Mutagenesis Advance Access originally published online on March 8, 2005
Mutagenesis 2005 20(2):115-124; doi:10.1093/mutage/gei015
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Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2005
Hepatic DNA adduct dosimetry in rats fed tamoxifen: a comparison of methods
Carcinogen–DNA Interactions Section, National Cancer Institute, Building 37, Room 4032 NIH, 37 Convent Drive MSC-4255, Bethesda, MD 20892-4255, USA, 1Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey, SM2 5NG, UK, 2Division of Biochemical Toxicology, National Center for Toxicological Research, HFT-110, Jefferson, AR 72079, USA and 3Cancer Biomarkers and Prevention Group, The Biocentre, University of Leicester, Leicester LE1 7RH, UK
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Liver homogenates from rats fed tamoxifen (TAM) in the diet were shared among four different laboratories. TAM–DNA adducts were assayed by high pressure liquid chromatography–electrospray tandem mass spectrometry (HPLC–ES-MS/MS), TAM–DNA chemiluminescence immunoassay (TAM–DNA CIA), and 32P-postlabeling with either thin layer (32P-P–TLC) or liquid chromatography (32P-P–HPLC) separation. In the first study, rats were fed a diet containing 500 p.p.m. TAM for 2 months, and the values for measurements of the (E)-
-(deoxyguanosin-N2-yl)-tamoxifen (dG-N2-TAM) adduct in replicate rat livers varied by 3.5-fold when quantified using ‘in house’ TAM–DNA standards, or other approaches where appropriate. In the second study, rats were fed 0, 50, 250 or 500 p.p.m. TAM for 2 months, and TAM–DNA values were quantified using both ‘in house’ approaches as well as a newly synthesized [N-methyl-3H]TAM–DNA standard that was shared among all the participating groups. In the second study, the total TAM–DNA adduct values varied by 2-fold, while values for the dG-N2-TAM varied by 2.5-fold. Ratios of dG-N2-TAM:(E)--(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-N2-N-desmethyl-TAM) in the second study were 
1:1 over the range of doses examined. The study demonstrated a remarkably good agreement for TAM–DNA adduct measurements among the diverse methods employed. |
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Current clinical standard-of-care recommendations for estrogen receptor-positive breast cancer include the use of tamoxifen (TAM, Nolvadex®) as adjuvant therapy to be given for 5 years post-operatively (1
–5). This regimen has been found to reduce the incidence of contralateral breast cancer in breast cancer survivors by 47% (6). When TAM was used prophylactically, new breast cancers in high-risk women were reduced by 38% (7). However, treatment with TAM is not without risks, as epidemiological evidence links the drug to increases in endometrial (7,8) and rare uterine cancer (9). In TAM-exposed rats, hepatic TAM–DNA adduct formation (10) and liver tumor incidence (11) are correlated with dose, indicating that classical genotoxicity is probably the underlying mechanism for the observed liver cancers (12,13). However, whether a similar mechanism is involved in the etiology of human endometrial cancer is currently unclear.
The hypothesis that TAM-associated human endometrial cancers may occur through a genotoxic mechanism would be strengthened if it were possible to identify the women at risk through the detection of TAM–DNA adducts (Figure 1) in human endometrial and/or myometrial tissue. Currently, the presence of TAM–DNA adducts in endometrial tissue is a subject of controversy, since conflicting observations have been reported in the literature (14–20). Since some of the disagreement centers on methodological differences (21), the current study was designed to compare the application of several different methods for the determination of TAM–DNA adducts in rat liver DNA.
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The methods used in this study for TAM–DNA adduct determination comprise three different approaches: mass spectrometry, immunoassays and 32P-postlabeling (32P-P). Electrospray ionization tandem mass spectrometry (ES-MS/MS) coupled with on-line sample preparation and high-performance liquid chromatography (HPLC) was used to measure the two major adducts, (E)-
-(deoxyguanosin-N2-yl)-tamoxifen (dG-N2-TAM) and (E)--(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-N2-N-desmethyl-TAM). The TAM–DNA chemiluminescence immunoassay (TAM–DNA CIA) employed an antiserum elicited against DNA modified with dG-N2-TAM that has low (20%) cross-reactivity for dG-N2-N-desmethyl-TAM. 32P-P, which involves the 5'-32P-phosphorylation of digested 3'-nt, was used in three laboratories with the separation of the adducts either by HPLC (32P-P-HPLC) or thin layer chromatography (TLC) (32P-P-TLC). In addition to the two major adducts, the 32P-P-based methods were frequently able to detect additional minor adducts, including, possibly, (E)--(deoxyguanosin-N2-yl)-N,N-didesmethyltamoxifen (dG-N2-N,N-didesmethyl-TAM). In order to validate these methods against each other, rats were fed TAM in the diet for 2 months, using either a single dose (500 p.p.m. TAM) or a dose–response (50, 250 and 500 p.p.m. TAM) protocol. Coded liver nuclei samples were shared among the different participants, and each laboratory performed DNA extraction. DNA was assayed using the standard protocol employed in each laboratory, which in some instances involved the use of an ‘in house’ TAM–DNA adduct standard. For the dose–response study, participants also used an [N-methyl-3H]TAM-modified calf thymus DNA standard that was synthesized in one laboratory and supplied to all the participants. These experiments were designed to provide a critical evaluation of the several different methods employed for the determination of TAM–DNA adducts, which were performed in different laboratories under uniform conditions.
