Formation of Tamoxifen−DNA Adducts in Human Endometrial

An increased risk of developing endometrial cancer has been observed in women receiving tamoxifen (TAM) endocrine therapy and chemoprevention...
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Chem. Res. Toxicol. 2005, 18, 889-895

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Formation of Tamoxifen-DNA Adducts in Human Endometrial Explants Exposed to r-Hydroxytamoxifen Sung Yeon Kim,† Naomi Suzuki,† Y. R. Santosh Laxmi,† Barbara P. McGarrigle,‡ James R. Olson,‡ Moheswar Sharma,§ Minoti Sharma,§ and Shinya Shibutani*,† Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214, and Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263 Received January 27, 2005

An increased risk of developing endometrial cancer has been observed in women receiving tamoxifen (TAM) endocrine therapy and chemoprevention. The genotoxic damage induced by TAM metabolites may be involved in the development of endometrial cancer. To investigate the capability of endometrial tissues to form TAM-DNA adducts, primary cultured human endometrial explants were exposed to R-hydroxytamoxifen (R-OHTAM) and used for quantitative analysis of TAM-DNA adducts, using 32P-postlabeling/HPLC analysis. A trans isoform of R-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM) was detected as the major adduct in eight of nine endometrial explants exposed to 100 µM R-OHTAM at levels of 7.7 ( 5.3 (mean ( SD) adducts/107 nucleotides. Approximately 25- and 37-fold lower amounts of the cis form of dGN2-TAM and another trans isoform were also detected. The dG-N2-TAM adduct (3.3 adducts/ 107 nucleotides) was detected in one of three endometrial explants exposed to 25 µM R-OHTAM. No TAM-DNA adducts were detected in any unexposed tissues. These results indicate that TAM-DNA adducts are capable of forming through O-sulfonation and/or O-acetylation of R-OHTAM in the endometrium. The endometrial explant culture can be used as a model system to explore the genotoxic mechanism of antiestrogens for humans.

Introduction (TAM)1

The antiestrogen tamoxifen (structure in Figure 1) is widely used as an adjuvant chemotherapeutic agent for breast cancer patients and as a chemopreventive agent for healthy women at high risk for this disease (1-3). However, the increased risk of developing endometrial cancer was observed in women receiving longterm TAM treatment (4-9). TAM has been listed as a human carcinogen by the International Agency for Research on Cancer (10). The cellular mechanism underlying TAM-induced carcinogensis has not been defined (11-13). The carcinogenic effects may be due to the estrogenic activity of TAM through the estrogen receptor; TAM acts as a partial estrogen agonist and induces signal transduction, resulting in promotion of cellular proliferation (14, 15). Alternatively, TAM is a potent hepatocarcinogen in rats (16, 17) and TAM-DNA adducts have been identified in the rat liver (18-20). The genotoxic mechanism of TAM has recently been reviewed (21). TAM is metabolized by phase I enzymes to several reactive species including R-hydroxytamoxifen (R-OH* To whom correspondence should be addressed. Tel: 631-444-8018. Fax: 631-444-3218. E-mail: [email protected]. † State University of New York at Stony Brook. ‡ State University of New York at Buffalo. § Roswell Park Cancer Institute. 1 Abbreviations: dN, 2′-deoxynucleoside; dG, 2′-deoxyguanosine; dGP, 2′-deoxyguanosine 3′-monophosphate; TAM, tamoxifen; N-desTAM, N-desmethyltamoxifen; TAM N-oxide, tamoxifen N-oxide; 4-OHTAM, 4-hydroxytamoxifen; R-OHTAM, R-hydroxytamoxifen; dGN2-TAM, R-(N2-deoxyguanosinyl)tamoxifen; dG-N2-N-desTAM, R-(N2deoxyguanosinyl)-N-desmethyltamoxifen.

