4-Hydroxytamoxifen Gives DNA Adducts by Chemical Activation, but

Martin R. Osborne,*,† Warren Davis,† Alan J. Hewer,† Ian R. Hardcastle,‡ and. David H. Phillips†. Section of Molecular Carcinogenesis, Haddo...
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Chem. Res. Toxicol. 1999, 12, 151-158

151

4-Hydroxytamoxifen Gives DNA Adducts by Chemical Activation, but Not in Rat Liver Cells Martin R. Osborne,*,† Warren Davis,† Alan J. Hewer,† Ian R. Hardcastle,‡ and David H. Phillips† Section of Molecular Carcinogenesis, Haddow Laboratories, and CRC Centre for Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Received August 17, 1998

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumors in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and in rat hepatocytes in culture. These arise primarily from its metabolite R-hydroxytamoxifen, and may also arise, in part, from another metabolite, 4-hydroxytamoxifen. We have prepared two model compounds for the potential reactive metabolite formed from 4-hydroxytamoxifen in rat liver. One of these was its R-acetoxy ester. This was much more reactive than that from tamoxifen, and could not be isolated in pure form. It reacted with DNA in the same way that R-acetoxytamoxifen did, to give adducts which were isolated by hydrolysis and chromatography, and identified as alkyldeoxyguanosines. The second derivative was R,β-dehydro-4-hydroxytamoxifen. This also reacts with DNA in vitro, to give the same products as those from R-acetoxy-4-hydroxytamoxifen. Reaction probably proceeds through the same resonancestabilized carbocation in either case. However, when primary cultures of rat hepatocytes were treated with either 4-hydroxytamoxifen, 4,R-dihydroxytamoxifen, or R,β-dehydro-4-hydroxytamoxifen at a concentration of 10 µM, no adducts could be detected in their DNA by the 32Ppostlabeling technique. Similarly, no adducts could be found in the liver DNA of female Fischer F344 rats treated orally (at 0.12 mmol kg-1) with the same substances. If 4-hydroxytamoxifen is metabolized to 4,R-dihydroxytamoxifen in rat liver, then either this substance is not converted to reactive esters or they are rapidly detoxified. Tamoxifen1 (Scheme 1, 1) is an anti-estrogen which is widely used in the treatment of breast cancer. It is also being tested as a tumor preventive agent in healthy women (1, 2). However, it shows evidence of genotoxicity in some systems, notably, in inducing DNA adducts (35) and cancer (6-9) in the liver of rats. The principal mechanism of DNA bonding is believed to be as follows. Tamoxifen is oxidized by cytochrome P450 to R-hydroxytamoxifen. This is converted by a sulfotransferase into its sulfate ester which is unstable under physiological conditions and loses the sulfate group to yield a carbocation. This is stabilized by conjugation with the poxyphenylvinyl group; it is highly reactive, and alkylates DNA (10-12). We have prepared R-acetoxytamoxifen (2) as a model compound for such a reactive ester metabolite, and have shown that it reacts with DNA to give several tamoxifen-deoxyguanosine and tamoxifen-deoxyadenosine adducts (13, 14). * To whom correspondence should be addressed. E-mail: martino@ icr.ac.uk. † Section of Molecular Carcinogenesis. ‡ CRC Centre for Cancer Therapeutics. 1 Abbreviations: tamoxifen, (Z)-1-{4-[2-(dimethylamino)ethoxy]phenyl}-1,2-diphenyl-1-butene; R-hydroxytamoxifen, (E)-4-{4-[2-(dimethylamino)ethoxy]phenyl}-3,4-diphenyl-3-buten-2-ol; R-acetoxytamoxifen, (E)-2-acetoxy-4-{4-[2-(dimethylamino)ethoxy]phenyl}-3,4-diphenyl-3butene; 4,R-dihydroxytamoxifen, (E)-4-{4-[2-(dimethylamino)ethoxy]phenyl}-4-(4-hydroxyphenyl)-3-phenyl-3-buten-2-ol; R,β-dehydro-4-hydroxytamoxifen, 1-{4-[2-(dimethylamino)ethoxy]phenyl}-1-(4-hydroxyphenyl)-2-phenyl-1,3-butadiene; tamoxifen quinone methide, 4-(1-{4[2-(dimethylamino)ethoxy]phenyl}-2-phenylbut-2-enylidene)cyclohexa2,5-dien-1-one; ODS, octadecyl silica.

