Chem. Res. Toxicol. 2006, 19, 421-425
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Absence of DNA Adduct in the Leukocytes from Breast Cancer Patients Treated with Toremifene Atsushi Umemoto,*,† Chun-Xing Lin,† Yuji Ueyama,† Kansei Komaki,† Y. R. Santosh Laxmi,‡ and Shinya Shibutani‡ Department of Oncological and RegeneratiVe Surgery, Institute of Health Biosciences, The UniVersity of Tokushima Graduate School, Tokushima 770-8503, 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 NoVember 2, 2005
Tamoxifen (TAM) causes cancer in rat liver and human endometrium, whereas the carcinogenicity of its chlorinated analogue toremifene (TOR) has not been observed. To elucidate the genotoxicity of TOR, the capability of forming DNA adducts by TOR was examined in the leukocytes of patients treated with TOR. Leukocytes were collected from 27 breast cancer patients (57.7 ( 11.4 years old) taking TOR (40 mg/day for 25 patients, 80 mg/day for one patient, and 120 mg/day for one patient; average duration, ∼12 months) and 20 untreated breast cancer patients (58.2 ( 12.3 years old). The DNA extracted was analyzed by 32P-postlabeling/high-performance liquid chromatography. No DNA adducts were detected in the leukocytes of either TOR-treated or nontreated patients. Our results contrast to the previous observation detecting TAM-DNA adducts in several patients treated with TAM, indicating that TOR is less genotoxic to humans. Introduction (TAM)1
Tamoxifen has been widely used as a first-line endocrine therapy for patients with advanced and recurrent breast cancer (1, 2) and as a preventive agent for healthy women at a high risk of developing of this disease (3, 4). Despite its high validity on the breast cancer therapy, various adverse effects including a development of endometrial cancer (2, 3, 5) have been recognized. TAM induced a potent hepatocarcinoma in the rat liver (6, 7); however, the uterus, in which the estrogen receptor is highly expressed, is not a target organ (8). TAM forms DNA adducts abundantly in the rat liver but not in the uterus (9). R-(N2Deoxyguanosinyl)tamoxifen (dG-N2-TAM) and R-(N2-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N2-N-desTAM) were the major hepatic DNA adducts in rodents treated with TAM (10-12), indicating that the formation of TAM-DNA adducts may be responsible for developing hepatocarcinoma. Toremifene (TOR; the structure shown in Figure 1), a chlorinated analogue of TAM, has shown a similar clinical efficacy to TAM on breast cancer therapy (13). Although TOR has similar estrogenic/antiestrogenic properties to TAM (1416), this drug promoted none or only a trace of DNA adducts in the rat liver (17-19), indicating that TOR is less genotoxic in the rat. In humans, TAM-induced endometrial cancer has different histological and molecular biological characteristics from ordinary endometrial cancer. TAM users were more likely to * To whom correspondence should be addressed. Tel: +81-88-633-7143. Fax: +81-88-633-7144. E-mail:
[email protected]. † University of Tokushima. ‡ State University of New York at Stony Brook. 1 Abbreviations: dG , 2′-deoxyguauosine 3′-monophosphate; PEI, 3′P polyethyleneimine; PBS, phosphate-buffered saline; TAM, tamoxifen; TOR, toremifene; R-OHTAM, R-hydroxytamoxifen; R-OHTOR, R-hydroxytoremifene; dG-N2-TAM, R-(N2-deoxyguanosinyl)tamoxifen; dG-N2-NdesTAM, R-(N2-deoxyguanosinyl)-N-desmethyltamoxifen; HPLC, highperformance liquid chromatography; tR, retention time.
Figure 1. Structures of TAM and TOR.
have malignant mixed mesodermal tumors or endometrial sarcomas and immunohistochemically p53-positive and estrogen receptor-negative staining (20). Endometrial stage III and IV cancers occurred more frequently in TAM users than in nonusers; therefore, the 3 year survival for TAM users was much worse than the nonusers (20). The difference between TAMinduced and sporadic endometrial cancers suggests that TAMinduced cancer may be developed through the TAM-specific genotoxic events. A high frequency of mutations was detected at codon 12 of the K-ras gene in the endometrium of patients treated with TAM but not TOR (21); the mutational spectrum was consistent with that observed in rodents treated with TAM (22, 23) and site-specific mutagenesis studies (24, 25). Therefore, the K-ras mutation was expected due to the genotoxic effect of TAM, not through the estrogenic potential of TAM on estrogen receptor (26). TAM-DNA adducts were removed relatively slowly from rat liver (18, 27-29). If TAM-DNA adducts are not readily repaired (30), mutations may occur at adduct sites and initiate the development of cancer. TAM-DNA adducts have been detected in endometrial tissues of breast cancer patients treated with TAM, using 32Ppostlabeling analysis (31, 32) and accelerator mass spectrometry (33), while other research groups were unable to detect TAMDNA adducts in the endometrial tissues (34-36) and lymphocytes of patients treated with TAM (37, 38). We have recently detected TAM-DNA adducts in the leukocytes from breast cancer patients receiving TAM by a sensitive 32P-postlabeling/ high-performance liquid chromatography (HPLC) analysis (39)
10.1021/tx0503045 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/09/2006
422 Chem. Res. Toxicol., Vol. 19, No. 3, 2006
Umemoto et al.