TAM), N-desmethyltamoxifen (N-desTAM), tamoxifen N-oxide (TAM N-oxide), and 4-hydroxytamoxifen (4OHTAM) (Figure 1). R-Hydroxylation of TAM metabolites, followed by O-sulfonation and/or O-acetylation, constitutes a major pathway capable of forming DNA adducts (22-24). R-OHTAM is O-sulfonated by rat and human hydroxysteroid sulfotransferase (25, 26) and reacts with the exocyclic amino group of guanine in DNA forming two trans (fr-1 and fr-2) and two cis (fr-3 and fr-4) diastereoisomers of R-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM) (Figure 1) (18, 24, 27). dG-N2-TAM and R-(N2-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N2N-desTAM) were observed as major hepatic DNA adducts in rodents exposed to TAM using mass spectroscopic and 32 P-postlabeling/HPLC analyses (28-30). dG-N2-TAM adducts display a high miscoding and mutagenic potential in mammalian cells (31, 32). A high frequency of mutations has been observed in the liver DNA of λ/lac I transgenic rats treated with TAM (33). High frequent mutations were also detected at codon 12 of the K-ras gene in the endometrium of patients treated with TAM but not TOR (34); the mutational spectrum was consistent with that observed in our mutagenesis study (32). Because TAM has an estrogenic potential similar to TOR (35, 36), the higher mutational frequency observed in the K-ras gene in the TAM-treated patients was expected due to the genotoxic effect of TAM. TAM-DNA adducts have been observed to be removed relatively slowly from rat liver (37-39). If TAM-DNA adducts are not readily repaired (40), mutations may occur at adducted sites and initiate the development of cancer.

10.1021/tx050019l CCC: $30.25 © 2005 American Chemical Society Published on Web 04/16/2005

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Figure 1. Formation of TAM-DNA adducts via R-hydroxylation of TAM metabolites.

There is a controversy regarding the detection of TAM-DNA adducts in the human tissues (41). TAMDNA adducts have been detected in endometrial tissues of breast cancer patients treated with TAM, using 32Ppostlabeling analysis (42-44) and accelerator mass spectrometry (45). However, other research groups have been unable to detect TAM-DNA adducts in the endometrial tissues (46-48) and lymphocytes of TAM-treated women (49, 50). Umemoto et al. (51) have recently detected a low level of TAM-DNA adducts in leukocytes from breast cancer patients receiving TAM by a sensitive 32P-postlabeling/HPLC analysis and pointed out the lower sen-

sitivity of methods used by the groups (49, 50) who could not detect adducts. TAM-DNA adducts were also detected in the uterus, ovary, liver, and brain cortex from cynomolgus monkey treated with TAM, using a newly developed 32P-postlabeling/HPLC method (52) as well as by a chemiluminescence immunoassay and liquid chromatography tandem mass spectroscopy (53). The present study was designed to elucidate whether TAM-DNA adducts can be produced from R-OHTAM in primary cultured human endometrium. We found, using 32P-postlabeling/HPLC analysis, that significant amounts of TAM-DNA adducts were formed in the tissues.

