Further characterization of the DNA adducts formed in rat liver after the administration of tamoxifen, N-desmethyltamoxifen or N,N-didesmethyltamoxifen
Karen Brown1,
Robert T. Heydon,
Rebekah Jukes,
Ian N.H. White and
Elizabeth A. Martin2
MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK
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Abstract
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The present study compares the formation of DNA adducts, determined by 32P-postlabelling, in the livers of rats given tamoxifen and the N-demethylated metabolites N-desmethyltamoxifen and N,N-didesmethyltamoxifen. Results show that after 4 days treatment (0.11 mmol/kg i.p.), similar levels of DNA damage were seen after treatment with either tamoxifen or N-desmethyltamoxifen [109 ± 40 (n = 3) and 100 ± 33 (n = 4) adducts/108 nucleotides, respectively], even though the concentration of tamoxifen in the livers of tamoxifen-treated rats was about half that of N-desmethyltamoxifen in the N-desmethyltamoxifen-treated animals (51 ± 16 and 100 ± 8 nmol/g, respectively). Administration of N,N-didesmethyltamoxifen to rats resulted in a 5-fold lower level of damage (19 adducts/108 nucleotides, n = 2). Following 32P-postlabelling and HPLC, hepatic DNA from rats treated with tamoxifen and its metabolites showed distinctive patterns of adducts. Treatment of rats with N,N-didesmethyltamoxifen gave a major product that co-eluted with one of the minor adduct peaks seen in the livers of rats given tamoxifen. Following dosing with N-desmethyltamoxifen, the major product co-eluted with one of the main peaks seen following treatment of rats with tamoxifen. This suggests that tamoxifen can be metabolically converted to N-desmethyltamoxifen prior to activation. However, analysis of the 32P-postlabelled products from the reaction between
-acetoxytamoxifen and calf thymus DNA showed two main peaks, the smaller one of which (~15% of the total) also co-eluted with that attributed to N-desmethyltamoxifen. This indicates that N-desmethyltamoxifen and N,N-didesmethyltamoxifen are activated in a similar manner to tamoxifen leading to a complex mixture of adducts. Since an HPLC system does not exist that can fully separate all these 32P-postlabelled adducts, care has to be taken when interpreting results and determining the relative importance of individual adducts and the metabolites they are derived from in the carcinogenic process.
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Introduction
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The anti-oestrogen tamoxifen {trans-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene} is currently undergoing evaluation as a chemopreventive agent in women at increased risk of developing breast cancer (1). Recent results in healthy, high risk women from the NSABP P1 study demonstrated that this drug resulted in a 49% reduction in breast cancer incidence (2). However, tamoxifen treatment is associated with an increased incidence of endometrial cancer (3,4) and long-term administration to rats results in a dose-dependent increase in hepatic tumours (5,6). Tamoxifen requires metabolic activation to electrophilic species before it can react with DNA, forming adducts detectable using the 32P-postlabelling assay (7,8). The major phase I metabolites of tamoxifen in both rat and human liver are N-desmethyltamoxifen and 4-hydroxytamoxifen (Figure 1
; 9,10). However, one of the main DNA adducts detected in the livers of tamoxifen-dosed rats is thought to arise as a consequence of
-hydroxylation, followed by hydroxysteroid sulphotransferase-mediated sulphate conjugation (1113). This adduct, which co-elutes with the major product from the in vitro reaction between
-sulphate tamoxifen or
-acetoxytamoxifen and DNA, contains tamoxifen in the trans form covalently linked via the
carbon to the exocyclic amino group of deoxyguanosine (dG-N2-tam) (14,15). This same adduct has recently been identified in DNA extracted from the livers of tamoxifen-treated rats (16). A second major product was N-desmethyltamoxifen linked covalently to the amino group of deoxyguanosine in a similar manner (16). Previously we have shown that 4-hydroxytamoxifen also forms other minor adducts in rat liver (17).
In this study, we have conducted further detailed investigations into the adducts formed by tamoxifen in rat liver. Using an HPLC system developed previously to separate 32P-postlabelled tamoxifen adducts (17), we have identified those formed as a result of activation of tamoxifen and its metabolites N-desmethyl and N,N-didesmethyltamoxifen. This has been achieved using authentic standards of tamoxifendeoxyguanosine adducts and by dosing rats with the above compounds. Results show that the major peaks contain adducts formed via
-hydroxylation of tamoxifen and N-desmethyltamoxifen whilst minor peaks contain N-desmethyltamoxifen and N,N-didesmethyltamoxifen-derived adducts.
