Identification and Quantification of Tamoxifen-DNA Adducts in the

A new HPLC gradient system was developed for 32P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated...
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Chem. Res. Toxicol. 2001, 14, 1006-1013

Identification and Quantification of Tamoxifen-DNA Adducts in the Liver of Rats and Mice Atsushi Umemoto,*,† Kansei Komaki,† Yasumasa Monden,† Masato Suwa,‡ Yoshikazu Kanno,‡ Masayuki Kitagawa,‡ 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 Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651 Received January 17, 2001

A new HPLC gradient system was developed for 32P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of R-(N 2-deoxyguanosinyl)tamoxifen (dG3′P-N 2-TAM), R-(N 2-deoxyguanosinyl)N-desmethyltamoxifen (dG3′P-N 2-N-desmethyl-TAM), and R-(N 2-deoxyguanosinyl)tamoxifen N-oxide (dG3′P-N 2-TAM N-oxide) were prepared by reacting either R-acetoxytamoxifen, R-acetoxy-N-desmethyltamoxifen or R-acetoxytamoxifen N-oxide with 2′-deoxyguanosine 3′monophosphate, and used as standard markers for 32P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG3′P-N 2-TAM, dG3′P-N 2-N-desmethylTAM and dG3′P-N 2-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N 2-TAM (fr-2) and trans-dG3′P-N 2-Ndesmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N 2-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG3′P-N 2-TAM, dG3′P-N 2-N-desmethyl-TAM and dG3′P-N 2-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N 2TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/108 nucleotides) were 3.8-fold higher than mice (56.2 adducts/108 nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from R-hydroxylation of tamoxifen.

Introduction (Z)-1-{4-[2-(Dimethylamino)ethoxy]phenyl}-1,2-diphenyl-1-butene (tamoxifen) is a widely used anticancer drug for patients with breast cancer, However, long-term use of tamoxifen causes endometrial cancer in the treated patients (1, 2), as well as healthy women enrolled in chemopreventive trials (3). Although many anticancer drugs are often mutagenic and carcinogenic due to their DNA adduct formations (4), the treatment period of tamoxifen for the patients is exceptionally very long (3-5 years). Therefore, careful evaluation of the chronic use of this drug is required. Tamoxifen causes liver cancer in rats (5, 6), but not in mice or humans. This may be * 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. † Second Department of Surgery. ‡ Pharmaceuticals Group. § Laboratory of Chemical Biology.

based on a species-specific difference in bioactivation and detoxification activities of tamoxifen in organs (7). Tamoxifen-DNA adducts were detected in rats at high levels in the liver, but only at a trace level in the uterus (8). This suggests that tamoxifen liver cancer may be induced via DNA adduct formation. The investigation of DNA modifications induced by tamoxifen in animal models may help to clarify the mechanism of tamoxifen carcinogenesis in humans. To identify DNA adducts in animals treated with tamoxifen, the syntheses of plausible DNA adducts are necessary, because it is very difficult to analyze directly the minimal amount of DNA adducts from animal tissue by mass spectrometry and NMR spectroscopy. In our previous study, two types of tamoxifen-DNA adducts, R-(N 2deoxyguanosinyl)tamoxifen 3′-monophosphate (dG3′P-N 2TAM)1 and R-(N 2-deoxyguanosinyl)tamoxifen N-oxide 3′monophosphate (dG3′P-N 2-TAM N-oxide) were prepared as the authentic standards. It was demonstrated that in

10.1021/tx010012d CCC: $20.00 © 2001 American Chemical Society Published on Web 07/13/2001

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Figure 1. Structures of the tamoxifen-dG3′P adduct standards used in the present study.