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Preparation of [N-methyl-3H]TAM–DNA standard
Calf thymus DNA, TAM and enzymes were obtained from Sigma (Poole, UK). [N-Methyl-3H]TAM (85 Ci/mmol) was obtained from Amersham Biosciences (London, UK). Unlabeled
-hydroxytamoxifen (-hydroxy-TAM) was synthesized as described (22). A microsome suspension was prepared from the livers of female Fischer F344 rats by centrifugation (23). The protein concentration was 28 mg/ml. HPLC was performed on a Waters apparatus, using a ‘Jupiter’ octadecylsilane (ODS) column, 250 x 4.6 mm (Phenomenex, Macclesfield, UK). Tritium activity was measured in a Packard liquid scintillation counter at a counting efficiency of 35%. For the preparation of [N-methyl-3H]-hydroxy-TAM, metabolism was carried out as reported previously (24). [N-Methyl-3H]TAM (0.9 µg, 200 µCi) was dissolved in 50 µl acetone and mixed with 1 ml water, 0.4 ml 0.1 M KH2PO4 (pH 7.4), 0.2 ml 0.05 M MgSO4, 70 µl NADPH (29 mg/ml) and 0.1 ml rat liver microsome suspension. After incubation for 1 h at 37°C, the mixture was extracted with ethyl acetate (4 x 1.4 ml), and the extract was dried over Na2SO4, evaporated and dissolved in 1 ml 30% acetonitrile. The mixture was separated on an ODS column by elution with 0.05 M ammonium formate containing acetonitrile (from 30 to 70% in 40 min) at 0.8 ml/min. The eluate at 22–28 min contained [N-methyl-3H]-hydroxy-TAM, as shown by re-chromatography of samples mixed with unlabeled -hydroxy-TAM as a marker. Unchanged TAM eluted at 40–48 min and was recovered for a second metabolism. The total yield of [N-methyl-3H]-hydroxy-TAM was 3 µCi (1.5%). It was diluted with unlabeled -hydroxy-TAM (0.1 mg) and partitioned into ethyl acetate, which was dried over sodium sulfate and evaporated at low pressure.
For acetylation and reaction with DNA, the [N-methyl-3H]-hydroxy-TAM was incubated in 10 µl acetic anhydride and 50 µl pyridine at room temperature overnight. Chromatography of a sample on an ODS column showed 91% conversion to -acetoxy-TAM. The reagents were evaporated at low pressure, and the residue was dissolved in 20 ml ethanol. The solution was diluted with 40 ml water and added to calf thymus DNA (1.7 g) in 440 ml 0.22 M sodium cacodylate (pH 6). The mixture was left overnight at 37°C. Sodium chloride (1 M, 50 ml) was added and the unreacted TAM derivatives were removed by extraction with ether (4 x 300 ml). The DNA was precipitated with ethanol and washed with ethanol and ether.
For analysis, the [N-methyl-3H]TAM–DNA (4 mg) was dissolved in 1.5 ml water and hydrolyzed to nucleosides at 37°C by the sequential addition of DNase (0.3 mg, 17 h), snake venom phosphodiesterase (0.08 U, 6 h) and alkaline phosphatase (7 U, 17 h). dG-N2-TAM (3 µg), obtained in another experiment (25), was added as a marker, and the mixture separated on an ODS column and eluted with 0.05 M ammonium formate containing acetonitrile (from 10 to 55% in 30 min) at 0.8 ml/min (25). The normal nucleosides were quantified by their UV absorbance. dG-N2-TAM was eluted and quantified by its radioactivity.
Animal care and dietary TAM exposure
Two separate experiments, a single dose study and a dose–response study, were conducted in female Fischer F344/NHsd rats (Frederick, MD). Animal care was provided in accordance with the standards established by the Association for Assessment and Accreditation for Laboratory Animal Care. The experimental protocols were approved by the NCI Animal Care and Use Committee. The rats were fed TAM (Sigma, St Louis, MO) in a powdered purified rat diet (AIN-76A, Research Diets, Inc., New Brunswick, NJ) and fasted 24 h before euthanasia. The liver was harvested for DNA isolation and stored frozen. In the single dose study, five 8-week-old female rats were fed a purified rat diet containing 500 p.p.m. TAM for 8 weeks. In the dose–response study, eleven 8-week-old female rats were fed the same purified diet containing 0 (n = 2 rats), 50 (n = 3), 250 (n = 3) or 500 (n = 3) p.p.m. TAM for 8 weeks.
