Affiliations of authors: Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR (FAB, MIC, DRD); Veterans Affairs Medical Center, Minneapolis, MN (DRP, DMG); Institute of Cancer Research, Surrey, U.K. (AH, DHP); Imperial College London, Biological Chemistry, Faculty of Medicine, London, U.K. (PLC); Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Lisboa, Portugal (GGC, MMM)
Correspondence to: Frederick A. Beland, PhD, Division of Biochemical Toxicology, HFT-110, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079 (e-mail: fbeland{at}nctr.fda.gov)
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ABSTRACT |
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INTRODUCTION |
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Although several investigators have reported the presence of tamoxifenDNA adducts in human endometrium (1316), there has been controversy regarding these data (19). The high-performance liquid chromatography (HPLC) data reported by Hemminki et al. (13), for example, contained numerous peaks that were present in both the tamoxifen-treated and untreated patients. Hence, it may have been fortuitous that one of these peaks co-eluted with the rat liver tamoxifenDNA adduct that was used as a positive control. Furthermore, as noted by Orton and Topham (20), there was poor reproducibility among the chromatograms from tamoxifen-treated samples, and when adducts were assessed by thin-layer chromatography (TLC), the same adduct was detected in tamoxifen-treated and control samples. The endometrial tamoxifenDNA adducts reported by Shibutani et al. (14,15) were identified on the basis of their HPLC retention times, which were similar to those of well-characterized tamoxifenDNA adduct standards; however, a number of the major adducts detected had a Z configuration about the triphenylethylene ring structure. This structural feature implies that, upon tamoxifenDNA adduct formation, isomerization occurred across the tamoxifen double bond. Such an isomerization seems unlikely because it does not take place upon formation of the major tamoxifenDNA adducts detected in tissues from experimental animals (21,22), including uteri from nonhuman primates (23), which retain the original configuration about the triphenylethylene ring structure that is found in tamoxifen. Finally, the data reported by Martin et al. (16) were obtained by using DNA that was not hydrolyzed or chromatographed before it was analyzed by accelerator mass spectrometry, making it unclear whether the [14C] that was measured represented covalently bound tamoxifenDNA adducts or noncovalently bound metabolites of tamoxifen that were intercalated within the DNA.
With the exception of the study by Martin et al. (16), all analyses reported to date of human endometrial DNA from tamoxifen-treated patients have used a 32P-postlabeling method to detect tamoxifenDNA adducts. Although this method is quite sensitive, it does not allow an unequivocal characterization of tamoxifenDNA adducts. We recently developed an assay for quantifying tamoxifenDNA adducts that couples on-line sample preparation with HPLC and electrospray ionizationtandem mass spectrometry [HPLCES-MS/MS; (24)]. By using a deuterated (E)--(deoxyguanosin-N2-yl)-tamoxifen (dG-Tam) adduct as an internal standard and multiple reaction monitoring, we have obtained high chemical specificity for dG-Tam and (E)-
-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-desMeTam), the predominant DNA adducts detected in experimental animals treated with tamoxifen (25,26). In our initial studies, we used this methodology to assess the tamoxifenDNA adduct levels in SpragueDawley rats (24) and cynomolgus monkeys (27) treated with tamoxifen. We have now used this technique to assay endometrial DNA from women who received tamoxifen therapy. For comparison, the same DNA samples were analyzed by 32P-postlabeling assays.
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MATERIALS AND METHODS |
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Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (BisTris), salmon testis DNA, proteinase K, RNase A, RNase T1, DNase I, snake venom phosphodiesterase I, and bacterial alkaline phosphatase were obtained from Sigma Chemical (St. Louis, MO). The following enzymes were used for 32P-postlabeling analyses: spleen phosphodiesterase (Calbiochem-Novabiochem, La Jolla, CA, or Worthington Biochemical, Lakewood, NJ), micrococcal nuclease (Roche Applied Science, Indianapolis, IN), nuclease P1 (Sigma), and T4 polynucleotide kinase (PNK; Epicentre Technologies, Madison, WI). Carrier-free [-32P]ATP was obtained from ICN Biomedicals (Irvine, CA).