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Materials and methods
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Chemical methods: general procedures
1H NMR spectra were determined at 250 MHz using a Bruker 250 ARX spectrometer. Chemical shifts are expressed using the
system of units relative to the internal standard, Me4Si. Routine mass spectra of synthesized compounds were obtained with an Autospec Ultima-Q (Micromass, Manchester, UK) spectrometer. Column chromatography was performed on silica gel for flash chromatography (4063 µm; BDH, Poole, UK). Tamoxifen was a gift from Dr J. Topham (Zeneca plc, Macclesfield, UK) and N,N-didesmethyltamoxifen was a gift from Dr Ian Hardcastle (Institute of Cancer Research, Sutton, UK). Calf thymus DNA, 2'-deoxyguanosine 3'-monophosphate, tricaprylin, proteinase K, RNase A and RNase T1 were from Sigma Chemical Co. (Poole, UK). Chemical reagents and solvents of analytical grade were purchased from Aldrich Chemical Co. (Poole, UK) or Fisher Scientific Ltd (Loughborough, UK) unless otherwise stated.
-Acetoxytamoxifen was synthesized by the published procedure (14).
Synthesis of N-desmethyltamoxifen
N-Desmethyltamoxifen was synthesized from tamoxifen by a novel route using a method adapted from Montzka et al. (18) for the demethylation of tertiary methylamines. To a refluxing solution of tamoxifen (2.30 g, 6.20 mmol) in toluene (100 ml) was added 2,2,2-trichloroethyl chloroformate (2.5 ml, 30 mmol) and potassium carbonate (1.0 g). After 4 h the mixture was cooled to room temperature and extracted with 4 M NaOH (100 ml), 2 M HCl (100 ml), then water (100 ml), dried (potassium carbonate) and concentrated. Crystallization of the residue from heptane afforded the intermediate (3.0 g, 91% yield). Using positive ion electrospray mass spectrometry a protonated molecular ion (M+H)+ was observed with m/z 534 corresponding to the N-demethylated trichloroethyl carbamate intermediate.
The carbamate intermediate (2.5 g, 4.69 mmol) was dissolved in diglyme (50 ml), then 90% acetic acid (150 ml), and zinc (2.5g, 38.24 mmol) was added and the mixture stirred at room temperature overnight. The reaction mixture was extracted with toluene (3x100 ml), dried (Na2SO4), concentrated and subjected to column chromatography on silica gel (20x2.5 cm) using an initial mobile phase of 100% chloroform to elute any unreacted intermediate, followed by CH2Cl2/methanol/ammonia (95:5:0.3) to elute the product. Crystallization from heptane gave white crystals of N-desmethyltamoxifen acetate as the pure trans isomer (1.50 g, 77% yield). 1H-NMR (CDCl3)
0.92 (t, 3H, CH2CH3), 1.7 (s, 1H, D2O exchangeable NH), 2.0 (s, 3H, acetate CH3COOH), 2.5 (q, 2H, CH2CH3), 2.55 (s, 3H, NCH3), 3.05 (t, 2H, CH2CH2N), 4.0 (t, 2H, CH2CH2N), 6.55 (d, 2H, H-3,5 of C-C6H4-O), 6.8 (d, 2H, H-2,6 of C-C6H4-O), 7.07.5 (m, 10H, ArH). Using positive ion electrospray a protonated molecular ion was observed at m/z 358 representing the molecular weight minus the acetate moiety.
Reaction of
-acetoxytamoxifen with 2'-deoxyguanosine 3'-monophosphate
2'-Deoxyguanosine 3'-monophosphate (5 mg) was reacted overnight at 37°C with
-acetoxytamoxifen (10 mg) in 2 ml 100 mM TrisHCl buffer (pH 8.0). The reaction mixture was centrifuged and the supernatant extracted with 3x1 ml butanol. The pooled extracts were separated by reverse phase HPLC on a Hypersil ODS column (5µ, 250x4.6 mm i.d.) with a 0.05 M ammonium formate (pH 5.4) to methanol gradient (080% in 50 min then to 100% over 10 min at a flow rate of 1 ml/min). Four adduct peaks were collected and subject to analysis by mass spectrometry using a Quattro BIO-Q tandem quadrupole instrument (Micromass, Manchester, UK) fitted with an electrospray source operating in the positive ion mode. Each adduct peak was 32P-postlabelled by treating 10 µl aliquots as 10 µg DNA digest and separated using the HPLC system developed by Martin et al. (17).