Isolation of liver nuclei and preparation of sample for interlaboratory trial
Upon dissection, livers were removed and immediately placed into lysing solution (330 mM sucrose, 1 mM potassium phosphate (pH 7.4), 1% Triton X-100 and 2 mM EGTA) on ice. Tubes were kept on ice at all times. The tissue was homogenized using an Ultra Turrax IKA Labortechniks Homogenizer, at a dial setting of 4, until solid tissue chunks were no longer visible. The tubes were then centrifuged at 2500 g for 15 min at 4°C. The supernatant was removed. The nuclei were washed with 50 ml of washing solution (identical to lysing solution but without the Triton X-100). The tubes were then centrifuged at 2500 g for 15 min at 4°C. The supernatant was removed and the nuclear pellets were stored at –20°C. For the single dose study, five randomly numbered samples containing 0.5 ml of isolated nuclei from rats fed 500 p.p.m. TAM were sent to each of the participating laboratories. For the dose–response study, 11 randomly numbered samples containing 0.1 ml of isolated nuclei were sent to each of the same laboratories.
TAM–DNA chemiluminescence immunoassay (Poirier lab)
TAM and calf thymus DNA were purchased from Sigma. Opaque 96-well high-binding plates came from Greiner Labortechnik (PGC Scientific, Frederick, MD). Biotinylated anti-rabbit IgG and streptavidin–alkaline phosphatase were supplied by BioGenex (San Ramon, CA). I-Block (Casein) and CDP-Star with Emerald II were from Tropix (Bedford, MA). Reacti-Bind DNA coating solution was obtained from Pierce (Rockford, IL). CIA wash buffer was obtained from KD Medical (Columbia, MD). Phosphate buffered saline (PBS) was from GibcoBRL (Grand Island, NY).
DNA was isolated from liver nuclei by non-organic extraction (DNA Extraction Kit, Serologicals Corporation, Norcross, GA) and quantified by ultraviolet spectrophotometry at A260.
The TAM–DNA CIA employed rabbit antiserum elicited against DNA modified to 2.4% with -acetoxy-TAM, and has been described previously (26). In brief, TAM–DNA (2.4% modified, 8.2 pg DNA) containing 0.6 fmol of the dG-N2-TAM adduct in Reacti-Bind DNA coating solution was used to coat each well of a 96-well microtiter plate, and the coated plates were stored at –20°C. At the time of assay, nonspecific binding was reduced by incubating with 300 µl casein solution/well (0.33% I-Block in PBS, 0.05% Tween-20 and 0.1% NaN3). For each assay, an ‘in-house’ TAM–DNA standard (4.8 adducts/106 nt) was serially diluted from 6.63 to 0.0091 fmol dG-N2-TAM/well; carrier DNA was added so that all standard curve wells had the same DNA concentration as the wells containing biological samples. Equal volumes of anti-TAM–DNA (final dilution 1:1 000 000) in casein solution and either TAM–DNA standard plus carrier in PBS or biological sample DNA in PBS were mixed and incubated together 15–20 min before adding 100 µl to each microtiter well. After a 90-min incubation and washing, biotinylated anti-rabbit IgG (100 µl, 1:2500 dilution in casein solution) was added, followed by streptavidin–alkaline phosphatase (100 µl, 1:1500 dilution in casein solution) and 100 µl of CDP-Star containing Emerald II enhancement solution. The light emission was measured at 542 nm using a TR717 Microplate Luminometer (PE Applied Biosystems, Foster City, CA) at 20 min and 18 h after the addition of CDP-Star.
For the ‘in-house’ TAM–DNA standard curve, 50% inhibition was at 3.0 ± 0.6 fmol dG-N2-TAM (mean ± SE, n = 4)/well. Since up to 20 µg DNA could be analyzed per well, the limit of detection was calculated to be 10 amol of dG-N2-TAM adduct/µg DNA or 0.3 adducts/108 nt.
Determination of TAM–DNA adducts by 32P-P–TLC (Phillips lab)
DNA was isolated from hepatic nuclei by a phenol–chloroform extraction method using conditions described previously (27). 32P-P analysis was carried out as described in earlier publications (10,27,28). Briefly, aliquots of DNA (4 µg) were digested for 20 h with micrococcal nuclease (0.14 U, Sigma) and spleen phosphodiesterase (0.6 mU, Merck Biosciences Ltd) at 37°C, followed by nuclease P1 (0.24 U) for 1 h. The digests were then subjected to 32P-P by incubation with 50 µCi carrier-free [
-32P]ATP and polynucleotide kinase (6 U) for 30 min. Resolution of the labeled adducts was then carried out on PEI-cellulose thin-layer plates using the following solvents: D1, 2.3 M sodium phosphate, pH 5.8; D2, 2.27 M lithium formate, 5.52 M urea, pH 3.5; D3, 0.52 M LiCl, 0.325 Tris–HCl, 5.52 M urea, pH 8.0. Chromatograms were scanned for radioactivity using an InstantImager (Canberra Packard, Pangbourne, UK). Relative levels of DNA modification were calculated from the levels of radioactivity in the DNA adduct spots detected on the chromatograms and from the specific activity of the [-32P]ATP used in the labeling procedure (29).