DNA Adduct Standards
dG-Tam (24) and dG-desMeTam (26) were prepared as previously described. The deuterated adduct, (E)--(deoxyguanosin-N2-yl)-N,N-bis(trideuteriomethyl)tamoxifen (dG-Tam-d6), which was used as an internal standard in the HPLCES-MS/MS analyses, was synthesized from
-hydroxytamoxifen-d6 and isolated as previously described (24). For quantification purposes, we assumed that the molar extinction coefficients of dG-desMeTam and dG-Tam-d6 were identical to that previously determined for dG-Tam (24). Stock solutions containing known concentrations of dG-Tam, dG-desMeTam, or dG-Tam-d6 in methanol were prepared and stored at 20 °C in vials sealed with teflon septa.
Tissue Samples and DNA Isolation
Written informed consent was obtained from each woman who provided a sample for this study. The study was reviewed and approved by institutional review boards at the National Center for Toxicological Research (U.S. Food and Drug Administration, Jefferson, AR); the Veterans Affairs Medical Center, Minneapolis, MN; the University of Minnesota, Minneapolis; and the Imperial College London, London, U.K.
Endometrial tissue was obtained from five women (mean age = 47 years, range = 3757 years) who had taken tamoxifen (20 mg/day for 660 months) and were undergoing surgical procedures at the Fairview-University Medical Center, University of Minnesota. The tissues were provided by the Tissue Procurement Facility of the University of Minnesota Comprehensive Cancer Center. We also obtained endometrial tissue from five women (mean age = 42 years, range = 3255 years) who had undergone hysterectomies for benign or malignant uterine or ovarian abnormalities at the same institution and had never taken tamoxifen. Nine of these tissue samples were histologically normal; the exception was a sample of malignant endometrium from a tamoxifen-treated woman. We also obtained endometrial tissue samples from three women (mean age = 65 years, range = 5575 years) who had taken tamoxifen (20 mg/day for 1537 months) and were undergoing surgical procedures at St. Helier Hospital, Carshalton, Surrey, U.K., and from three women (mean age = 48 years, range = 4354 years) who had not taken the drug and had undergone hysterectomies at the same institution. The endometrial tissues from the U.K. were macroscopically normal.
We obtained two breast tissue samples that were taken from the contralateral breasts of two breast cancer patients who had undergone bilateral mastectomy at the Fairview-University Medical Center. One breast tissue sample came from a 51-year-old woman who had taken tamoxifen (20 mg/day) for 24 months, and the other came from a 40-year-old woman who had not taken tamoxifen. Both breast tissue samples were histologically normal. All tissue samples were stored at 70 °C until DNA isolation, and all DNA samples were stored at 70 °C until DNA adduct analysis.
The U.S. tissue samples (100200 mg) were frozen in liquid nitrogen, pulverized with a Bessman tissue pulverizer (Fisher Scientific, Pittsburgh, PA), transferred to a glass homogenizer, homogenized in the presence of 1 mL of lysis buffer (500 mM TrisHCl [pH 8.0], 20 mM EDTA, 10 mM NaCl, 1% sodium dodecyl sulfate), incubated with 1.4 mg of proteinase K at 37 °C for 4 hours, and deproteinized by precipitation with saturated NaCl and centrifugation at 15 000g for 30 minutes at 4 °C. The DNA was then precipitated with ethanol, collected by centrifugation at 15 000g for 15 minutes at 4 °C, dissolved in 400 µL of 5 mM BisTris and 0.1 mM EDTA (pH 7.1), incubated at 37 °C for 2 hours with 270 µg of RNase A and 250 U of RNase T1, extracted with organic solvents (28), and precipitated with NaCl and ethanol. The DNA was collected by centrifugation at 20 000g for 10 minutes at 4 °C, dissolved in 100200 µL of 5 mM BisTris and 0.1 mM EDTA (pH 7.1), and quantified by UV spectroscopy. DNA from the U.K. samples was isolated as described in Beland et al. (28).