and pointed out that the lower sensitivity of methods used by the groups (37, 38) failed to detect TAM-DNA adducts. In the present study, leukocyte DNA obtained from patients treated with TOR was analyzed using our sensitive 32P-postlabeling/ HPLC. We have found that TOR did not promote DNA adducts, indicating that this drug is a safer antiestrogen than TAM.
Table 1. Information of Patients with Breast Cancer TOR-Treated
Materials and Methods Chemicals. Proteinase K, potato apyrase, and 2′-deoxyguauosine 3′-monophsphate (dG3′P) 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 Amersham Biosciences Co. (Piscataway, NJ), respectively. [γ-32P]adenosine 5′-triphosphate (7000 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). A polyethyleneimine (PEI)-cellulose sheet (Polygram Cell 300 PEI) was purchased from Machery-Nagel (Du¨ren, Germany). Dextran T70 was purchased from Extrasynthese S. A. (Genay Cedex, France). TOR was a gift from Nippon Kayaku Co., Ltd. Preparation of TOR-Derived DNA Adducts. R-Acetoxy-TOR was synthesized as described previously (40). dG3′P (0.5 mg) or calf thymus DNA (10 µg) was reacted at 37 °C for 1 h with R-acetoxy-TOR (0.5 mg) in 200 µL of 100 mM Tris-HCl buffer (pH 7.5). The reaction sample for dG3′P was extracted twice with 200 µL of butanol. The butanol fractions were combined, backextracted with 50 µL of distilled water, and evaporated to dryness. The DNA was recovered by phenol/chloroform extraction and used for 32P-postlabeling/HPLC analysis. Preparation of Human Leukocyte DNA. Blood samples (2025 mL) were collected from 27 breast cancer patients (57.7 ( 11.4 years old) taking TOR (40 mg/day for 25 patients, 80 mg/day for one, and 120 mg/day for one; average duration, ∼12 months) and 20 untreated breast cancer patients (58.2 ( 12.3 years old) who received neither TAM nor other hormonal therapies (Table 1). This study was approved by the Ethical Committee of School of Medicine, the University of Tokushima. Written informed consent was obtained from all Japanese donors. The heparinized blood was suspended in one volume of 3% of dextran in phosphate-buffered saline (PBS, pH 7.4), and a test tube was allowed to stand for 3 h. The leukocytes in the upper layer were collected, washed three times with PBS, and stored at -80 °C until use. DNA Isolation. The DNA was isolated using our published methods with a slight modification (41). Briefly, human leukocytes samples were thawed and suspended in 0.5 mL of 1.0% SDS/10 mM EDTA/20 mM Tris-HCl (pH 7.4) and homogenized with Polytron. The homogenate was incubated at 37 °C for 30 min with RNase A (200 µg/mL) and RNase T1 (34 units/mL). After the addition of proteinase K (500 µg/mL), the homogenate was further incubated at 37 °C for 30 min. The extractions were performed with 1 volume 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 the addition of 0.1 volume of 5 M NaCl, DNA was precipitated by 1 volume of cooled ethanol. By inverting the tube gently, the DNA lump was moved 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 DNA impurities 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 as 1.0 ODS260 nm ) 50 µg/mL and adjusted to 1 mg/mL. 32P-Postlabeling/HPLC Analysis for the Detection of Leukocyte DNA Adducts. Leukocyte DNA (18 µg) was digested to deoxynucleoside 3′-monophosphates with micrococcal nuclease (0.6 U/µg DNA) and spleen phosphodiesterase (0.006 U/µg DNA) in 0.1 M sodium succinate/0.05 M calcium chloride (pH 6.0) at 37
patient
age (years)
duration (months)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 mean ( SD
43 57 43 67 69 72 69 70 62 41 68 78 49 48 56 59 43 54 44 52 51 58 54 57 81 47 67 57.7 ( 11.4
7 4 8 39 9 8 8 3 3 6 18 18 11 18 18 36 25 20 2 32 2 2 12 8 15 1 1 12.4 ( 10.8
dose (mg/day) 40 80 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 120 40 40 40 40 40 40
other drugs none ADMa none none none none 5-FUb none none CPAc none none none none 5′-DFURd none 5′-DFUR 5′-DFUR none none none none none none none none none
Control patient
age (years)
other drugs
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 mean ( SD
61 44 64 39 81 54 50 54 75 74 74 58 45 59 62 50 39 68 48 64 58.2 ( 12.3
none none none none none 5-FU CPA none UFTe none none none none 5′-DFUR none none none none CMFf 5′-DFUR
a ADM, adriamycin. b 5-FU, 5-fluorouracil. c CPA, cyclophosphamide. 5′-DFUR, doxifluridine. e UFT, tegafur/uracil. f Cyclophosphamide/methotrexate/5-fluorouracil.