HPLC analysis was also conducted on DNA obtained from the single dose study for the purpose of determining the relative amounts of dG-N2-TAM and dG-N2-N-desmethyl-TAM, but not for the overall quantitation of total adduct levels. Before performing HPLC, labeled digests of adducts were chromatographed on PEI-cellulose in solvent D1 only (see above). Material was eluted from the origin with 4 M pyridinium formate, pH 4.5. HPLC analysis of TAM–DNA adducts was carried out using the system as described previously (30) with modifications (28): the HPLC column used was a Jupiter 5 µ ODS (250 x 4.6 mm) column from Phenomenex (Macclesfield, Cheshire, UK); the solvent system was 82% 2 M ammonium formate, pH 4.0 (solvent A), 18% acetonitrile:methanol (6:1, v/v) (solvent B) for 40 min followed by a linear gradient of 18–45% solvent B for 20 min. Flow rate was 1 ml/min.
Determination of TAM–DNA adducts by 32P-P–HPLC (Brown lab)
DNA was extracted from hepatic nuclei using Qiagen 100G columns (Qiagen, Ltd, Crawley, UK) according to the manufacturer's protocol. Adducts were quantified using a 32P-P–HPLC assay that incorporates nuclease P1 enrichment and separates adducts by HPLC (30,31). Extracted DNA samples and the [N-methyl-3H]TAM–DNA standard (10 µg) were hydrolyzed to nucleoside 3'-monophosphates by digesting with 0.35 U micrococcal nuclease (Sigma) and 20 mU calf spleen phosphodiesterase (Merck Biosciences Ltd) for 16 h at 37°C. Each digest was then incubated with nuclease P1 (22.5 µg, Sigma) at 37°C for 1 h. 5'-Phosphorylation was performed using 125 µCi [-32P]ATP (>5000 Ci/mmol, 10 mCi/ml; Amersham) and 12.5 U of 3'-phosphatase-free T4 polynucleotide kinase (Roche) for 1 h at 37°C. 32P-Postlabelled DNA adducts were separated and quantified directly using an HPLC system consisting of a Varian 210 Prostar solvent delivery module and 410 autosampler with an on-line radiochemical detector (LabLogic, ß-ram) fitted with a solid-phase cell (500 µl), and data analysis was performed using Laura, a Microsoft Windows package (LabLogic). Typically, 9.1 µg of each digest was injected and separation was performed on a Hypersil BDS C18 column (5 µm, 250 x 4.6 mm) using a gradient of (A) 2 M ammonium formate, 0.27 mM EDTA, pH 4.0 and (B) acetonitrile:methanol (6:1) at a flow rate of 1 ml/min. Initial conditions were 20% (B) for 40 min, increased linearly to 45% over a further 25 min. Total TAM–DNA adducts were quantified by the integration of all detectable peaks, apart from those due to excess ATP, and adduct levels were corrected using the [N-methyl-3H]TAM–DNA standard. For the dose–response study, each extracted DNA sample was subjected to 32P-P–HPLC on three separate occasions, while each sample was analyzed four times for the single dose study.
Determination of TAM–DNA adducts by 32P-P–HPLC (Beland lab)
DNA was isolated from the rat liver nuclei using slight modifications of the procedure described (32). 32P-P–HPLC analyses were conducted with 9.3–16.4 µg DNA, depending upon the particular sample, using procedures described previously (33), with the following solvent conditions: 0–10 min, isocratic elution with 58% solvent A [1.2 M ammonium formate, 10 mM ammonium phosphate (pH 4.5)] and 42% solvent B [24% acetonitrile in 1.2 M ammonium formate, 10 mM ammonium phosphate (pH 4.5)]; 10–30 min, a linear gradient to 83% solvent B; 35–40 min, a linear gradient to 100% solvent B; 40–60 min, isocratic elution with 100% solvent B. The flow rate was 1 ml/min. The samples were quantified using an ‘in-house’ TAM–DNA standard modified to 560 adducts/108 nt as reported previously (34). Individual levels are reported for dG-N2-TAM and dG-N2-N-desmethyl-TAM. The total adduct level is slightly more than the sum of the two because there was also a small peak eluting early, which was included in the quantitation. The sample values were adjusted for comparison with the [N-methyl-3H]TAM–DNA standard (660 adducts/108 nt) described above.