HPLCES-MS/MS Assay To Detect TamoxifenDNA Adducts
DNA samples (40100 µg) were hydrolyzed to deoxyribonucleosides (29) and analyzed by HPLCES-MS/MS as previously described (24). Briefly, each hydrolyzed DNA sample was loaded onto a reverse-phase trap column [Luna C18(2), 2 mm x 30 mm, 3-µm particle size; Phenomenex, Torrance, CA]. After the trap column was washed, the flow was reversed and the sample was eluted through an analytical column [Luna C18(2), 2 mm x 150 mm, 3-µm particle size; Phenomenex] with a solution of 0.1% formic acid and acetonitrile (73% : 27%) at a flow rate of 200 µL/minute and into a Quattro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, U.K.) equipped with an electrospray interface, with a source block of 120 °C and a desolvation temperature of 400 °C. Nitrogen was used as the desolvation (750 L/hour) and the nebulizing gas; argon was used as the collision gas (collision cell pressure = 1.5 µbar). Adducts were detected and identified by multiple reaction monitoring (dwell time = 0.3 seconds, span = 0.02 d, interchannel delay = 0.03 seconds) and quantified by comparison to the internal standard dG-Tam-d6. Positive ions were acquired for the (M+2H)2+(BH+2H)2+ transitions of dG-Tam (m/z 319
261), dG-desMeTam (m/z 312
254), and the internal standard, dG-Tam-d6 (m/z 322
264). The cone voltage was 15 V, and the collision energy was 9 eV for all three transitions. As positive controls, the following samples were analyzed concurrently: liver (
10 dG-Tam per 108 nucleotides) and uterus (
0.9 dG-Tam per 108 nucleotides) DNA from cynomolgus monkeys treated daily for 30 days with tamoxifen at 2 mg/kg of body weight (27), liver DNA (
500 dG-Tam per 108 nucleotides and
625 dG-desMeTam per 108 nucleotides) from female SpragueDawley rats treated daily for 7 days with tamoxifen at 20 mg/kg of body weight (24), and salmon testis DNA (
560 dG-Tam per 108 nucleotides) reacted in vitro with
-acetoxytamoxifen (24). The limit of detection, based on a signal-to-noise ratio of 3, was approximately two adducts per 109 nucleotides for dG-Tam and two adducts per 108 nucleotides for dG-desMeTam.
32P-Postlabeling Assay To Detect TamoxifenDNA Adducts
Each of the human endometrial and breast DNA samples was also assessed by 32P-postlabeling assays. In addition, a DNA sample from the liver of a male F344 rat that had received tamoxifen at 45 mg/kg of body weight daily by gavage for 14 days (30) was included as a positive control. We used two different protocols to digest and 32P-postlabel each DNA sample. Method A was based on a protocol resulting from an interlaboratory trial on 32P-postlabeling methodologies (31). Briefly, each DNA sample (10 µg) was digested with 60 mU of spleen phosphodiesterase (Calbiochem) and 240 mU of micrococcal nuclease for 2 hours at 37 °C. The digest was further incubated with 443 mU of nuclease P1 for 1 hour, then with 50 µCi of [-32P]ATP and 6 U of T4 PNK for 30 minutes at 37 °C, and the 32P-labeled digest was applied to a TLC plate. Chromatography was then conducted, and adducts were detected and quantified as described previously (32). Method B was based on procedures described by Shibutani et al. (14,33). Briefly, DNA (10 µg) was digested with 150 mU of spleen phosphodiesterase (Worthington) and up to 15 U of micrococcal nuclease for 2 hours at 37 °C, followed by incubation with 1 U of nuclease P1 for 1 hour at 37 °C. The digest was then extracted with butanol (14), the butanol was evaporated, and the residue was dissolved in water and incubated with 30 µCi of [
-32P]ATP and 30 U of T4 PNK for 40 minutes at 37 °C. Each 32P-labeled digest was subjected to TLC by using 1.7 M sodium phosphate [pH 6.0] as the D1 solvent (14) and the D2 and D3 solvents described in (32).
The tamoxifenDNA adducts were also analyzed by HPLC according to the procedure described in Shibutani et al. (14). Briefly, each 32P-labeled digest was applied to a TLC plate and eluted with 1.7 M sodium phosphate (pH 6.0). 32P-Labeled products were recovered from the origin, loaded onto a reverse-phase column (Jupiter C18, 4.6 mm x 250 mm, 5-µm particle size; Phenomenex), and eluted with a linear gradient of 10%70% methanol in 200 mM ammonium formate (pH 4.0), over 30 minutes at a flow rate of 1 mL/minute. Adducts were detected and quantified with a flow scintillation analyzer (Canberra Packard, Pangbourne, U.K.). The limit of detection, based on a signal that was twice the background radioactivity, was approximately one adduct per 109 nucleotides for dG-Tam and dG-desMeTam for both the TLC and HPLC analyses.