Tamoxifen-DNA Adducts in Endometrial Explants

Materials and Methods Chemicals. [γ-32P]ATP (specific activity, 6000 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL). Polyethylenimine-cellulose plates were purchased from MacheryNagel (Duren, Germany). TAM, proteinase K, and potato apyrase were purchased from Sigma (St. Louis, MO). Nuclease P1 and 3′-phosphatase-free T4 polynucleotide kinase were obtained from Roche Applied Sciences (Indianapolis, IN). RNase A, RNase T1, micrococcal nuclease, and spleen phosphodiesterase were obtained from Worthington Biochemical Co. (Freehold, NJ). Surgical Specimens. Human endometrial tissue specimens, removed at hysterectomy, were procured under IRB approved protocols from the Tissue Procurement Facility at Roswell Park Cancer Institute, with the donor’s consent, but without the patients’ identities. Endometrial tissue (approximately 6.0 mm cube) was obtained from individuals, approximately 35-45 years of age, who had no previous history of TAM exposure. These endometrial tissue samples did not overlap with those collected for the TAM studies (54). Endometrial Explant Culture. The surgical specimens were prepared and cultured under sterile conditions similar to the method described previously (54). The time period between the surgical removal and the explant culture was within 2 h. Each sample of fresh endometrial tissues, microscopically uninvolved in disease, was placed in 3% FBS-DMEM/F-12 medium containing 17β-estradiol (10 nM) and cut into uniform explants with a sterile scalpel blade. The pieces were immediately transferred at a concentration of 8-10 pieces per well (30-67 mg/well) to a 24 well plate (Costar, Cambridge, MA) containing 1 mL medium/well with 25 or 100 µM R-OHTAM or vehicle (0.1% ethanol). The explants were incubated for 24 h at 37 °C in a humidified 5% CO2-air environment. Digestion of DNA Samples. The explant DNA was extracted using a Mannheim-Boehringer DNA isolation kit following the manufacturer’s protocol. Following established protocol in our laboratory (52, 55), 5 or 10 µg of DNA was digested at 37 °C for overnight in 100 µL of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2, using 15 units of micrococcal nuclease and 0.15 units of spleen phosphodiesterase. Subsequently, 1.0 unit of nuclease P1 was added and the reaction mixture was incubated at 37 °C for 1 h. To enrich TAMmodified nucleotides, samples were dissolved in 100 µL of distilled water and extracted twice with 200 µL of butanol. The butanol fraction was back-extracted with 50 µL of distilled water, dried, and then used for analysis of TAM-DNA adducts. Approximately 95% of TAM-DNA adducts were recovered by butanol extraction. 32P-Postlabeling/HPLC Analysis. The DNA digests were incubated at 37 °C for 40 min with 20 µCi of [γ-32P]ATP and 3′-phosphatase-free T4 polynucleotide kinase (20-30 units) and then incubated with apyrase (50 milliunits) for another 30 min, as described previously (52). The labeled samples were developed for 16 h on a 10 cm × 10 cm of polyethylenimine-cellulose thin-layer plates using 2.3 M sodium phosphate buffer, pH 6.0, with a paper wick (52). 32P-labeled products remaining on the TLC plate were recovered, using 4 M pyrimidinium formate, pH 4.3, and evaporated to dryness. Recovery of 32P-labeled products was ∼84%. The 32P-labeled products were subjected to a Hypersil BDS C18 analytical column (0.46 cm × 25 cm, 5 µm, Shandon), eluted at a flow rate of 1.0 mL/min with a linear gradient of 0.2 M ammonium formate and 20 mM H3PO4, pH 4.0, containing 20-30% acetonitrile for 40 min, 30-50% acetonitrile for 5 min, followed by an isocratic condition of 50% acetonitrile for 15 min. The radioactivity was monitored using a radioisotope detector (Berthold LB506 C-1, ICON Scientific Inc.) linked to a Waters 990 HPLC instrument. Known amounts (0.152, 0.0152, 0.00152, or 0.000152 pmol) of dG-N2-TAMmodified oligodeoxynucleotide prepared by a phosphoramidite chemical procedure (56) were mixed with 5 µg of calf thymus DNA (15200 pmol) and served as a standard (1 adduct/105 nucleotides, 1 adduct/106 nucleotides, 1 adduct/107 nucleotides,

Chem. Res. Toxicol., Vol. 18, No. 5, 2005 891 Table 1. Level of TAM-DNA Adducts in Human Endometrial Explants Exposed to r-OHTAMa dG-N2-TAM (adducts/107nucleotides) cis form R-OHTAM specimen (µM) 1 2 3 4 5 6 7 8 9 10

0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 25 0 25 100 0 25 100

trans form fr-1 fr-2 ND 0.22 ( 0.04 ND ND ND 0.29 ( 0.05 ND ND ND ND ND ND ND ND ND ND ND ND 0.11 ( 0.03 ND ND ND

ND 3.28 ( 0.87 ND ND ND 5.83 ( 1.32 ND 6.30 ( 2.13 ND 9.29 ( 2.26 ND 5.97 ( 1.11 ND 5.06 ( 0.55 ND ND ND 3.26 ( 0.32 5.89 ( 0.56 ND ND 20.24 ( 1.83

fr-3 and fr-4 ND 0.39 ( 0.12 ND ND ND ND ND 0.30 ( 0.03 ND 0.43 ( 0.10 ND 0.23 ( 0.05 ND ND ND ND ND ND 0.14 ( 0.05 ND ND ND

total ND 3.89 ND ND ND 6.12 ND 6.60 ND 9.72 6.20 ND 5.06 ND ND ND 3.26 6.14 ND ND 20.24

a Data are expressed as mean values ( SD from two or three analyses.

or 1 adduct/108 nucleotides). As described previously (55), the amount of TAM adducts detected increased linearly depending on the amounts of oligodeoxynucleotide used. The detection limit of this assay was ∼8 adducts/1010 nucleotides for 10 µg of DNA.