Determination of TAM–DNA adducts by HPLC–ES-MS/MS (Beland lab)
DNA was isolated from the rat liver nuclei using slight modifications of the procedure described (32). DNA samples were hydrolyzed to nucleosides and analyzed for dG-N2-TAM and dG-N2-N-desmethyl-TAM by HPLC–ES-MS/MS as described (34). Briefly, each hydrolyzed DNA sample was loaded onto a reversed-phase trap column [Luna C18(2), 2 x 30 mm, 3 µm, 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, Phenomenex] with 73% 0.1% formic acid and 27% acetonitrile at 200 µl/min into the mass spectrometer. A Quattro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, UK), equipped with an electrospray interface, was used with a source block of 120°C and a desolvation temperature of 400°C. Nitrogen was the desolvation (750 l/h) and nebulizing gas and argon was the collision gas, at a collision cell pressure of 1.5 µbar. Positive ions were acquired in the multiple reaction monitoring (MRM) mode (dwell time of 0.3 s and interchannel delay of 0.03 s) for the doubly charged (M+2H)2+
(BH+2H)2+ transitions of dG-N2-TAM (m/z 319261), dG-N2-N-desmethyl-TAM (m/z 312254), and the internal quantitation standard dG-N2-TAM-d6 (m/z 322264). In addition, the singly charged (M+H)+(TAM-H)+ transitions of dG-N2-TAM (m/z 637370), dG-N2-N-desmethyl-TAM (m/z 623356), and dG-N2-TAM-d6 (m/z 643376) were monitored. The cone voltage was 15 V and the collision energy was 10 eV for the doubly charged transitions; for the singly charged transitions, the cone voltage was 55 V and the collision energy was 25 eV.
Two assays were conducted. A total of 9.3–16.4 µg DNA were hydrolyzed for the first assay and 93–164 µg DNA were hydrolyzed for the second assay. Only a small portion of the total sample was used for the second HPLC–ES-MS/MS assay. The total adducts represent the sum of dG-N2-TAM and dG-N2-N-desmethyl-TAM, as these were the only peaks quantified. The data presented are the mean of both assays. For the HPLC–ES-MS/MS analyses of the ‘in-house’ TAM–DNA standard and the [N-methyl-3H]TAM–DNA, the only peaks evident were the dG-N2-TAM.
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Analysis of the [N-methyl-3H]TAM–DNA standard
The [N-methyl-3H]TAM–DNA standard was prepared by incubation of [N-methyl-3H]TAM with rat liver microsomes, conversion of the resulting [N-methyl-3H]
-hydroxy-TAM to [N-methyl-3H]-acetoxy-TAM, and subsequent reaction with calf thymus DNA. When this DNA was digested and chromatographed on an ODS column (Figure 2), the peaks obtained for dG-N2-TAM isomers were the principal (trans or E, TG1) adduct with 330 c.p.m. and the minor (cis or Z, TG2) adduct with 65 c.p.m.; in addition, 126 c.p.m. (32%) remained as unidentified or incompletely hydrolyzed adducts.
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The specific activity of [N-methyl-3H]
-hydroxy-TAM was originally 11 c.p.m./pmol, but some tritium was lost during incubation, and so to establish the specific activity of the adducts, the specific activity was determined for -hydroxy-TAM, which was obtained from the hydrolysis of -acetoxy-TAM during the incubation. The ether extract obtained after reaction with DNA was evaporated to dryness and dissolved in acetonitrile, and a sample was analyzed on the ODS column by elution with 0.05 M ammonium formate/35% acetonitrile. The [3H]-hydroxy-TAM isolated in two such runs amounted to 2.76 nmol (as estimated by UV absorbance) and gave 21684 c.p.m., for a specific activity of 7.8 c.p.m./pmol (10.1 mCi/mmol). The level of tritiated adducts in the [N-methyl-3H]TAM–DNA was therefore considered to be 4.2 trans-adducts/106 nt, or 6.6 total adducts/106. This TAM–DNA was shared among the different laboratories and used as a standard in the dose–response study to calculate the assay values obtained from the different methods. The [N-methyl-3H]TAM–DNA standard was assayed by each of the laboratories to ascertain how direct measurement of the 3H content compared with TAM–DNA adduct determination performed in each laboratory using the currently employed ‘in house’ approach. The values differed over a 3-fold range overall. The [N-methyl-3H]TAM–DNA standard gave a value of 7.96 ± 0.45 adducts/106 nt (mean ± SE, n = 3) when measured by HPLC–ES-MS/MS. A value of 7.16 ± 0.38 adducts/106 nt (mean ± range, n = 2) was obtained by 32P-P–TLC. The TAM–DNA CIA determined 3.97 ± 0.27 adducts/106 nt (mean ± range, n = 2), while the 32P-P–HPLC (Brown lab) showed 5.09 ± 1.29 adducts/106 nt (mean ± SE, n = 3), and the 32P-P–HPLC (Beland lab) gave 12.2 adducts/106 nt (n = 1).
Different methodological approaches
Owing to aspects intrinsic to each assay, the four methods used in this study, HPLC–ES-MS/MS, TAM–DNA CIA and 32P-P–TLC or 32P-P–HPLC did not always measure exactly the same adducts in the rat liver samples.