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RESULTS |
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HPLCES-MS/MS Analyses
Each of the DNA samples was enzymatically hydrolyzed to deoxyribonucleosides, mixed with the internal standard dG-Tam-d6, and subjected to HPLCES-MS/MS assays to detect dG-Tam and dG-desMeTam, the predominant tamoxifenDNA adducts found in experimental animals and/or reported to be present in human endometrial DNA samples. Figure 1 shows representative multiple reaction monitoring chromatograms for control DNA from salmon testis (A) and for endometrial DNAs from women who had (C) or had not (B) taken tamoxifen. The upper row of chromatograms presents the multiple reaction monitoring traces for the internal standard, dG-Tam-d6. The lower row of chromatograms presents the corresponding multiple reaction monitoring traces for dG-Tam. Figure 1, A, shows the multiple reaction monitoring traces obtained for 100 µg of salmon testis DNA plus 5 pg of dG-Tam and 25 pg of dG-Tam-d6. The amount of dG-Tam in this sample is equivalent to a DNA adduct level of 2.5 tamoxifenDNA adducts per 108 nucleotides. We detected peaks corresponding to the internal standard dG-Tam-d6 (retention time = 8.18 minutes; upper row) and to dG-Tam (retention time = 8.19 minutes; lower row). Endometrial DNA samples from women who had (Fig. 1, C, top) or had not (Fig. 1, B, top) taken tamoxifen also had peaks corresponding to the internal standard dG-Tam-d6 (retention time = 8.14 minutes). We detected a peak in the 8.0- to 8.5-minute region of the m/z 319261 multiple reaction monitoring traces of endometrial DNA from women who had taken tamoxifen (Fig. 1, C, bottom). This peak (retention time = 8.248.25 minutes), which consistently eluted after the peak corresponding to dG-Tam-d6, was also detected in endometrial DNA samples obtained from women who had not taken tamoxifen (Fig. 1, B, bottom). The same peak was also observed in the breast DNA sample from a woman who was treated with tamoxifen as well as in the breast DNA sample from a woman who did not receive tamoxifen (not shown). Because tamoxifen-treated and control samples gave essentially identical profiles, we conclude that neither dG-Tam (less than two adducts per 109 nucleotides) nor dG-desMeTam (less than two adducts per 108 nucleotides) was present in any of the endometrial DNA samples or in the single breast DNA sample from women who received tamoxifen therapy.
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32P-Postlabeling Analyses
To confirm the results obtained by HPLCES-MS/MS, each of the human endometrial and breast DNA samples was assessed by 32P-postlabeling analyses. As a positive control, a DNA sample from the liver of a male F344 rat that had received 45 mg/kg of tamoxifen daily by gavage for 14 days (30) was also analyzed. When analyzed by method A, the rat liver sample gave rise to one major and several minor spots by TLC (data not shown) and to a single major peak by HPLC that eluted at 26 minutes (Fig. 2, A). This HPLC peak contained both dG-Tam and dG-desMeTam, which co-eluted in a single peak under the HPLC solvent gradient conditions used. Similar results were obtained with method B (data not shown). The level of adduct recovery, which in previous experiments (32) has been shown to be greater than 90% when using method A, was essentially the same with both 32P-postlabeling protocols.
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DISCUSSION |
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Our results differ from those published by Hemminki et al. (13), who were the first to report the detection of tamoxifenDNA adducts, at a level of 2.7 adducts per 109 nucleotides, in endometrial DNA samples from women treated with tamoxifen. This adduct level is near the limit of detection of our HPLCES-MS/MS method but clearly within the limit of sensitivity of the 32P-postlabeling procedures we used. However, Orton and Topham (20) have noted that the chromatograms shown by Hemminki et al. (13) contained high levels of background radioactivity, which could account for some of their findings. In addition, Hemminki et al. did not use synthetic DNA adduct standards for comparison. By contrast, our HPLCES-MS/MS analyses included a well-characterized deuterated adduct standard to facilitate adduct identification and quantitation.
Our results also differ from those of Shibutani et al., who, using an improved 32P-postlabeling procedure coupled with HPLC separation, reported the presence of tamoxifenDNA adducts in eight of 16 endometrial tissue samples from tamoxifen-treated women (14,15). The adducts were identified on the basis of co-chromatography with synthetic standards, and in seven of the eight positive samples, dG-Tam was estimated to occur at 1.618 adducts per 108 nucleotides. The presence of dG-Tam at these levels in the samples we analyzed would definitely have been detected by our HPLCES-MS/MS methodology.