mouse liver the epimers of trans-dG3′P-N 2-TAM and trans-dG3′P-N 2-TAM N-oxide were present as the first and the third major adducts, respectively (9). However, the second major adduct remains to be identified. In the rat liver, Rajaniemi et al. (10) suggested that N-desmethyltamoxifen-deoxyguanosine adduct was another major adduct, although this conclusion was based upon MS alone. Brown et al. (11) showed that following i.p. dosing with N-desmethyltamoxifen, the major hepatic DNA adduct coeluted with one of the main peaks seen following treatment of rats with tamoxifen. They suggested that tamoxifen can be metabolically converted to N-desmethyltamoxifen prior to activation. Recently, a trans-form of R-(deoxyguanosin-N 2-yl)-N-desmethyltamoxifen (dG-N 2-N-desmethyl-TAM) was demonstrated to be one of the major adducts in rat liver (12, 13). More recently, we characterized all four stereoisomers of the 1 Abbreviations: dG, 2′-deoxyguanosine; dG 3′P , 2′-deoxyguanosine 3′-monophosphate; dG-N 2-N-desmethyl-TAM, R-(N 2-deoxyguanosinyl)N-desmethyltamoxifen; dG3′P-N 2-N-desmethyl-TAM, R-(N 2-deoxyguanosinyl)-N-desmethyltamoxifen 3′-monophosphate; dG3′P-N 2-TAM, R-(N 2deoxyguanosinyl)tamoxifen 3′-monophosphate; dG-N 2-TAM N-oxide, R-(N 2-deoxyguanosinyl)tamoxifen N-oxide; dG3′P-N 2-TAM N-oxide, R-(N 2-deoxyguanosinyl)tamoxifen N-oxide 3′-monophosphate; fr, fraction; DMSO, dimethyl sulfoxide; LC, liquid chromatography; MS, mass spectrometry.

trans and cis forms of dG-N 2-N-desmethyl-TAM by MS and NMR which were generated in the reaction of R-acetoxy-N-desmethyltamoxifen with 2′-deoxyguanosine (dG) (14). In addition, N-desmethyltamoxifen adducts were detected in calf thymus DNA reacted with Racetoxy-N-desmethyltamoxifen by 32P-postlabeling/HPLC using dG 3′-monophosphate-N 2-N-desmethyltamoxifen adducts as standards. In the present study, we developed a new HPLC condition that enabled separation of the stereoisomers of three types of tamoxifen-deoxynucleotides for identification and quantification of the DNA adducts in rat and mouse livers.

Materials and Methods Caution. R-Acetoxytamoxifen, R-acetoxy-N-desmethyltamoxifen, and R-acetoxytamoxifen N-oxide may be genotoxic in humans and should be handled with care. Chemicals. Tamoxifen citrate, proteinase K, potato apyrase, 2′-deoxyguanosine 3′-monophosphate (dG3′P), and carboxymethyl cellulose were purchased from Sigma Chemical Co. (St. Louis, MO). 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 and T4 polynucleotide kinase were obtained from Yamasa Shoyu Co. (Choshi,

