and may contribute to the initiation of endometrial cancer.

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Acknowledgment. This study was supported by Grant-in-Aid 10671119 from the Ministry of Education, Science, and Culture of Japan and National Institute of Environmental Health Sciences Grant ES09418.

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References

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(1) Jordan, V. C. (1994) The development of tamoxifen for breast cancer therapy. In Long-Term Tamoxifen Treatment for Breast Cancer (Jordan, V. C., Ed.) pp 3-26, University of Wisconsin Press, Madison, WI. (2) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans (1996) Some Pharmaceutical Drugs, Vol. 66, International Agency for Research on Cancer, Lyon, France. (3) Fisher, B., Constantino, J. P., Wickerham, D. L., Redmond, C. K., Kavanah, M., Cronin, W. M., Vogel, V., Robidoux, A., Dimitrov, N., Atkins, J., Daly, M., Wieand, S., Tan-Chiu, E., Ford, L., and Wolmark, N. (1998) Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl. Cancer Inst. 90, 1371-1388. (4) Powles, T., Eeles, R., Ashley, S., Easton, D., Chang, J., Dowsett, M., Tidy, A., Viggers, J., and Davey J. (1998) Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomized chemoprevention trial. Lancet 352, 98101. (5) Fisher, 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. (6) Rutqvist, L. E., Johansson, H., Signomklao, T., Johansson, U., Fornander, T., and Willing, N., (1995) Stockholm Breast Cancer Study Group Adjuvant tamoxifen therapy for early stage breast cancer and second primary malignancies. J. Natl. Cancer Inst. 87, 645-651. (7) Greaves, P., Goonetilleke, R., Nunn, G., Topham, J., and Orton, T. (1993) Two-year carcinogenecity study of tamoxifen in Alderley Park Wister-derived rats. Cancer Res. 53, 3919-3924. (8) Hard, G. C., Iatropoulos, M. J., Jordan, K., Radi, L., Kaltenberg, O. P., Imondi, A. R., and Williams, G. M. (1993) Major difference in the hepatocarcinogenecity and DNA adduct forming ability

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(22)

(23)

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(27)

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A. Umemoto et al., unpublished results.