For the HPLC–ES-MS/MS assay, based on mass spectrometric determination of chemical structure, representative MRM chromatograms of synthetic dG-N2-TAM-d6, dG-N2-TAM, and dG-N2-N-desmethyl-TAM are shown in Figure 3. Chromatogram A presents the traces corresponding to MRM of the transitions from the singly charged protonated nucleoside molecule [(M+H)+] to the singly charged fragment [(TAM-H)+] for 25 pg of the internal standard dG-N2-TAM-d6 (m/z 643376). Chromatograms B and C are the corresponding singly charged MRM traces for 25 pg dG-N2-TAM (m/z 637370) and 25 pg dG-N2-desmethyl-TAM (m/z 623356). Figure 3 also shows the chromatograms corresponding to MRM of the transitions from the doubly charged protonated nucleoside molecule [(M+2H)2+] to the doubly charged protonated purine base [(BH+2H)2+] for dG-N2-TAM-d6 (m/z 322264; chromatogram D), dG-N2-TAM (m/z 319261; chromatogram E) and dG-N2-N-desmethyl-TAM (m/z 312254; chromatogram F). A comparison of the peak areas from these chromatograms indicates that there is 20- to 30-fold greater sensitivity when monitoring the transitions of the doubly charged molecules of dG-N2-TAM and dG-N2-TAM-d6 (chromatograms D and E) compared with the singly charged molecules (chromatograms A and B). Similarly, there is 10-fold greater sensitivity when monitoring the doubly charged molecule of dG-N2-N-desmethyl-TAM (chromatogram F) compared with the singly charged molecule (chromatogram C).
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The TAM–DNA antiserum used in the CIA was elicited against a TAM–DNA sample containing essentially only the dG-N2-TAM (26
), and maximum recognition is therefore directed against this adduct. In a previous investigation, a sample of rat liver DNA modified only with dG-N2-N-desmethyl-TAM was recognized 20% as well as the original immunogen, DNA modified with dG-N2-TAM (R.L.Divi, D.H.Phillips, M.R.Osborne and M.C.Poirier, unpublished data). It was therefore concluded that the TAM–DNA CIA has minimal recognition for the dG-N2-N-desmethyl-TAM. As part of this study, DNA modified with the dG-N2-N,N-didesmethyl-TAM adduct was assayed by TAM–DNA CIA and showed no inhibition at 183 000 amol adduct/µg DNA, compared with the 50% inhibition at 10 amol adduct/µgDNA for the dG-N2-TAM adduct, demonstrating that the antibody lacks cross-reactivity for the minor adduct. Therefore, in rat liver DNA samples assayed by TAM–DNA CIA, the values obtained reflect essentially only the dG-N2-TAM adduct.
Using the 32P-P–TLC followed by HPLC, members of the Phillips lab first quantified the total binding from separations on TLC by combining all visible adduct spots (Figure 4A). Subsequently, the relative amounts of dG-N2-TAM and dG-N2-N-desmethyl-TAM were determined by HPLC analyses (Figure 4B). When 32P-P–HPLC was performed by the Brown (Figure 5A) and Beland (Figure 5B) laboratories, total TAM–DNA adducts were quantified by integration of all the detectable peaks. Individual levels of trans isomers of dG-N2-TAM and dG-N2-N-desmethyl-TAM were determined by integration of the appropriate peaks previously shown to correspond to these adducts (30,35–37). The HPLC tracings for data from the three laboratories were quite similar (Figures 4B, 5A and B) and the two major adducts were readily visible.
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Dosing of rats with 500 p.p.m. TAM
In the first study, rats (n = 5) were fed 500 p.p.m. TAM for 2 months at the NIH, and liver nuclei were shared among the different laboratories. Each laboratory supplied unexposed control rats when required, and each laboratory extracted DNA. Where appropriate, each laboratory used an ‘in-house’ TAM–DNA standard. Liver DNA was examined for TAM–DNA adduct formation using the HPLC–ES-MS/MS (Beland lab), 32P-P–HPLC (Brown lab), 32P-P–TLC (Phillips lab) and TAM–DNA CIA (Poirier lab) assays.
The results for HPLC–ES-MS/MS analysis of liver DNA from a rat fed 500 p.p.m. TAM for 2 months are shown in Figure 6. The internal standard dG-N2-TAM-d6 (chromatograms A and D), dG-N2-TAM (chromatograms B and E) and dG-N2-N-desmethyl-TAM (chromatograms C and F) are clearly present with retention times of 8.69, 8.69 and 8.35 min, respectively, and as had been observed with the synthetic adducts, there was greater sensitivity when monitoring the doubly charged molecules (chromatograms D–F), compared with the singly charged molecules (chromatograms A–C). In the trace monitoring the m/z 637370 transition (chromatogram B), there were additional signals at 13.52 and 14.01 min; similarly, in the trace monitoring the m/z 623356 transition (chromatogram C), there were additional signals at 12.94 and 13.33. Umemoto et al. (38) have reported that the Z isomers of dG-N2-TAM and dG-N2-N-desmethyl-TAM have longer HPLC retention times than their corresponding E isomers (38). As such, the additional signals detected in chromatogram B are probably due to (Z)-dG-N2-TAM and those in chromatogram C are probably due to (Z)-dG-N2-N-desmethyl-TAM. Since synthetic standards were not available for the Z isomers, these signals were not included in the quantitation of the TAM–DNA adduct levels.