Animals and treatment
Female F344 rats (Harlan Olac Ltd, Oxford, UK), aged 6 weeks, received 0.11 mmol/kg tamoxifen, N-desmethyltamoxifen or N,N-didesmethyltamoxifen daily by i.p. injection for 4 days. Dosing solutions were made up in tricaprylin (0.11 mmol/ml). The animals were killed 24 h after the last dose. Liver tissue was removed, immediately frozen in liquid nitrogen and stored at 80°C.
DNA isolation and 32P-postlabelling
Liver DNA was extracted by the method of Gupta (19) using proteinase K digestion, phenol/chloroform extraction and digestion with RNase A and RNase T1. DNA purity and concentration were established spectrophotometrically. 32P-postlabelling using nuclease P1 enhancement was carried out as previously described (20) and the tamoxifen adducts were separated using a HPLC system with on-line radiochemical detection (17). Mean levels of adducts ± SD are expressed as adducts/108 nucleotides.
HPLC and LC-ESI-MS analysis of tamoxifen, N-desmethyltamoxifen and metabolites
Liver (100 mg) from female F344 rats was homogenized in ice-cold methanol/DMSO (95:5 v/v, 900 µl). Samples were centrifuged (14 000 g for 10 min at 4°C) and the supernatant (50 µl) analysed by HPLC using the system previously described (21).
For on-line LC-ESI-MS analysis, the column outlet was connected to a Platform II electrospray mass spectrometer (Micromass), operating in the positive ion mode. Capillary and high voltage electrode potentials were 3.5 and 0.24 kV, respectively, and the source temperature 95°C. A Hewlett Packard 1100 solvent delivery system with autoinjector was used. The metabolites were separated isocratically on a Hypersil BDS C18 column (5µ, 250x4.6 mm i.d.) using a mobile phase of methanol/0.5 M ammonium acetate (70:30 v/v) at a flow rate of 1 ml/min. The flow leaving the column was split in the ratio 1:6 with one part entering the electrospray.
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Results
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HPLC separation of 32P-postlabelled adducts formed from in vitro reaction of
-acetoxytamoxifen with calf thymus DNA and 2'-deoxyguanosine 3'-monophosphate
We have previously shown that following long-term administration of tamoxifen in the diet to Wistar rats (35 mg/kg/day), 32P-postlabelled hepatic DNA can be separated into at least 12 different tamoxifen adduct peaks using the present HPLC system (17). These were numbered in their elution order, with the major adduct peaks 5 and 6 accounting for approximately 45 and 24%, respectively, of the total DNA adducts in the liver after 6 months dosing (17). In the present study, with only 4 days dosing, some of the minor adduct peaks were not detected. Reaction of the model ester trans-
-acetoxytamoxifen with calf thymus DNA in vitro gave three 32P-postlabelled adducts (Figure 2A
) which eluted during the isocratic phase and several minor adducts which eluted after 40 min. The major product of this reaction, which accounts for 78% of the total adducts formed, co-elutes with in vivo peak 6 and has previously been identified by Osborne et al. as a trans isomer of dG-N2-tam (14). This group further report that stereoisomers of the cis form of dG-N2-tam and an adenine adduct are also generated in smaller yields (22). The second major peak which co-elutes with peak 5 contributes ~15% of the total adduct level and could contain a diastereoisomer of trans-dG-N2-tam, whilst the minor peak eluting at 23 min which co-elutes with in vivo peak 3 could be an adenine adduct.

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Fig. 2. HPLC separation of 32P-postlabelled calf thymus DNA reacted in vitro and hepatic DNA digests from rats treated with tamoxifen, N-desmethyltamoxifen or N,N-didesmethyltamoxifen. (A) Calf thymus DNA reacted in vitro with trans- -acetoxytamoxifen (0.5 µg). Traces (B)(E) represent hepatic DNA from female F344 rats dosed daily (0.11 mmol/kg i.p.) for 4 days: (B) tamoxifen; (C) N-desmethyltamoxifen; (D) N,N-didesmethyltamoxifen; (E) tricapylin vehicle. Adducts were separated on a C18 Hypersil BDS column (250x4.6 mm i.d.) with a gradient of 80% 2 M ammonium formate, pH 4.0, 20% acetonitrile:methanol (6:1 v/v) for 40 min followed by a linear gradient of 2045% acetonitrile:methanol (6:1 v/v) over 20 min.