It has been proposed (5, 15) that another metabolite of tamoxifen, 4-hydroxytamoxifen (Scheme 2, 4), also forms DNA adducts. By analogy with tamoxifen, one would expect that further metabolism of this would give 4,R-dihydroxytamoxifen (5), though this has not yet been detected as a metabolite. After esterification, this would alkylate DNA. The intermediate carbonium ion (7a) would be stabilized by the hydroxyl group, and could be more accurately represented by a quinone methide structure (7b). The neutral quinone methide 9 was postulated as an intermediate (15, 16) but has not yet been prepared. Experimentally, 4-hydroxytamoxifen can be covalently bound to DNA in vitro after activation by various means: with a peroxidase (17), with a rat uterus extract in the presence of peroxide (18), with rat liver microsomes (19, 20), or by oxidation with silver oxide (20) or manganese dioxide (21). The mechanism of these reactions has not been determined. In one case, the resulting DNA adduct was characterized. Marques and Beland (21) isolated a deoxyguanosine adduct from DNA treated with 4-hydroxytamoxifen and manganese dioxide, and identified it as the R-substituted derivative 10 (a mixture of trans and cis adducts, 10a and 10b, respectively). They considered the quinone methide 9 to be the reactive intermediate. To understand the mechanism better, we prepared the metabolite 4,R-dihydroxytamoxifen by chemical synthesis, and showed that it reacts with DNA in vitro to a small extent (22). In this paper, we report the preparation of a model ester, the R-acetoxy derivative of 4-hydroxy-

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Scheme 1. Structures of (1) Tamoxifen, (2) r-Acetoxytamoxifen, and (3) 4,r-Diacetoxytamoxifen

Scheme 2. Structures of (4) 4-Hydroxytamoxifen, (5) 4,r-Dihydroxytamoxifen, (6) r-Acetoxy-4-hydroxytamoxifen, (7) the Derived Carbonium Ion, (8a) Trans and (8b) Cis Isomers of r,β-Dehydro-4-hydroxytamoxifen, (9) Tamoxifen Quinone Methide, and (10a and 10b) the Probable Structures of Deoxyguanosine Adducts Derived from 4-Hydroxytamoxifena

a

Ph, phenyl; Ar, 4-[2-(dimethylamino)ethoxy]phenyl.

tamoxifen (6), and comparison of its reactivity with that of the corresponding tamoxifen derivative (2). We have been unable to isolate the quinone methide 9, but have prepared a tautomer of it, R,β-dehydro-4-hydroxytamoxifen (8), and studied its behavior in solution and its reaction with DNA. We also looked (by the 32P-postlabeling method) for DNA adducts formed from 4-hydroxytamoxifen, 4,R-dihydroxytamoxifen, and R,β-dehydro4-hydroxytamoxifen in rat liver and in rat hepatocytes in culture, to determine whether the increased reactivity of the 4-hydroxylated derivatives was also seen in intact rat cells.

Experimental Procedures Tamoxifen, 4-hydroxytamoxifen, salmon testis DNA, tricaprylin, collagenase IV, proteinase K, RNase A, RNase T1, and water-saturated phenol were purchased from Sigma Chemical Co. (Poole, Dorset, U.K.). R-Acetoxytamoxifen (13) and 4,Rdihydroxytamoxifen (22) were prepared as described previously. All operations with tamoxifen derivatives were carried out in darkness or subdued light where possible. Mass spectroscopy was carried out using a Finnigan TSQ 700 triple-quadrupole mass spectrometer fitted with an electrospray ion source as described previously (14). Proton magnetic resonance spectra were obtained on a Bruker AC250 spectrometer.