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The data for comparison of the four methods, HPLC–ES-MS/MS, 32P-P–HPLC, 32P-P–TLC, and TAM–DNA CIA, are shown in Table I. The dG-N2-TAM values among the four different methods varied by 3.5-fold, and the dG-N2-N-desmethyl-TAM, detectable by three of the four methods, varied by 3-fold. In this experiment, the dG-N2-TAM was shown to be 35–44% of the total of dG-N2-TAM plus dG-N2-N-desmethyl-TAM. For the two 32P-P-based methods, additional adducts were found such that the value for the total TAM–DNA adducts was 16.00 ± 0.16 adducts/106 nt for 32P-P–TLC and 9.51 ± 0.59 adducts/106 nt for 32P-P–HPLC (Brown lab).
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Dose–response experiment
In the second study comparing the dose–response experiment, rats were fed 0 (n = 2), 50, 250 and 500 p.p.m. TAM (n = 3 per dose) for 2 months, and liver nuclei DNA samples were prepared and assayed by 32P-P–TLC (Phillips lab), HPLC–ES-MS/MS (Beland lab), 32P-P–HPLC (Brown and Beland labs) and TAM–DNA CIA (Poirier lab). With the exception of the HPLC–ES-MS/MS, the shared [N-methyl-3H]TAM–DNA standard was used to calculate the results shown in Table II, and Figures 7 and 8.
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The values for dG-N2-TAM assayed by HPLC–ES-MS/MS, 32P-P–HPLC (Beland and Brown labs) and TAM–DNA CIA are presented in Figure 7. There was a strong dose–response for all the methods, and the inter-method variability was
2.5-fold, with the 32P-P–HPLC and HPLC–ES-MS/MS performed in the Beland laboratory having the highest values and the 32P-P–HPLC performed in the Brown laboratory having the lowest values. The HPLC–ES-MS/MS values were quantified using a deuterated internal standard, while data for the other assays were obtained by calculating against the shared [N-methyl-3H]TAM–DNA standard. For the dose–response study, 32P-P–TLC values from the Phillips laboratory did not include analyses of the specific adducts.
The values for total TAM–DNA adducts using HPLC–ES-MS/MS, 32P-P–TLC and 32P-P–HPLC are shown in Figure 8. Similar to Figure 7, the HPLC–ES-MS/MS values were quantified against a deuterated internal standard, while the other assays were calculated against the [N-methyl-3H]TAM–DNA standard. The variability among assays was 2-fold overall. It is important to note that for total adduct quantitation (Figure 8) the HPLC–ES-MS/MS data include only the two major adducts, while the 32P-P-based methods include the major adducts (dG-N2-TAM and dG-N2-N-desmethyl-TAM) and additional minor adducts, including, possibly, dG-N2-N, N-didesmethyl-TAM.
Fraction of specific TAM–DNA adducts
Both experiments provided opportunities to examine the relationships between quantities of dG-N2-TAM and dG-N2-N-desmethyl-TAM formed at the different dose levels. It was of particular interest to note whether the proportions of these adducts varied by dose. The data, shown in the last column of Tables I and II as the percentage of the total combined adducts (dG-N2-TAM plus dG-N2-N-desmethyl-TAM) that was represented by dG-N2-TAM, indicated that there was no difference in the proportion of the two major adducts at doses between 50 and 500 p.p.m. In addition, it appears that the two adducts were formed in approximately equal proportions in the second experiment, although dG-N2-TAM comprised 35–44% of the total in the first experiment.
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Discussion |
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In light of the current controversy concerning the formation of TAM–DNA adducts in human female reproductive organs (21
), this study was designed to explore the reproducibility of TAM–DNA adduct determination by diverse methods, including immunoassay, 32P-P and mass spectrometry approaches, performed in different laboratories. To maximize uniformity, rats were fed TAM in one laboratory and liver nuclei were prepared and aliquots were sent to the participants. In addition, a calf thymus DNA sample was modified in vitro with radiolabeled TAM to give a [N-methyl-3H]TAM–DNA standard with a modification level of 6.6 adducts/106 nt determined by radioactivity. This standard was used to normalize the sample values obtained from all the methods except the HPLC–ES-MS/MS assays. The sum total of all of the data obtained here allows us to conclude several things. First, all the methods employed gave very similar adduct values for the rat liver DNA. This was the case whether or not the data were calculated against the [N-methyl-3H]TAM–DNA standard. Second, in the livers of rats fed TAM (50–500 p.p.m.) for a substantial period of time (8 weeks) the two major adducts, dG-N2-TAM and dG-N2-N-desmethyl-TAM, were present in approximately equal amounts. Finally, the [N-methyl-3H]-TAM–DNA standard is a useful validation tool, and can be made available in small quantities for the calibration of additional methods, and comparison with the data described here.