Another concern regarding the data reported by Shibutani et al. (14,15) pertains to the configuration of the adducts detected. The predominant peaks detected by Shibutani et al. in several of the human endometrial DNA samples co-eluted with dG-Tam adduct standards that had a Z configuration. This finding differs from what has been observed in experimental animals (2123) and in in vitro reactions (25,26), where the adducts have predominantly an E configuration. Furthermore, even when the major dG-Tam adducts detected by Shibutani et al. had an E configuration, the ratio of epimers varied substantially among the samples, which again is a result not obtained in experimental animals (2123). Although the reasons for the high percentage of adducts with a Z configuration and the unusual ratio of epimers are not known, it is possible that the multiple enrichment and enzymatic steps involved in the 32P-postlabeling methodology used by Shibutani et al. could increase the likelihood of obtaining spurious results. The HPLCES-MS/MS technique we used has considerably fewer manipulations than 32P-postlabeling and, when we analyzed identical DNA samples from tamoxifen-treated cynomolgus monkeys by HPLCES-MS/MS, the results were very similar to those obtained by 32P-postlabeling (23) and chemiluminescence immunoassay (27).
An additional method that has been used to detect tamoxifenDNA adducts is accelerator mass spectrometry (16). Although this method can detect as few as approximately two tamoxifenDNA adducts per 1010 nucleotides (16), a detection limit substantially better than the ones we report for the HPLCES-MS/MS or 32P-postlabeling methods, the identity of the adducts was not established, and it is possible that the material being measured by accelerator mass spectrometry was non-covalently bound tamoxifen metabolites, as opposed to tamoxifenDNA adducts.
In addition to HPLCES-MS/MS, we used two different 32P-postlabeling methods to assess tamoxifenDNA adducts in the endometrial DNA samples. Method A complies with recommended protocols for 32P-postlabeling (31) and has been shown to have a 32P-postlabeling efficiency greater than 90% with tamoxifenDNA adducts (32). Because the efficiency of this procedure when applied to the analysis of human endometrial samples has been questioned (34), we also used method B, which was used by Shibutani et al. to detect the presence of tamoxifenDNA adducts in human endometrium (14,15). We found that the 32P-postlabeling efficiency of tamoxifenDNA adducts formed in rat liver did not differ between our method (method A) and method B. Furthermore, regardless of whether the 32P-postlabeling was conducted by method A or method B or whether the chromatography was performed by TLC or HPLC, we found no evidence for the presence of dG-Tam or dG-desMeTam in the human endometrial DNA samples.
In addition to dG-Tam and dG-desMeTam, other DNA adducts formed by tamoxifen metabolites such as 4-hydroxytamoxifen (35) and -hydroxy-N,N-didesmethyltamoxifen (36) have been characterized. At present, our HPLCES-MS/MS methodology is not configured to detect tamoxifenDNA adducts from 4-hydroxytamoxifen or
-hydroxy-N,N-didesmethyltamoxifen; however, there was no indication that these adducts were present in any of the endometrial DNA samples when they were assayed by 32P-postlabeling
In conclusion, we found no evidence with either detection method (i.e., HPLCES-MS/MS or 32P-postlabeling) for the formation of tamoxifenDNA adducts in the endometrium of women who had taken tamoxifen. These findings are consistent with our earlier results (17,18) and suggest that the initiation of endometrial cancer by tamoxifen is not mediated by a genotoxic mechanism involving the formation of dG-Tam or dG-desMeTam.
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NOTES |
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Supported in part by a postgraduate research program administered by the Oak Ridge Institute for Science and Education (Oak Ridge, TN), by research grants from Programa PRAXIS XXI and Programa Operacional "Ciência, Tecnologia, Inovação" (POCTI), Fundação para a Ciência e a Tecnologia (FCT), Portugal; by a fellowship from Subprograma Ciência e Tecnologia, FCT, Portugal (to G. Gamboa da Costa); by Biomedical Research Funds from the U.S. Department of Veterans Affairs; and by Cancer Research UK.
A portion of these data was presented at the 93rd Annual Meeting of the American Association for Cancer Research, March 2002, San Francisco, CA.
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Manuscript received October 29, 2003; revised May 7, 2004; accepted May 21, 2004.
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