Results To investigate the capability of human endometrial explants to form TAM-DNA adducts, endometrial explants were incubated with or without R-OHTAM and the DNA was used for 32P-postlabeling analysis. Human endometrial tissues were obtained from 35 to 45 year old women who had no history of TAM exposure, cut into several uniform blocks, and incubated for 24 h to 25 or 100 µM R-OHTAM and without R-OHTAM. Samples were coded during the analysis, and only after analysis was the R-OHTAM status revealed. Using 32P-postlabeling/HPLC, endometrial explant DNA samples were analyzed (Table 1). TAM-DNA adducts were detected in one out of three endometrial explants exposed to 25 µM R-OHTAM (Figure 2B) and in eight out of nine endometrial explants exposed to 100 µM R-OHTAM (Figure 2C,E). No DNA adducts were detected in any of the control samples (Figure 2A). Using this method, standards of fr-1 and fr-2, each trans isoforms of dG3′P-N2-TAM, dG3′P-N2-N-desTAM, and a mixture of their cis forms (fr-3 and fr-4) can be resolved (Figure 2D). The retention time of the major TAM-DNA adduct observed in explants exposed to 100 µM R-OHTAM was identical to fr-2 of the trans form of dG3′P-N2-TAM. As compared with known amounts of standard (1 adduct/ 105 nucleotides and 1 adduct/106 nucleotides), the level of dG3′P-N2-TAM adduct was 7.73 ( 5.31 adducts/107 nucleotides, ranging from 3.28 to 20.2 adducts/107 nucleotides (Table 1). The same adduct was detected in one of three endometrial explants exposed to 25 µM R-OHTAM at the level of 3.26 adducts/107 nucleotides (Figure 2B). To confirm the identity of the TAM-DNA adduct, the

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Figure 2. 32P-postlabeling/HPLC analysis of TAM-DNA adducts in human endometrial explants. DNA samples (5 µg) from untreated endometrial explants and from explants exposed to 25 or 100 µM R-OHTAM were digested by enzymes, labeled with 32P, and partially purified using a TLC plate and were subjected to HPLC, as described in the Materials and Methods. The radioactivity was monitored by a radioisotope detector linked to the HPLC instrument. (A) Untreated specimen 9; (B) specimen 9 exposed to 25 µM R-OHTAM; (C) specimen 3 exposed to 100 µM R-OHTAM; (D) standards containing stereoisomeric trans and cis forms of 32P-labeled dG-N2-TAM and dG-N2-N-desTAM; (E) specimen 9 exposed to 100 µM R-OHTAM; (F) cochromatography of standards (D) and specimen 9 exposed to 100 µM R-OHTAM (E). The level of TAM-DNA adducts in specimens 3 and 9 was shown in Table 1.

DNA sample from specimen 9 (Figure 2E) was coinjected with the 32P-labeled authentic standard (Figure 2D). The major TAM-DNA adduct was coeluted with a trans form (fr-2) of dG3′P-N2-TAM (Figure 2F). Among the nine endometrial explants exposed to 100 µM R-OHTAM, another trans form (fr-1) of dG3′P-N2-TAM adduct was detected as a minor adduct in three samples (specimens 1, 3, and 9); the level of adduct was 0.21 ( 0.09 adducts/107 nucleotides (0.11-0.29 adducts/107 nu-

cleotides). The cis form of dG3′P-N2-TAM adduct was also detected in five of the nine samples (0.30 ( 0.12 adducts/ 107 nucleotides). The total amount of TAM-DNA adducts was 7.99 ( 5.20 adducts/107 nucleotides.

Discussion When primary cultured human endometrial tissues were exposed to R-OHTAM, TAM-DNA adducts were