Reverse phase liquid chromatography was carried out on a Waters (Watford, U.K.) apparatus. In most experiments, we used a 4.6 mm × 250 mm column (Fisons, Loughborough, U.K.) packed with ODS (Nucleosil, 5 µm). The solvent (0.8 mL/min) was a water/acetonitrile mixture, containing 0.05 M ammonium formate throughout and either (A) 0 to 75% MeCN over the course of 50 min, (B) 45 to 75% MeCN over the course of 20 min, and then 75% MeCN, or (C) 15 to 45% MeCN over the course of 30 min, and then 45% MeCN. For separation of the two isomers of R,β-dehydro-4-hydroxytamoxifen, a “Jupiter” ODS column (4.6 mm × 250 mm; Phenomenex, Macclesfield, U.K.) was used, eluting with methanol/water (6:4) containing 0.03 M ammonium formate (pH 7) at 0.8 mL/min (system D). In each case, tamoxifen derivatives were detected in the eluate by their ultraviolet absorbance at 254 nm. Preparation of Acetates. These were prepared by acetylation of 4,R-dihydroxytamoxifen (5) in acetic anhydride and pyridine, as described for R-acetoxytamoxifen (13). The reaction gave both mono- and diacetates. Excess reagent (4 mg of dihydroxytamoxifen with 10 µL of acetic anhydride) gave predominantly 4,R-diacetoxytamoxifen (3). The mass spectrum (positive ion) of this showed ions at m/z 510 (MNa+, 51%) and 488 (MH+, 100%), as expected for the diacetate C30H33O5N. Acetylation with less reagent (2.4 mg of dihydroxytamoxifen with 2 µL of acetic anhydride, 22 °C, 18 h) gave a mixture of products. This was separated by chromatography on silica or