between toremifene and tamoxifen in female Crl:CD(BR) rats. Cancer Res. 53, 4534-4541. Martin, E. A., Heydon, R. T., Brown, K., Brown, J. E., Lim, C. K., White, I. N., and Smith, L. L. (1998) Evaluation of tamoxifen and R-hydroxytamoxifen 32P-post-labelled DNA adducts by the development of a novel automated on-line solid-phase extraction HPLC method. Carcinogenesis 19, 1061-1069. Phillips, D. H., Carmichael, P. L., Hewer, A., Cole, K. J., and Poon, G. K. (1994) R-Hydroxytamoxifen, a metabolite of tamoxifen with exceptionally high DNA-binding activity in rat hepatocytes. Cancer Res. 54, 5518-5522. Dasaradhi, L., and Shibutani, S. (1997) Identification of tamoxifen-DNA adducts formed by R-sulfate tamoxifen and R-acetoxytamoxifen. Chem. Res. Toxicol. 10, 189-196. 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, 66-71. Phillips, D. H., Potter, G. A., Horton, M. N., Hewer, A., CroftonSleigh, C., Jarman, M., and Venitt, S. (1994) Reduced genotoxicity of [D5-ethyl]-tamoxifen implicates R-hydroxylation of the ethyl group as a major pathway of tamoxifen activation to a liver carcinogen. Carcinogenesis 15, 1487-1492. Shibutani, S., Suzuki, N., Terashima, I., Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (1999) Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol. 12, 646-653. Terashima, I., Suzuki, N., and Shibutani, S. (1999) Mutagenic potential of R-(N2-deoxyguanosinyl)tamoxifen lesions, the major DNA adducts detected in endometrial tissues of patients treated with tamoxifen. Cancer Res. 59, 2091-2095. Poon, G. K., Walter, B., Lønning, 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. Lien, E. A., Solheim, E., and Ueland, P. M. (1991) Distribution of tamoxifen and its metabolites in rat and human tissues during steady-state treatment. Cancer Res. 51, 4837-4844. 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 R-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D5ethyl]tamoxifen. Carcinogenesis 16, 683-688. Rajaniemi, H., Rasanen, I., Koivisto, P., Peltonen, K., and Hemminki, K. (1999) Identification of the major tamoxifen-DNA adducts in rats liver by mass spectroscopy. Carcinogenesis 20, 305-309. Lim, C. K., Yuan, Z. X., Lamb, J. H., White, I. N., De Matteis, F., and Smith, L. L. (1994) A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes. Carcinogenesis 15, 589-593. Reddy, M. V., and Randerath, K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551. Umemoto, A., Kajikawa, A., Tanaka, M., Hamada, K., Seraj, M. J., Kubota, A., Nakamura, M., Kinouchi, T., Ohnishi, Y., Yamashita, K., and Monden, Y. P (1994) Presence of mucosa-specific DNA adduct in human colon: possible implication for colorectal cancer. Carcinogenesis 15, 901-905. Mani, C., and Kupfer, D. (1991) Cytochrome P-450-mediated activation and irreversible binding of the antioestrogen tamoxifen to proteins in rats and human liver: possible involvement of flavin-containing mono-oxygenases in tamoxifen activation. Cancer Res. 51, 6052-6058. Mani, C., Hodgson, E., and Kupfer, D. (1993) Metabolism of the antimammary cancer antiestrogenic agent tamoxifen. II. Flavincontaining monooxygenase-mediated N-oxidation. Drug Metab. Dispos. 21, 657-661. McCague, R., and Seago, A. (1986) Aspects of metabolism of tamoxifen by rat liver microsomes. Identification of a new metabolite: E-1-[4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenyl-1-buten-3-ol N-oxide. Biochem. Pharmacol. 35, 827-834. Potter, G. A., McCague, R., and Jarman, M. (1994) A mechanistic hypothesis for DNA adduct formation by tamoxifen following hepatic oxidative metabolism. Carcinogenesis 15, 439-442. Shibutani, S., Dasaradhi, L., Terashima, I., Banoglu, E., and Duffel, M. W. (1998) R-Hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts. Cancer Res. 58, 647-653. Shibutani, S., Shaw, P. M., Suzuki, N., Dasaradhi, L., Duffel, M. W., and Terashima, I. (1998) Sulfation of R-hydroxytamoxifen

DNA Adducts from R-Acetoxytamoxifen N-Oxide catalyzed by human hydroxysteroid sulfotransferase results in tamoxifen-DNA adducts. Carcinogenesis 19, 2007-2011. (29) Davis, W., Venitt, S., and Phillips, D. H. (1998) The metabolic activation of tamoxifen and R-hydroxytamoxifen to DNA-binding species in rat hepatocytes proceeds via sulfation. Carcinogenesis 19, 861-866. (30) Sanchez, C., Shibutani, S., Dasaradhi, L., Bolton, J. L., Fan, P. W., and McClelland, R. A. (1998) Lifetime and reactivity of an ultimate tamoxifen carcinogen: The tamoxifen carbocation. J. Am. Chem. Soc. 120, 13513-13514.

Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1089 (31) Umemoto, A., Monden, Y., Komaki, K., Suwa, M., Kanno, Y., Lin, C., Mimura, S., and Shibutani, S. (1999) Novel tamoxifen-DNA adduct found in leucocytes of breast cancer patients. Proc. Am. Assoc. Cancer Res. 40, 193. (32) Pearl, M. L., Suzuki, N., Terashima, I., Sugarman, S. M., Grollman, A. P., and Shibutani, S. (1999) DNA adducts detected in the endometrial tissues of women treated with tamoxifen. Proc. Am. Soc. Clin. Oncol. 18, 359.

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