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To confirm that the products in peaks 5 and 6 contained tamoxifen linked to deoxyguanosine,
-acetoxytamoxifen was reacted with 2'-deoxyguanosine 3'-monophosphate. This incubation yielded four products separable by HPLC, which were collected and analysed by electrospray mass spectrometry. Each peak gave a protonated molecular ion with m/z 717 (Figure 3
), consistent with the 3'-monophosphate of the tamoxifendeoxyguanosine adduct shown by Osborne et al. (14). The adducts were 32P-postlabelled and analysed by HPLC with on-line radiochemical detection. The major post-labelled products were found to co-elute with either peak 5 or peak 6 formed in the livers of tamoxifen-treated rats. Minor products were also detected after 40 min.

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Fig. 3. Positive ion electrospray mass spectrometry of a tamoxifendeoxyguanosine 3'-monophosphate adduct peak. Each of four peaks isolated by HPLC gave a protonated molecular ion with m/z 717 consistent with the known dG-N2tamoxifen adducts.
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HPLC separation of 32P-postlabelled rat liver DNA from F344 rats treated with tamoxifen, N-desmethyltamoxifen and N,N-didesmethyltamoxifen
Figure 2B
shows 32P-postlabelled liver DNA digests (10 µg DNA) from rats administered tamoxifen for 4 days. This gives the typical pattern of tamoxifen adducts, with the two major peaks being 5 and 6. Also detected are three additional minor peaks previously unreported (I, II and III). Following treatment with N-desmethyltamoxifen, two main peaks were detected which co-eluted with adduct peaks 3 and 5 formed in tamoxifen-treated animals (Figure 2C
). Since peak 5 was also observed in
-acetoxytamoxifen-treated calf thymus DNA (see above), this peak obviously contains at least two products. A third minor adduct which co-elutes with peak II was also detectable. Following treatment of rats with N,N-didesmethyltamoxifen two hepatic DNA adducts were induced, the main one co-eluting with peak 3 and the minor one, previously undetected, is here named peak 1a (Figure 2D
). Peak 1a elutes between peaks 1 and 2 seen in rats dosed long-term with tamoxifen (17). The level of hepatic DNA adducts induced by N-desmethyltamoxifen (100 ± 33 adducts/108 nucleotides, n = 4) was comparable with that found following tamoxifen administration (109 ± 40 adducts/108 nucleotides, n = 3). In contrast, N,N-didesmethyltamoxifen was activated to a lesser extent yielding significantly lower levels of adducts (19 adducts/108 nucleotides, n = 2).
Tamoxifen and N-desmethyltamoxifen metabolites in the livers of treated rats
After 4 days tamoxifen treatment (0.11 mmol/kg) the level of N-desmethyltamoxifen detectable in rat liver extracts is higher than tamoxifen itself. Following dosing with an equimolar concentration of N-desmethyltamoxifen, this compound accumulated in the liver giving concentrations approximately twice that present in tamoxifen-dosed rats (Table I
). No tamoxifen or monohydroxylated metabolites were detected after N-desmethyltamoxifen treatment, confirming that any adducts formed must contain an N-demethylated tamoxifen moiety (Figure 4
). The concentration of 4-hydroxylated metabolites was similar in both groups of animals. Using LC-ES-SIM-MS analysis, four peaks with [M+H]+ ions at m/z 374, corresponding to mono-oxygenated derivatives of N-desmethyltamoxifen, were detected in the livers of both tamoxifen- and N-desmethyltamoxifen-dosed animals. If it is assumed that these N-demethylated metabolites elute in the same order as their corresponding tamoxifen metabolites (m/z 388) then A, B, C and D are N-demethylated forms of trans-
-hydroxytamoxifen, cis-
-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen, respectively.
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Table I. Hepatic concentrations of tamoxifen and metabolites in F344 rats treated with tamoxifen or N-desmethyltamoxifen (0.11 mmol/kg daily) by i.p. injection for 4 days
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Discussion
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The products of the reaction between
-acetoxytamoxifen and the exocyclic amino function of deoxyguanosine in calf thymus DNA in vitro can exist as four stereoisomers (15). In this study the main adduct, a trans form of dG-N2-tam, accounts for 78% of the total adducts and co-elutes with peak 6 formed in DNA extracted from the livers of tamoxifen-treated rats (Figure 2A and B
). The relative importance of each isomer in the carcinogenic mechanism of tamoxifen is currently unknown. As it remains to be confirmed whether the present HPLC system can resolve diastereoisomers of 32P-postlabelled dG-N2-tam adducts, it is not known whether all four isomers are formed in vivo or whether there is some stereoselectivity in the reaction between the carbocation and double-stranded DNA.