Although the DNA adduct values induced by TAM in these rat livers were relatively high, limits of detection have been presented for each method, and there is every reason to believe that these methods will measure lower levels of TAM–DNA adducts with accuracy. For the HPLC–ES-MS/MS, linearity was shown for TAM–DNA modified between 1.5 adducts in 100 nt and 8 adducts/108 nt (34). In addition, good correspondence was reported for TAM–DNA adducts measured in DNA samples prepared from the organs of monkeys fed TAM for a month and assayed by TAM–DNA CIA, 32P-P–HPLC and HPLC–ES-MS/MS (39,40). In the monkey studies TAM–DNA adduct values were >100-fold lower than those reported here for rat liver.
The pathways of activation that lead to the TAM–DNA adduct formation in rat liver, a target organ for TAM-induced tumor formation, have been widely studied. The principal pathway is now generally recognized to involve -hydroxylation by cytochrome P450 3A2 (41), followed by -sulfonation by a single isoform of sulfotransferase 2A2 (ST2A2) (42), leading to a highly reactive species that reacts with exocyclic amino groups in guanine and adenine bases in DNA (13). In rats, ST2A2 is expressed almost exclusively in liver (43), and the fact that little, if any, DNA adduct formation occurs in rat extrahepatic tissues (10,37) (D.H.Phillips, A.S.Hewer and M.R.Osborne, unpublished data) is probably due to the absence of ST2A2.
In the present study, the [N-methyl-3H]TAM–DNA standard was prepared to provide an independent means of determining the level of TAM adduction. As expected, most of the radioactivity on this DNA was associated with dG-N2-TAM (Figure 2). Absolute determination of this adduct level by radiolabeling can only be assumed if there is no loss of tritium during incubations; however, in the present case, there was evidence that this had occurred. Therefore, adjustments to the calculations for the level of DNA modification were made. The estimated levels of modification for the [N-methyl-3H]-TAM–DNA standard, determined by HPLC–ES-MS/MS and 32P-P-TLC, were very similar to the levels estimated by 3H incorporation, suggesting that both these methods have very high dG-N2-TAM adduct recovery, with no evidence of loss of materials during the analytical procedures. Both the TAM–DNA CIA and the 32P-P–HPLC from the Brown laboratory appeared to underestimate the adduct level in this sample, while the 32P-P–HPLC from the Beland laboratory provided a value about twice that found by radiolabeling.
In a previous interlaboratory comparison involving two of the participants of the present trial, similar results were obtained by 32P-P and TAM–DNA CIA using rat liver DNA samples from TAM-treated rats, although at that time a TAM-modified DNA standard was not available (26). In a comparison of tissue from TAM-exposed primates by TAM–DNA CIA, HPLC–ES-MS/MS and 32P-P, the last by an investigator who did not participate in the present trial, similar results were obtained by all methods (39,40). Thus, the results of these studies and the present trial indicate that investigators who were involved in this trial, and also those who were not, have methods of analysis that have similar detection limits and similar rates of adduct recovery. All these data support the hypothesis that discrepancies in the detection of TAM–DNA adducts in human tissues (21) cannot be explained by the different rates of TAM–DNA adduct recovery in different laboratories as suggested by Kim et al. (44).
A significant proportion of the TAM–DNA adducts formed in rat liver had lost one of the TAM N-methyl groups, and evidence suggests that N-demethylation could occur either before or subsequent to -hydroxylation (28). The ratios of dG-N2-TAM : dG-N2-N-desmethyl-TAM observed in the present trial are very similar to previous observations in liver DNA from rats administered TAM by gavage (36,37,42). N-Demethylation is a major pathway of TAM metabolism in humans, such that after prolonged treatment, the major circulating form of the drug is N-desmethyl-TAM. Since this metabolite contributes to more DNA adducts in rat liver than the parent compound, it might be reasonable to expect that if TAM forms DNA adducts in human tissues, a significant proportion of these would be derived from N-desmethyl-TAM. However, although some investigators have reported TAM–DNA adducts in human tissues (17–19,45), presence of dG-N2-N-desmethyl-TAM has not been proven.
Overall, the results of this study should inspire confidence that multiple different methodological approaches to the measurement of TAM–DNA adducts provide consistent values well within the same order of magnitude. Although the TAM–DNA CIA measures primarily the dG-N2-TAM, all of the other methods successfully documented the presence of the two major adducts, dG-N2-TAM and dG-N2-N-desmethyl-TAM. The [N-methyl-3H]TAM–DNA standard provides an additional tool for interlaboratory assay validation, and has been employed successfully here to demonstrate the concordance of TAM–DNA values in DNA obtained from rats fed TAM for 2 months.
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Acknowledgments |
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We thank I.R.Hardcastle, Institute for Cancer Research, Sutton, UK for synthesizing the
-hydroxy-TAM. This study was supported by Cancer Research UK, and the intramural research programs of the National Center for Toxicological Research and the National Cancer Institute, NIH. |
Notes |
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* To whom correspondence should be addressed. Tel: +1 301-402-1835; Fax: +1 301-402-8230; Email: poirierm{at}exchange.nih.gov
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References |
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