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Japan) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. [γ-32P]Adenosine 5′-triphosphate (>7000 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). Polyethyleneimine-cellulose sheet (POLYGRAM CELL 300 PEI) was purchased from Machery-Nagel (Du¨ren, Germany). Preparation of Tamoxifen-dG3′P Standards. dG3′P-N 2TAM, R-(N 2-deoxyguanosinyl)-N-desmethyltamoxifen 3′-monophosphate (dG3′P-N 2-N-desmethyl-TAM) and dG3′P-N 2-TAM N-oxide were prepared by reacting dG3′P with R-acetoxytamoxifen, R-acetoxy-N-desmethyltamoxifen or R-acetoxytamoxifen N-oxide, as reported previously (9, 14-16) (Figure 1). The trans and cis stereoisomers of each nucleotide were isolated by HPLC. These dG3′P-adducts were characterized by LC/MS and by comparing the dephosphorylated products of these dG3′P adducts with the corresponding dG adduct standards, dG-N 2-TAM, dGN 2-N-desmethyl-TAM and dG-N 2-TAM N-oxide. Animal Treatment. Female DBA/2 mice and female Fischer 344 rats (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 bedding was changed twice per week. They were kept under constant 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 MF (Oriental Yeast, Co., Ltd., Tokyo, Japan) and tap water ad libitum. Tamoxifen citrate was administered daily to female DBA/2 mice (6 week-old) and female F344 rats (6 week-old) by intragastric instillation at doses of 120 and 45 mg/kg, respectively, for 7 days. The vehicle was 0.5% (w/v) carboxymethyl cellulose sodium salt in water. 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 Isolation. DNA was isolated using the following the method reported previously (17). Briefly, liver samples (0.25 g) were thawed and suspended in 3.0 mL of 1.0% SDS/10 mM EDTA/20 mM Tris-HCl (pH 7.4) and homogenized with Polytron (Kinematica CH-6010, Switzerland). The homogenate was incubated at 37 °C for 30 min with RNase A (200 µg/mL) and RNase T1 (34 units/mL). After addition of proteinase K (500 µg/mL), the homogenate was further incubated at 37 °C for 30 min. Extractions were performed with 1 vol. each of phenol [saturated with 0.1 M Tris-HCl (pH 8.0)], a 1:1 mixture of phenol/sevag (chloroform/isoamyl alcohol, 24:1) and sevag successively. After addition of 0.1 vol of 5 M NaCl, DNA was precipitated by 1 vol of cooled ethanol. By inverting the tube gently, the DNA lump was fished out and washed twice with 70% cold ethanol to remove the salt. The DNA was dissolved in 1 mL of 0.01 × SSC/1 mM EDTA (1 × SSC ) 0.15 M NaCl/ 0.015 M sodium citrate). To remove the impurities of the DNA samples further, the DNA solution was again treated with RNases followed by proteinase K and the above organic solvent extractions were repeated. The concentration of the DNA was estimated spectrophotometrically (OD260 1.0 ) 50 µg/mL) and adjusted to 1 mg/mL. 32P-Postlabeling of Hepatic DNA and Synthetic Tamoxifen-dG3′P Adducts. Each DNA sample (10 µg) was digested to deoxynucleoside 3′-monophosphate with micrococcal nuclease and spleen phosphodiesterase at 37 °C for 3.5 h. The adducts were enriched with nuclease P1 at 37 °C for 1 h (18). The digest was then converted to 5′-32P-labeled deoxynucleoside 3′,5′bisphosphate by T4 polynucleotide kinase-catalyzed transfer of [32P]phosphate from [γ-32P]ATP (∼100 µCi) at 37 °C for 1 h. For the synthetic tamoxifen-dG3′P standards, 32P-postlabeling reactions were directly started after treatment with T4 polynucleotide kinase. After labeling, the mixture was further treated with apyrase at 37 °C for 45 min. The 32P-labeled nucleoside bisphosphate adducts were purified by development on PEIcellulose sheets with 1.7 M sodium phosphate (pH 6.0) at 22 °C for 15 h. The adducts which remained around the origin were cut out (2 cm from the origin to the top) and extracted with 4 M pyrimidium formate (pH 4.5) twice (1.5 mL and 1.0 mL). Nearly 100% of the radioactivity of the standard adducts was recovered

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Figure 2. HPLC separation of 32P-postlabeled tamoxifen-dG3′P adduct standards and liver DNA adducts from rats and mice. (A) Tamoxifen-dG3′P adduct standards; (a) fr-1 of trans-dG3′PN 2-N-desmethyl-TAM, (b) fr-1 of trans-dG3′P-N 2-TAM, (c) fr-2 of trans-dG3′P-N 2-N-desmethyl-TAM, (d) fr-2 of trans-dG3′P-N 2TAM, (e) fr-1 of trans-dG3′P-N 2-TAM N-oxide, (f) fr-2 of transdG3′P-N 2-TAM N-oxide, (g) fr-3 and 4 of cis-dG3′P-N 2-N-desmethylTAM, (h) fr-3 and 4 of cis-dG3′P-N 2-TAM, (i) fr-3 and 4 of cisdG3′P-N 2-TAM N-oxide. (B) Liver DNA (0.8 µg) from a female F344 rat treated with tamoxifen citrate (45 mg/kg) by gavage daily for 7 days. (C) Liver DNA (2.0 µg) from a control rat. (D) Liver DNA (4.0 µg) from a female DBA/2 mouse treated with tamoxifen citrate (120 mg/kg) by gavage daily for 7 days. (E) Liver DNA (2.0 µg) from a control mouse. All peak numbers correspond to those listed in Table 1. by this extraction step. The extract was filtrated by Millipore Millex-HA filter (0.45 µm) and evaporated to dryness under