DNA Adducts of 4-Hydroxytamoxifen on ODS (system B), yielding three products in a ratio of about 5:1:4. The first of these was the phenyl ester, R-hydroxy-4acetoxytamoxifen.This was unreactive (half-life in water of about 72 h) and of no further interest. The second was the required aliphatic ester, R-acetoxy-4-hydroxytamoxifen (Scheme 2, 6). It was immediately dried over sodium sulfate and stored in ether solution at -18 °C. It was stable under these conditions, but rapidly hydrolyzed by water; the reactivity and small yield precluded complete characterization. The third product was 4,Rdiacetoxytamoxifen (3). To determine the rate of hydrolysis, an HPLC-purified sample of each R-acetoxy compound (0.2 mg) was incubated at 37 °C in 15 mL of water/acetonitrile (85:15, with 0.08 M sodium phosphate at pH 7). Samples (1 mL) were taken and kept at -20 °C prior to analysis by chromatography on ODS (system B). The amount of remaining ester was estimated from the area of its peak on the ultraviolet absorption trace, compared with those of the hydrolysis products. (Z)-1-(4-Acetoxyphenyl)-1-[4-(2-chloroethoxy)phenyl]-2phenyl-1,3-butadiene (12). A mixture of the vinyl bromide 11 (22) (0.94 g, 2 mmol), vinyltributyltin (0.6 g, 3 mmol), tetrakistriphenylphosphine palladium(0) (0.08 g, 0.1 mmol), and toluene (40 mL) was degassed and heated under reflux for 16 h under argon. It was filtered through Celite, eluted with ether (2 × 10 mL), and evaporated. The residue was purified by column chromatography (silica; 3:10 hexane/ether). Crystallization (ethanol) gave 12 as a white solid (0.583 g, 70%). 1H NMR spectrum (CDCl3): δ 2.23 (3, s, OAc), 3.74 (2, t, J ) 5.7 Hz, CH2Cl), 4.10 (2, t, J ) 5.9 Hz, CH2O), 4.91 (2, d, J ) 17.2 Hz, vinyl H), 5.15 (2, d, J ) 11.8 Hz, vinyl H), 6.55 (2, d, J ) 8.0 Hz, ArH), 6.75-6.80 (3, m, vinyl H and ArH), 7.07-7.28 (9, m, ArH). Anal. Calcd for C26H23ClO3: C, 74.55; H, 5.53; Cl, 8.46. Found: C, 74.29; H, 5.70; Cl, 8.55. (E) and (Z)-1-[4-(2-Chloroethoxy)phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1,3-butadiene (13). Acetate 12 (0.101 g, 0.24 mmol), a saturated sodium hydrogen carbonate solution (1 mL), and methanol (10 mL) were stirred for 48 h. Then the mixture was diluted with ether (30 mL) and washed with water (2 × 30 mL). The aqueous portion was extracted with ether (20 mL). The organic portions were combined, dried (MgSO4), and evaporated to give 13 as an oil (0.145 g, 92%) which was used without further purification. 1H NMR spectrum (CDCl3): δ 3.74 (2, t, J ) 5.8 Hz, trans ClCH2), 3.86 (2, t, J ) 5.9 Hz, cis ClCH2), 4.11 (2, t, J ) 5.9 Hz, trans OCH2), 4.28 (2, t, J ) 5.9 Hz, cis OCH2), 4.88 (1, s, vinyl H), 4.96 (1, s, vinyl H), 5.03 (1, broad s, vinyl H), 5.12 (1, s, vinyl H), 5.16 (1, s, vinyl H), 5.31 (1, s, vinyl H), 6.48 (2, d, ArH), 6.57 (2, d, ArH), 6.71-6.94 (10, m, ArH), 7.12-7.26 (12, m, ArH). Mass spectrum (negative ion): m/z 375 (M - H, 100% relative intensity). 1-{4-[2-(Dimethylamino)ethoxy]phenyl}-1-(4-hydroxyphenyl)-2-phenyl-1,3-butadiene (r,β-Dehydro-4-hydroxytamoxifen, 8). A mixture of chloro compound 13 (0.145 g, 0.38 mmol) and a dimethylamine solution (10 mL; 30% ethanol) was heated to 100 °C in a sealed bomb for 16 h, than allowed to cool, diluted with ether (50 mL), washed with a sodium hydrogen carbonate solution (30 mL) and water (2 × 30 mL), dried (Na2SO4), and evaporated to give 8 as a yellow oil. 1H NMR spectrum (CDCl3): δ 2.67 [6, s, N(CH3)2], 3.17 (2, broad s, CH2N), 4.26 (2, broad s, CH2O), 4.88 (1, dd, Ja ) 17.3 Hz, Jb ) 1.6 Hz, vinyl H), 5.11 (1, dd, Ja ) 10.8 Hz, Jb ) 1.6 Hz, vinyl H), 6.48 (2, d, J ) 8.6 Hz, ArH), 6.66 (2, d, J ) 8.7 Hz, ArH), 6.69-6.83 (3, m, ArH and vinyl H), 7.11-7.20 (7, m, ArH), 8.44 (1, s, ArOH). Mass spectrum (negative ion): m/z 384 (M - H, 100% relative intensity). 8 was separated by liquid chromatography on an ODS column (system D) into two major components, with retention times of about 30 and 34 min and roughly equal in quantity (peak heights ratio of 46:54). These accounted for 82% of the UV-absorbing material eluted from the column. Separation of 2 mg of crude material in 35 runs yielded 1 mg of each isomer 8a and 8b, both consisting of 97% of one isomer. The UV spectrum of either isomer showed λmax values of 251.5 and 306 nm, establishing that their electronic structures were equiva-