d
°C for 3.5 h. The adducts were enriched with nuclease P1 (1.2 µg/ µg DNA) in 0.25 M sodium acetate/1 mM zinc chloride (pH 4.8) at 37 °C for 1 h (42). After incubation, 100 µL of water was added. The reaction samples were then extracted twice with 200 µL of butanol. The butanol fractions were combined, back-extracted with 50 µL of distilled water, and evaporated to dryness. The digests were then incubated at 37 °C for 1 h with ∼170 µCi of [γ-32P]ATP and 10 units of T4 polynucleotide kinase (pH 9.5). For the R-acetoxy-TOR-derived dG3′P adducts, 32P-postlabeling reactions were performed directly with T4 polynucleotide kinase treatment. The reaction mixture was further incubated with apyrase at 37 °C for 45 min. 32P-Labeled nucleoside bisphosphate adducts were partially purified by development on PEI-cellulose sheets with 2.3 or 1.7 M sodium phosphate buffer (pH 6.0) at 22 °C for ∼15 h.
No Toremifene-DNA Adducts in Human Leukocytes
Chem. Res. Toxicol., Vol. 19, No. 3, 2006 423
Figure 2. 32P-postlabeling/HPLC analysis of DNA adduct in the leukocytes from breast cancer patients treated with TOR. HPLC on-lined a radioisotope detector performed with a Develosil ODS-UG-5 column (5 µm, 150 mm × 2.0 mm) and the following solvents: (A) 2 M ammonium formate/20 mM phosphoric acid (pH 4.0) and (B) acetonitrile/methanol (6:1, v/v). The elution was carried out by the linear gradient: 0-40 min from 15 to 20% B; 40-80 min from 20 to 30% B; 80-85 min from 30 to 50% B at a flow rate of 0.2 mL/min. (A) dG3′p reacted with R-acetoxyTOR; (B) calf thymus DNA reacted with R-acetoxy-TOR; (C) TOR-treated patient #4 (18 µg of DNA); (D) TOR-treated patient #17 (18 µg of DNA); (E) control patient #C14 (18 µg of DNA); and (F) control patient #C15 (18 µg of DNA).
The adducts that were retained at the origin were cut out and extracted with 1 mL of 4 M pyrimidinium formate (pH 4.5) three times. The extract was filtrated by Millipore Millex-HA filter (0.45 µm) and evaporated to dryness. No loss of radioactivity occurred in the filtration step. The dried extract was dissolved in 20 µL of 4 M pyrimidinium formate, and an aliquot (18 µL) was subjected to HPLC (Hewlet Packard 1090 Liquid Chromatograph) equipped with a Develosil ODS-UG-5 column (5 µm, 150 mm × 2.0 mm) using the following solvents: (A) 2 M ammonium formate/20 mM phosphoric acid (pH 4.0) and (B) acetonitrile/methanol (6:1, v/v). The elution was carried out by the linear gradient: 0-40 min from 15 to 20% B; 40-80 min from 20 to 30% B; 80-85 min from 30 to 50% B at a flow rate of 0.2 mL/min. For on-line radioactivity monitoring of the eluate, a Beckman 171 Radioisotope Detector with a Teflon sample loop (cell volume, 200 µL) was used with mixing scintillation
cocktail, Atomlight (Packard), at a flow rate of 0.1 mL/min. All analyses were performed twice or more. The level of DNA adducts was estimated using the calibration data from the ratio of the peak area to radioactivity of [γ-32P]ATP (43). The detection limits were approximately 0.8 adducts/109 (S/N ) 3) for 18 µg of DNA sample.
Results When dG3′P was reacted directly with R-acetoxy-TOR and analyzed using 32P-postlabeling/HPLC, many adducts (peaks a-n in Figure 2A) including minor products were detected. Parts of the adducts (peaks c-e, g, h in Figure 2B) were observed in calf thymus DNA reacted with R-acetoxy-TOR at the level of 13.2-32.1 adducts/108 nucleotides, indicating that the dG residue in DNA is a target base modified by R-acetoxy-TOR.