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Table 1. Tamoxifen-DNA Adducts in Rat and Mouse Liver rat (N ) 4) peak no.

retention time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

29.6 31.6 32.8 34.2 38.0 40.1 41.5 43.5 47.1 51.1 54.4 59.4 61.3 63.0 67.6 70.3 72.6 75.8 76.0

a

adduct level (adducts/108 nucleotides)

structure

trans-dG-N2-N-desmethyl-TAM (fr-1) trans-dG-N2-TAM (fr-1) trans-dG-N2-N-desmethyl-TAM (fr-2) trans-dG-N2-TAM (fr-2) trans-dG-N2-TAM N-oxide (fr-1) trans-dG-N2-TAM N-oxide (fr-2)

(a) (b) (c) (d) (e) (f)

cis-dG-N2-N-desmethyl-TAM cis-dG-N2-TAM

(g) (h)

cis-dG-N2-TAM N-oxide

(i)

0.9 ( 0.1a 3.7 ( 0.6 1.6 ( 0.6 1.7 ( 0.1 7.7 ( 0.4 8.9 ( 1.2 72.2 ( 13.6 104.2 ( 17.0 0.2 ( 0.4 0.6 ( 0.5 ND 2.2 ( 0.2 ND ND 0.9 ( 0.7 4.9 ( 0.5 5.3 ( 0.6 1.0 ( 0.4 ND 215.9 ( 32.9

Mean ( SD. b Not detected. c Percentage to the total adduct level.

reduced pressure. No radioactivity was lost during the filtration step. HPLC Analysis of Labeled DNA Adducts. For detection of tamoxifen-DNA adducts from hepatic DNA samples, the dried extract was dissolved in appropriate amount of 4 M pyridinium formate and analyzed by HPLC (Hewlett-Packard 1090 Liquid Chromatograph) on a Develosil ODS-UG-5 column (5 µm, 150 × 2.0 mm, Nomura Chemical Co., Aichi, Japan). Solvent A was 0.2 M ammonium formate-20 mM phosphoric acid (pH 4.0); solvent B was acetonitrile/methanol (6:1, v/v). The elution was carried out at a flow rate of 0.2 mL/min using the following linear gradient: 0 to 40 min from 15 to 20% B; 40 to 80 min from 20 to 30% B; 80 to 85 min from 30 to 50%. For the on-line radioactivity detection of the eluate, the Beckman 171 radioisotope detector with a Teflon sample loop (cell volume, 200 µL) was used with the mixed scintillation cocktail, Atomlight (Packard) at a flow rate of 0.1 mL/min. To detect minor adducts, various amounts of sample were subjected to HPLC (0.2-4.0 µg DNA). The minimum detection limit for tamoxifenDNA adducts was 1.0 adducts/109 normal nucleotides.

Results Separation of Tamoxifen-dG3′P Standards. When a mixture of 32P-labeled tamoxifen-dG3′P standards was subjected to HPLC, the trans- and cis-isomers were separated as nine peaks (Figure 2A); six peaks represented a paired trans-epimers (fr-1 and fr-2) of dG3′P-N 2N-desmethyl-TAM (a and c), dG3′P-N 2-TAM (b and d), and dG3′P-N 2-TAM N-oxide (e and f), and three peaks represented an unseparated paired cis-epimers (fr-3 and fr-4) of dG3′P-N 2-N-desmethyl-TAM (g), dG3′P-N 2-TAM (h), and dG3′P-N 2-TAM N-oxide (i). In our previous study (9), since the retention time of the fr-2 of dG3′P-N 2-N-desmethylTAM was similar to that of the fr-1 of dG3′P-N 2-TAM, these two nucleotides could not be separated. However, the HPLC gradient system in the present study could resolve the two nucleotides. Therefore, our HPLC system can be used for identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice treated with tamoxifen. Identification of Tamoxifen-DNA Adducts in the Liver of Rats and Mice. When rats were treated with tamoxifen (45 mg/kg/day, p.o.) for 7 days, 15 DNA