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 153 lent, and distinct from that of 4-hydroxytamoxifen (λmax ) 244 and 286 nm). Reaction of Tamoxifen Derivatives with DNA. The compound was dissolved in 5 mL of ethanol and reacted with 10 or 20 mg of DNA in 10 mL of water (unbuffered). NaOAc (1 mL, 2.5 M) was added, and the mixture was extracted seven times with 15 mL of ether to remove unreacted material. The DNA was precipitated with 2 volumes of ethanol, washed, dried, and dissolved in 5 mL of water. Degradation to nucleosides was carried out with the enzymes DNase 1 [0.5 mg; 20 h in 1 mL of 10 mM Tris/10 mM MgCl2 (pH 7)], snake venom phosphodiesterase [Sigma type VIII-S, 0.05 unit; 6 h in 0.1 M Tris (pH 9)], and alkaline phosphatase (Sigma type III, 15 units, 17 h), all at 37 °C. The hydrolysate was separated by reverse phase chromatography (system A) in three portions of 2 mL. In Vivo Treatment of Rats. Female Fischer F-344 rats were divided into four groups consisting of three animals per group. Each group was orally dosed with tamoxifen, R-hydroxytamoxifen, 4-hydroxytamoxifen, or 4,R-dihydroxytamoxifen at 0.12 mmol kg-1. All of the compounds were dissolved in tricaprylin at a concentration of 1 mg/mL. A control group of three rats was given tricaprylin alone. Twenty-four hours after dosing, the rats were killed by dislocation of the neck. The livers were removed and stored at -80 °C before the DNA was isolated. Preparation and Treatment of Rat Hepatocytes. Hepatocytes were isolated from the livers of female Fischer F-344 rats (8-10 weeks old), according to standard procedures (23). Cell viabilities were consistently >80% as judged by trypan blue exclusion. Cells were established in primary culture as described previously (24). The cells were allowed to attach to the flask for 3 h at 37 °C in an atmosphere of 5% CO2 in the presence of 10% fetal calf serum. The medium was changed to exclude serum, and the cells were treated with dimethyl sulfoxide solutions of tamoxifen (1, final concentration of 10 µM), R-hydroxytamoxifen (1 µM), 4-hydroxytamoxifen (4, 10 µM), 4,Rdihydroxytamoxifen (5, 10 µM), or R,β-dehydro-4-hydroxytamoxifen (8, 10 µM). After incubation for 18 h, the cells were harvested and separated from the medium before DNA isolation. DNA Isolation. DNA was isolated and purified from rat liver or hepatocytes essentially by a published procedure (25). Cell pellets were suspended in EDTA (10 mM) containing sodium dodecyl sulfate (1%) and proteinase K (0.1%) (w/v) and incubated at 37 °C for 2 h. The mixture was then extracted sequentially with equal volumes of phenol, phenol/chloroform/3-methyl-1butanol (25:24:1), and chloroform/3-methyl-1-butanol (24:1). NaCl (5 M, 0.1 volume) and cold ethanol (2 volumes) were then added to the aqueous phase to precipitate the DNA. It was redissolved in 1 mM EDTA/50 mM Tris (pH 8) to which were added RNase A (0.15 mg/mL) and RNase T1 (0.75 unit/mL), and the mixture was incubated at 37 °C for 30 min. It was then extracted twice with equal volumes of chloroform/3-methyl-1butanol (24:1), and the DNA was reprecipitated as before. DNA was redissolved in 1.5 mM NaCl/0.15 mM sodium citrate and stored at -20 °C before postlabeling. 32P-Postlabeling.

DNA samples isolated from cells or tissue were subjected to the nuclease P1 enrichment method of postlabeling analysis (26). Aliquots of DNA (4 µg) were taken and evaporated to dryness using a Savant Speedvac SVC100 vacuum centrifuge. The DNA was digested overnight at 37 °C with micrococcal nuclease (0.14 unit) and spleen phosphodiesterase (1.2 mg, 1.2 µL) in sodium succinate (20 mM)/calcium chloride (10 mM) (pH 6.0, 0.8 µL) and distilled water (2.8 µL). Samples were then further digested for 1 h at 37 °C with nuclease P1 (0.15 unit, 0.96 µL) in sodium acetate (62.5 mM, 2.4 µL) and ZnCl2 (0.3 mM, 1.44 µL). 32P-Postlabeling of each sample was carried out by incubation at 37 °C for 30 min with [γ-32P]ATP (50 µCi, specific activity of approximately 4000 Ci mmol-1) and polynucleotide kinase (6 units, 0.6 µL) in bicine (14 mM, pH 9.0)/magnesium chloride (7 mM)/dithiothreitol (7 mM)/spermidine (0.7 mM) (1 µL).