N-Desmethyltamoxifen is a major metabolite of tamoxifen present in liver extracts at a higher concentration than the parent drug (Table I
). Relatively higher levels of N-desmethyltamoxifen have also been found both in the liver of rats (23) and in liver and uterine tissues of women (10) following long-term dosing with tamoxifen. Recent results of Rajaniemi et al. have suggested that in rats dosed with tamoxifen, the N-desmethyltamoxifen metabolite undergoes activation to a somewhat greater extent than tamoxifen itself (16). We have found that N-desmethyltamoxifen induces similar levels of adducts as tamoxifen. Dosing with this compound produces three 32P-postlabelled hepatic DNA adducts which co-elute with adduct peaks II, 3 and 5 in tamoxifen-dosed animals. The major component formed co-elutes with peak 5 and is presumably an N-demethylated derivative of trans-dG-N2-tam (Figure 2C
). N,N-Didesmethyltamoxifen is activated to a much lesser extent than tamoxifen or N-desmethyltamoxifen, producing 5-fold lower levels of adducts. An adduct co-eluting with peak 3 is a major product in the livers of N,N-didesmethytamoxifen-treated rats and a minor product in the livers of N-desmethyltamoxifen-treated rats. Therefore, the major component of this peak is probably an N,N-didemethylated form of trans-dG-N2-tam, arising via
-hydroxylation of N,N-didesmethyltamoxifen. An adduct corresponding to in vivo peak 3 was also produced following reaction of
-acetoxytamoxifen with calf thymus DNA. It is therefore likely that this peak may contain other products such as the adenine adduct reported by Osborne et al. (22). Only low levels of N,N-didesmethyltamoxifen are detected in the livers of rats given tamoxifen but in primates and women this metabolite accumulates in the liver (10,24). N,N-Didesmethyltamoxifen is a potent inhibitor of certain CYP-catalysed reactions and it has been suggested that this metabolite may limit the activation of tamoxifen to the
-hydroxylated reactive metabolite (24).
Past studies using 32P-postlabelling with polyethyleneiminecellulose TLC separation have failed to detect DNA damage in the livers of women given tamoxifen (20). With the greater resolution and sensitivity of the present analytical system, we have shown the importance of N-demethylated metabolites in tamoxifenDNA adduct formation. As with tamoxifen, the major adducts are formed as a consequence of
-hydroxylation, but it has previously been shown that activation of 4-hydroxytamoxifen can also result in DNA adduct formation through oxidation to a quinoine methide (25) or via radical intermediates (26). It is therefore likely that a similar route of activation could also take place with the 4-hydroxy metabolites of N-desmethyltamoxifen and N,N-didesmethyltamoxifen, leading to the potential formation of DNA adducts. We have shown that N-desmethyltamoxifen and N,N-didesmethyltamoxifen are activated in a similar manner to tamoxifen leading to a complex mixture of adducts which at the present time cannot be fully separated. Therefore, care has to be taken when interpreting results and determining the relative importance of individual adducts and the metabolites they are derived from in the carcinogenic process.
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Acknowledgments
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The authors would like to thank Prof. David Phillips and colleagues for helpful discussions and sharing their data with us prior to submission of this paper. We also thank Dr John Brown (Pharmaceutical Chemistry, University of Bradford, UK) for synthesizing
-hydroxytamoxifen, Dr J.Topham, (Zeneca, Macclesfield, UK) for supplying the tamoxifen and Dr Ian Hardcastle (Institute of Cancer Research, Sutton, UK) for generously supplying the N,N-didesmethyltamoxifen. We thank N.Ravi, A.M.Davies and J.Lamb for their expert technical support.
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Notes
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1 Present address: Lawrence Livermore National Laboratory,7000 East Avenue L452, CA 94551-9900, USA 
2 To whom correspondence should be addressedEmail: eam6{at}le.ac.uk 
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Received March 17, 1999;
revised June 17, 1999;
accepted June 17, 1999.