Tamoxifen-DNA Adducts in Endometrial Explants

apparently formed. The total amounts of TAM-DNA adducts were 3.26 and 7.99 adducts/107 nucleotides in the endometrial explants exposed to 25 and 100 µM R-OHTAM, respectively. The major TAM-DNA adduct was a trans form (fr-2) of dG3′P-N2-TAM, and the minor adducts were another trans isoform (fr-1) and cis form(s). This is direct evidence that TAM-DNA adducts are capable of being produced from R-OHTAM in human endometrial tissues supporting the previous observation that TAM-DNA adducts were detected in endometrium of women treated with TAM (43, 44). The level of each TAM and its metabolites in plasma of patients treated with TAM was 0.1-5.0 µM (57). The concentration of TAM and its metabolites in human tissues is approximately 10-60-fold higher than in serum (58). R-OHTAM is a minor metabolite, accounting for ∼0.1% of the administered TAM dose (59, 60). Because of the limited time (24 h) of culturing the primary endometrial explant, our present study may not reproduce the result found in the endometrium of patients with long-term exposure to low levels of R-OHTAM. Although the endometrial explant was exposed with a relatively higher level of R-OHTAM for a short time, the capability of forming TAM-DNA adducts was observed. This indicates that the endometrial explant system is a useful tool to investigate the genotoxic mechanism of antiestrogens. The level of TAM-DNA adducts varied significantly among individual endometrial explants, ranging from 0 to 20.2 adducts/107 nucleotides. A significant variation has been observed on not only the TAM metabolites in the culture media of human endometrial explants (54) but also the TAM-DNA adduct level in the endometrial samples from women treated with TAM (44). This variation may be due to the differences in the activity and expression of enzyme(s) involved in activating R-OHTAM and/or detoxifying R-OHTAM (61). The mechanism of forming TAM-DNA adducts in human endometrial tissues has not been extensively explored. In in vitro studies, R-OHTAM is O-sulfonated by rat and human hydroxysteroid sulfotransferases (HST) (25, 26) and reacts with the exocyclic amino group of guanine in DNA, forming two trans and two cis diastereoisomers of dG-N2-TAM (Figure 1) (18, 24, 27). This result was supported by the evidence that the formation of TAM-DNA adducts was increased in rat hepatocytes incubated with inorganic sulfate and was decreased when rat hepatocytes were treated with dehydroisoandrosterone-3-sulfate, an inhibitor of HST (62). The formation of TAM-DNA adducts was also inhibited by adding a HST antibody (63). On the other hand, TAM-DNA adducts were not formed from R-OHTAM when acetyl-CoA was used as a cofactor in reactions catalyzed by rat and human liver cytosol (63), indicating that the formation of TAM-DNA adducts occurs primarily through O-sulfonation, not O-acetylation, of ROHTAM. Phenol and estrogen sulfotransferases were detected in the endometrial tissues of women unexposed to TAM while no expression of the hHST was detected (64). This suggests that hHST may be readily induced by TAM treatment, as observed in rats (65). Unknown enzymes may also be involved in the activation of R-OHTAM in human endometrial tissues. Detailed analysis of enzymes expression, including sulfotransferases, in the human endometruim of women exposed to TAM

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is required to identify the enzyme involved in forming TAM-DNA adducts. Human endometrial tissue is capable of biotransforming TAM in the explant culture model (66). The formation of R-OHTAM, in addition to 4-OHTAM and N-desTAM, was observed in culture media as well as whole tissue lysates (54, 66). CYP3A4 is involved in the conversion of TAM to R-OHTAM (67-69) and was detected in viable endometrial explants using immunohistochemical analyses (66). The formation of dG-N2-TAM-DNA adducts was observed in the cultured human endometrial explants exposed to TAM using HPLC/fluorescence detection (54). However, using the same endometrial explant DNA samples, the presence of the TAM-DNA adduct was not confirmed using mass spectroscopy and 32P-postlabeling analysis (70); in this study, only seven DNA samples from endometrial explants exposed to either 10, 25, or 100 µM TAM were used for the analysis. Because the marked interindividual variation was observed, in the present study, in the levels of TAM-DNA adduct in the endometrial explants exposed to R-OHTAM, significant numbers of TAM-exposed DNA samples should be required to resolve the discrepancy. R-OHTAM has been detected in the plasma of women treated with TAM (71). In addition to the formation of R-OHTAM in endometrial tissues, R-OHTAM produced in other tissues may be transferred to the endometrium through the blood circulation and activated in the endometrial tissue, resulting in the formation of TAMDNA adducts. In conclusion, the present study showed direct evidence that the TAM-DNA adduct is capable of forming from R-OHTAM in human endometrial tissue. The endometrial explant culture can be used as an experimental system to explore the genotoxic mechanism of antiestrogens including TAM for humans.

Acknowledgment. This research was supported by Grants ES09418 (to S.S.) from the National Institute of Environmental Health Sciences and CA 86875 (to M.S.) from the National Cancer Institute. We gratefully acknowledge the Tissue Procurement Facility of Roswell Park Cancer Institute for providing the human endometrium specimens.

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