d

mouse (N ) 5)

ratioc (%)

positive ratiod

0.4 1.7 0.7 0.8 3.6 4.1 33.4 48.2 0.1 0.3

4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4

1.0

4/4

0.4 2.3 2.5 0.5

4/4 4/4 4/4 4/4

100.0

adduct level (adducts/108 nucleotides) 0.4 ( 0.2 2.6 ( 0.7 NDb 0.3 ( 0.2 1.7 ( 0.9 1.8 ( 0.9 14.8 ( 6.7 27.1 ( 13.6 0.5 ( 0.4 3.6 ( 2.7 0.1 ( 0.1 0.3 ( 0.1 0.1 ( 0.1 0.2 ( 0.1 0.2 ( 0.2 0.7 ( 0.3 1.2 ( 0.5 ND 0.6 ( 0.5 56.2 ( 26.5

ratio (%)

positive ratio

0.7 4.6

5/5 5/5

0.5 3.0 3.2 26.3 48.2 0.9 6.4 0.2 0.5 0.2 0.4 0.4 1.3 2.1

5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

1.1 100.0

5/5

Ratio of positive case to the total.

adducts were detected in the liver (Figure 2B and Table 1). Among them, three peaks corresponding to two transepimers (fr-1, peak 6; fr-2, peak 8) of dG3′P-N 2-TAM and a mixture of the cis-epimers (fr-3 and -4, peak 17) were observed at 40.1, 43.5, and 72.6 min, respectively. The fr-2 of dG3′P-N 2-TAM (peak 8) appeared as a major adduct showing a level of 104 adducts/108 nucleotides. This accounted for 48.2% of the total tamoxifen-DNA adducts. Fr-1 and fr-3 and -4 of dG3′P-N 2-TAM (peak 6) were 4.1 and 2.5% of the total adducts, respectively. Three stereoisomer peaks of dG3′P-N 2-N-desmethyl-TAM were also identified by co-chromatographic studies (Figure 3, panels A-E). The trans-epimer (fr-2) of dG3′P-N 2-N-desmethyl-TAM (peak 7) appeared as the second major adduct showing a level of 72.2 adducts/108 nucleotides that accounted for 33.4% of the total adducts. The level of another trans-epimer (fr-1, peak 5) and the cis epimers (fr-3 and -4, peak 16) accounted for only 3.6 and 2.3% of the total adducts, respectively (Table 1). dG-N 2-TAM N-oxide adducts were not detected when 0.2 µg of DNA was used (Figure 2B). However, when the 10-fold higher amounts of DNA (2.0 µg) were analyzed, two transepimers of the dG-N 2-TAM N-oxide (peaks 9 and 10) were detected at low levels (0.2 and 0.6% of the total adducts, respectively). Seven other minor adducts were also detected in the rat liver (Figure 2B and Table 1), but the amounts of the minor adducts were only 5.5% of the total DNA adducts. No tamoxifen-DNA adducts were detected in nontreated rat liver (Figure 2C). However, when mice were treated with tamoxifen (120 mg/kg/day, p.o.) for 7 days, 17 DNA adducts were detected in the liver (Figure 2D) (Table 1). As observed in rat liver, a trans-form (fr2) of dG3′P-N 2-TAM and a trans-form (fr-2) of dG3′P-N 2N-desmethyl-TAM were detected as major tamoxifenDNA adducts in mice liver. The level of fr-2 of dG3′P-N 2TAM was 27.1 adducts/108 nucleotides that accounted for 48.2% of the total adducts (Table 1). The level of fr-2 of dG3′P-N 2-N-desmethyl-TAM was 14.8 adducts/108 nucleotides that accounts for 26.3% of total adducts. Unlike rats, significant amounts of dG3′P-N 2-TAM N-oxide adducts were detected in the mice liver (Figure 2D). The