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A

B

Figure 1. Rate of hydrolysis of various esters in water/ acetonitrile at pH 7. The half-lives were as follows: 4,Rdiacetoxytamoxifen, 3.2 h; R-acetoxytamoxifen, 2.4 h; and R-acetoxy-4-hydroxytamoxifen, 7 min. Postlabeling analysis of salmon testis DNA was performed in the same way, except that the nuclease P1 digestion was omitted. TLC of DNA Adducts. Radiolabeled digests were applied to the origins of 10 cm × 10 cm polyethyleneimine-cellulose TLC plates (Macherey-Nagel, Duren, Germany). Multidirectional chromatography was carried out using the following solutions: D1, sodium phosphate (2.3 M, pH 5.8) overnight onto a paper wick outside the tank; D2, lithium formate (2.275 M) and urea (5.525 M) at pH 3.5; and D3, lithium chloride (0.52 M), TrisHCl (0.325 M), and urea (5.525 M) at pH 8.0. DNA adducts were detected as radioactive spots on the TLC plates, visualized using a Packard InstantImager (Canberra Packard, Pangbourne, Berks, U.K.). Quantification was carried out using the measured specific activity of the [γ-32P]ATP (25) with background subtraction.

Results Reactivity of Acetates. The reactivity of each acetate ester was estimated from its rate of hydrolysis in an aqueous medium [85:15 water/acetonitrile (pH 7)], and the results are shown in Figure 1. The R-acetoxy-group of 4,R-diacetoxytamoxifen (3) was hydrolyzed, giving R-hydroxy-4-acetoxytamoxifen. The half-life for this was 3.2 h, close to that of R-acetoxytamoxifen. The monoester, R-acetoxy-4-hydroxytamoxifen, was hydrolyzed much faster; the half-life in water at 37 °C was about 7 min. The main product of hydrolysis was not 4,R-dihydroxytamoxifen as might be expected, but a nonpolar compound (retention time of 26 min, system B) having an ultraviolet spectrum quite different from that of tamoxifen: λmax ) approximately 249 (A ) 0.040) and 312 nm (A ) 0.034). This retention time and UV spectrum were very similar to those of R,β-dehydro-4-hydroxytamoxifen (8, see below), which can arise by loss of acetic acid from the ester (Scheme 2). Reaction of 4,r-Diacetoxytamoxifen with DNA. 4,R-Diacetoxytamoxifen (3, 2.5 mg) was dissolved in 5 mL of ethanol and reacted with 20 mg of DNA in 10 mL of water for 24 h. The DNA was isolated and then degraded with enzymes to nucleosides. The conditions of degradation included an alkaline step (24 h at pH 9) which caused hydrolysis of the phenolic esters so that the resultant nucleoside adducts had a free 4-hydroxyl group. Upon analysis on an ODS column (system A), the mixture gave the elution profile shown in Figure 2B. Two principal adducts, DG1 and DG2, were seen eluting at 38 and 39

C

Figure 2. Elution of nucleosides from hydrolysates of DNA treated with (A) R-acetoxytamoxifen, (B) 4,R-diacetoxytamoxifen, or (C) R,β-dehydro-4-hydroxytamoxifen (chromatography in system A). The peaks at about 16, 18, and 22 min corresponded to deoxycytidine, deoxyguanosine and thymidine, and deoxyadenosine, respectively. The labeled peaks corresponded to nucleoside-drug adducts. The material eluted before them, at around 35 min, probably consisted of undigested di- or oligonucleotide adducts.

min, respectively. They were eluted earlier than the corresponding tamoxifen adducts (Figure 2A), owing to the addition of the polar phenolic group. DG1 and DG2 had very similar retention times, but could be separated by repeated chromatography on the same system, using a shallower gradient (15 to 45% MeCN over the course of 30 min). They were clearly adducts of 4-hydroxytamoxifen and deoxyguanosine, as shown by their ultraviolet spectra, which were similar to that of a mixture of 4,R-dihydroxytamoxifen and N2methylguanosine, but with slightly increased wavelength maxima (adducts, λmax ) 247 nm, with a shoulder at 280 nm at neutral pH; mixture, λmax ) 245 nm, with a shoulder at 275 nm). Their mass spectra (positive ion) were similar to each other. DG1 gave ions at m/z 675 (MNa+, 17% relative intensity), 653 (MH+, 92% relative intensity), 537 (MH+ - deoxyribose, 17% relative intensity), 393 (21% relative intensity), 354 (19% relative intensity), 349 (MNa22+, 26% relative intensity), and 338 (MHNa2+, 100% relative intensity). DG2 gave ions at m/z 675 (MNa+, 20% relative intensity), 653 (MH+, 100% relative intensity), 488 (unidentified, 70% relative intensity), 446 (37% relative intensity), 386 (M - dGuo, 26% relative intensity), and 118 (52% relative intensity). These are as expected for a 4-hydroxytamoxifen-deoxyguanosine adduct (C36H40N6O6). It is probable that DG1 and DG2 were the trans and cis isomers of R-(deoxygua-