424 Chem. Res. Toxicol., Vol. 19, No. 3, 2006
Leukocyte DNA obtained from 27 breast cancer patients (57.7 ( 11.4 years old) treated with TOR (Table 1) was analyzed using 32P-postlabeling/HPLC analysis. Twenty-five patients were treated with TOR (40 mg/day). The other two patients received 80 and 120 mg/day, respectively, due to their body weight. The average duration of TOR treatment was approximately 12 months. DNA adducts were not detected in any of the leukocyte DNA samples from TOR-treated patients (for example, see Figure 2C,D) with the detection limits, approximately 0.8 adducts/109 nucleotides for 18 µg of DNA sample. No DNA adducts were also observed in the 20 control patients (58.2 ( 12.3 years old) (Figure 2E,F).
Discussion TAM-DNA adducts were detected in six out of 47 leukocytes of breast cancer patients treated with TAM (20 mg/day) for an average period of 37 months (39). The major TAMDNA adducts were identified chromatographically as trans isomers of dG3′P-N2-TAM and dG3′P-N2-N-desTAM at the level of 0.9-8.6 adducts/109 nucleotides. Therefore, DNA adducts in the leukocytes may be a good surrogate marker of evaluating the risk of TAM for humans. In contrast, no DNA adducts were detected in the 27 leukocytes from breast cancer patients treated with TOR. Although the average duration of TOR (12 months) was shorter than that of the study with TAM (39), the doses used for 25 TOR-treated patients (40 mg/day) were at least two times higher than those for the TAM-treated patients; the other two patients were treated with TOR 80 and 120 mg/day, respectively. Therefore, the lack of detecting TOR-derived DNA adducts in the leukocytes may not be due to the short period of TOR duration. The presence of TAM-DNA adducts was observed only when sufficient amounts of leukocyte DNA samples (18-45 µg) were used; however, TAM-DNA adducts were not detected when 5 µg of leukocyte DNA sample was used (39). Using only 4 µg of lymphocyte DNA sample, Bartsch et al. did not detect TAM-DNA adducts in 25 patients treated with TAM (38). The use of such small amounts of DNA samples may decrease the detection limit, resulting in the failure of detecting TAM-DNA adducts. Therefore, the assay system used by this group was not sufficient for analysis of DNA adducts in the lymphocyte obtained from TOR-treated patients. TAM-DNA adducts are formed through R-hydroxylation of TAM (44, 45) and its metabolites followed to the O-sulfonation (46, 47). Similar metabolic activation could be conducted on TOR, a chlorinated analogue of TAM. In fact, the formation of R-hydroxytoremifene (R-OHTOR) by rat CYP 3A2 or human CYP 3A4 was higher than that of R-hydroxytamoxifen (ROHTAM) (48). However, the formation of DNA adducts from R-OHTOR in the reactions catalyzed by rat or human hydroxysteroid sulfotransferase (HST) (49, 50) was 2 orders of magnitude lower than that of R-OHTAM, indicating that R-OHTOR was a poor substrate for HST (40). In addition, the reactivity of R-acetoxy-TOR to DNA in vitro was much lower, as compared with that R-acetoxyTAM (40). The steric hindrance caused by a bulky chlorine atom positioned at the ethyl moiety of TOR may reduce the ability of HST to sulfonate the R-OHTOR and the reactivity of R-acetoxyTOR with DNA (40). The field effect caused by the electron-withdrawing chlorine atom may also diminish the effective elimination of the R-acetoxyl group from the R-carbon and therefore inhibit the formation of the carbocation intermediate that reacts with DNA (40). The lower genotoxicity of TOR may be attributable to the limited formation of DNA adducts induced by TOR.
Umemoto et al.
Supporting this mechanism, TOR promoted none or only a trace of DNA adducts in the rat liver (17-19) and was noncarcinogenic in rat (17, 18). TAM-DNA adducts have strong miscoding and mutagenic potential (24, 25) and resist the repair enzymes (30), indicating that TAM-DNA adducts may pose a potential risk of development of cancer in women treated with TAM. In fact, a high frequency of K-ras mutations was observed in the endometrium of patients treated with TAM but not in patients treated with TOR (21). Therefore, TOR is likely to be less genotoxic to the human endometrium than TAM. Clinical efficacy of TOR for breast cancer patients has been known to be similar to that of TAM (13). To avoid the secondary cancers caused by TAM, TOR may be a safer alternative for breast cancer therapy and chemoprevention. Acknowledgment. We thank Drs. Masato Suwa and Takashi Kawashiro for helpful discussions. This study was supported by the Japan Society for the Promotion of Science (10671119) and the National Institute of Environmental Health Sciences Grant ES09418.
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(34) (35)
(36)
(37) (38)
(39)
(40)
(41)
(42) (43) (44) (45)
(46)
(47) (48)
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