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Figure 3. Co-chromatography of rat and mouse liver DNA adducts with authentic standard adducts. (A) DNA adducts of rat liver; (B) co-chromatography of A with fr-1 of trans-dG3′P-N 2-N-desmethyl-TAM (standard peak a); (C) with fr-2 of trans-dG3′P-N 2-Ndesmethyl-TAM (peak c); (D) with fr-3 of cis-dG3′P-N 2-N-desmethyl-TAM (peak g); (E) with fr-4 of cis-dG3′P-N 2-N-desmethyl-TAM (peak g); epimers of cis-dG3′P-N 2-N-desmethyl-TAM could not be separated and formed a single peak g; (F) with fr-1 of trans-dG3′PN 2-TAM (peak b); (G) DNA adducts of mouse liver; (H) co-chromatography of G with fr-1 of trans-dG3′P-N 2-TAM N-oxide (peak e); (I) with fr-2 of trans-dG3′P-N 2-TAM N-oxide (peak f). The alphabetic designation of all standard peaks corresponds to those listed in Table 1.

presence of trans-forms of dG3′P-N 2-TAM N-oxide adducts were confirmed by coeluting 32P-labeled fr-1 (Figure 3H) and fr-2 (Figure 3I) of dG3′P-N 2-TAM N-oxide. The level of two trans-forms (fr-1 and fr-2) of this adduct were 0.5 and 3.6 adducts/108 nucleotides that accounted for 0.9 and 6.4% of the total tamoxifen-DNA adducts. The level of the cis-form was 0.6 adducts/108 nucleotides. The fr-2 of the dG3′P-N 2-TAM N-oxide adducts was the third most abundant adduct in the mouse liver. Except for dG3′PN 2-TAM, dG3′P-N 2-N-desmethyl-TAM, and dG3′P-N 2-TAM N-oxide adducts, eight other minor adducts were detected in the mice liver. Among them, adducts 11, 13, 14, and 19 were mouse-specific (Table 1). The quantity of the minor adducts was only 7.5% of the total DNA adducts. No adducts were observed in the liver of nontamoxifen treated mice (Figure 2E).

Discussion Treatment of tamoxifen induces large numbers of DNA adducts in animals (9, 19). One of the possible reasons

is that several metabolic activation pathways may be involved in the formation of tamoxifen-DNA adducts. Another reason is that stereoisomers of tamoxifen adducts may be generated from reactions of each activated form of tamoxifen with DNA. The former is suggested because several tamoxifen metabolites, such as the R-hydroxy, 4-hydroxy, 3-hydroxy, N-desmethyl, N,Ndidesmethyl, and N-oxide forms, were produced in the tamoxifen-treated patients and rats (20, 21). The first identified tamoxifen-DNA adduct was detected in rat liver as the trans-form of dG-N 2-TAM (22); however, the distinction of the epimers of two trans forms was not determined. Characterization of two trans-epimers and two cis-epimers of dG-N 2-TAM were performed by Dasaradhi and Shibutani. (15). The adduct formed via the 4-hydroxytamoxifen pathway was reported later, but accounted for minor DNA adducts in the rat liver (19, 23, 24). dG-N 2-TAM N-oxide was recently characterized in in vitro experiments in our laboratory (16). One of the trans-dG-N 2-TAM N-oxides (fr-2) was shown to be the

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Figure 4. Proposed metabolic activation pathways of tamoxifen leading to DNA adducts.

Figure 5. Tamoxifen-DNA adducts in rat and mouse livers (mean ( SD).

third most abundant adduct in the liver of tamoxifentreated mice (9). We also synthesized and identified four stereoisomers of dG-N 2-N-desmethyltamoxifen adducts by reacting R-acetoxy-N-desmethyltamoxifen with dG, dG3′P and DNA (14). To identify minor tamoxifen-DNA adducts formed in animals, use of standard markers is required. In the present study, three types of tamoxifendG3′P nucleotides (dG3′P-N 2-TAM, dG3′P-N 2-N-desmethylTAM and dG3′P-N 2-TAM N-oxide) including all four stereoisomers were prepared for this purpose. An HPLC gradient system that can be resolved above synthetic standards was established (Figure 2A). In the present study, many TAM-DNA adduct peaks were detected in HPLC in rats and mice liver. Two major adducts of trans-dG-N 2-TAM (fr-2) and trans-dG3′P-N 2N-desmethyl-TAM (fr-2) were identified as reported previously (9-13, 22). In addition, the trans-isomer (fr1) and cis-isomers (fr-3 and -4) of dG3′P-N 2-TAM, dG3′PN 2-N-desmethyl-TAM, and dG3′P-N 2-TAM N-oxide were detected as minor adducts in both animals except for the cis-dG-N 2-TAM N-oxide in rats. The percentage of dG3′PN 2-TAM, dG3′P-N 2-N-desmethyl-TAM and dG3′P-N 2-TAM

N-oxide adducts were 54.8, 39.3, and 0.4% in rats, and 53.5, 30.6, and 8.4% in mice, respectively. Other undetermined adducts may have resulted from other tamoxifen metabolites such as N,N-didesmethyltamoxifen, 4-hydroxytamoxifen, 4′-hydroxytamoxifen, and 3,4-dihydroxytamoxifen. dG-N 2-TAM N-oxide was the third most abundant adduct in the mouse liver (9), whereas only a trace of this adduct was detected in the rat liver. The N-oxide forms are produced by flavin-containing monooxygenase (25, 26). The activity of this enzyme is presumably higher in mice than rats, because the production of tamoxifen N-oxide, R-hydroxytamoxifen N-oxide and 4-hydroxytamoxifen N-oxide in mouse plasma was greater than in rat plasma (27, 28). Therefore, it may be reasonable that the formation of dG-N 2-TAM N-oxide was much higher in mice than rats. When rat hepatocytes were treated with tamoxifen N-oxide or R-hydroxytamoxifen N-oxide or when rats were treated with tamoxifen N-oxide, the pattern of DNA adduct formation in the hepatocytes and in the liver was similar to those formed by tamoxifen or R-hydroxytamoxifen (29). Therefore, N-oxygen may be

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lost prior to the formation of DNA adducts. If the N-oxide reduction is stronger in rat liver than mouse liver, this may be another reason for the lower production of dGN 2-TAM N-oxide in rat liver. To detect many small tamoxifen-DNA adducts, the tamoxifen doses for rats (45 mg/kg) and mice (120 mg/ kg) were determined using the highest levels reported previously (19, 30). These doses are about 100-300-fold more than the daily dose for breast cancer patients (20 mg/body). Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/108 nucleotides) was 3.8-fold higher than mice (56.2 adducts/108 nucleotides). This suggests that the extent of tamoxifen-DNA adduct formation in rat liver is about 10 times higher than that in mouse liver, and this may be the reason tamoxifen causes liver cancer in rats, but not in mice. This may be based on the different enzyme capabilities involved in activation and/or detoxification of tamoxifen (7, 31). For example, it was shown that the rate of formation of R-hydroxytamoxifen or R-sulfate tamoxifen is faster with liver microsomes of rats than that of mice (32, 33). In addition to the lower formation of tamoxifen-DNA adducts in mice liver, tamoxifen-DNA adducts in mice might be repaired more rapidly than in rats (34). R-Hydroxylated metabolites of N-desmethyltamoxifen and tamoxifen N-oxide as well as tamoxifen were detected in vitro and in vivo (20, 21, 35). dG-N 2-TAM is formed through successive R-hydroxylation of tamoxifen (36, 37), followed by O-sulfation (16, 38-40). Since all tamoxifen-DNA adducts identified in the present study resulted from the binding of the R-carbon of tamoxifen derivatives with the N 2-position of guanine, the same activation mechanism observed for dG-N 2-TAM may be involved in the formation of N-desmethyltamoxifen and tamoxifen N-oxide adducts (Figure 4). The total amount of dG-N 2 adducts (dG-N 2-TAM, dG-N 2-N-desmethylTAM, and dG-N 2-TAM N-oxide) accounted for 94.5 and 92.5% of DNA adducts formed in the livers of rats and mice, respectively (Figure 5).

Acknowledgment. This study was supported by Grants-in-Aids no. 10671119 from the Ministry of Education, Science, Sports and Culture of Japan and the National Institute of Environmental Health Sciences Grant ES09418.

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