DNA Adducts of 4-Hydroxytamoxifen

Figure 3. Autoradiographs of polyethyleneimine-cellulose thin layer plates after chromatography of 32P-labeled tamoxifennucleoside bisphosphates. The origins are at the bottom left corner, and the directions of development are as follows: D1, downward; D2, upward; and D3, to the right. The adducts were derived from salmon sperm DNA treated with (A) 4,R-dihydroxytamoxifen at pH 5, (B) R-acetoxy-4-hydroxytamoxifen, (C) R,βdehydro-4-hydroxytamoxifen, and (D) the product of oxidation of 4-hydroxytamoxifen with manganese dioxide.

nosin-N2-yl)-4-hydroxytamoxifen, respectively (10a and 10b in Scheme 2), i.e., identical to the tamoxifen adducts identified previously (13, 14) except for the addition of a hydroxyl group. We did not have enough material for complete identification by proton magnetic resonance spectroscopy. Reaction probably proceeds through a carbocation (Scheme 2, 7) which can rotate freely about the central bond, resulting in products with both trans and cis configurations. Reaction of r-Acetoxy-4-hydroxytamoxifen with DNA. Reaction of R-acetoxy-4-hydroxytamoxifen (6) was carried out in the same way, but on a smaller scale (2 mg of DNA and 20 µg of ester). The DNA was degraded to nucleosides and analyzed on an ODS column. The resulting elution profile (not shown) was similar to that

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from 4,R-diacetoxytamoxifen (Figure 2B), with products at 38 and 39 min, probably identical to the adducts DG1 and DG2 prepared earlier. In this case, DG1 and DG2 were equal in amount, suggesting that the alkylation proceeds through an intermediate having free rotation about the central double bond, such as the carbonium ion or quinone methide (7 or 9). The DNA was also analyzed by the 32P-postlabeling procedure, and the resulting autoradiograph is shown in Figure 3B. The pattern of adducts was identical to that obtained when DNA was incubated with 4,R-dihydroxytamoxifen in an acidic medium (Figure 3A; 22). The two prominent spots may correspond to the bisphosphates of adducts DG1 and DG2, but this has not yet been established. The level of adducts was about 1.6 per 1000 nucleotides. Synthesis and Properties of r,β-Dehydro-4-hydroxytamoxifen (Scheme 3). This derivative was prepared from the vinyl bromide 11 (22). Palladium coupling with vinyltributyltin in toluene at reflux afforded diene 12 in 70% yield. Hydrolysis of the acetate group with a sodium carbonate solution in methanol gave phenol 13 in 92% yield. Reaction of 13 with a dimethylamine solution in ethanol at 100 °C gave R,β-dehydro4-hydroxytamoxifen 8; this step was accompanied by cistrans isomerization. The isomers were separated by chromatography on ODS. They were easily interconverted. A sample of 8a was dissolved in neutral water/ methanol [65:35, containing 50 mM sodium phosphate and 17 mM ammonium formate (pH 7)] at 37 °C and examined at intervals by chromatography on ODS (system D). It was converted to an equilibrium mixture of 8a and 8b (49:51), with a half-life of 50 h. 8b gave the same mixture, with a half-life of 96 h. The process of interconversion presumably involves tautomerism from the dienol form to the quinone methide (Scheme 2, 9), through the intermediate carbocation 7. This quinone and carbocation can freely rotate to give either the cis or trans isomer. However, no change in UV spectrum was observed, nor were any new components seen on the chromatogram; so the quinone methide intermediate must be considerably less stable than either of the dienol forms.

Scheme 3. Synthesis of r,β-Dehydro-4-hydroxytamoxifena

a

(a) Vinyltributyltin, Pd(PPh3)4, toluene, reflux; (b) Na2CO3, water/methanol; (c) Me2NH, EtOH, 100 °C.

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Figure 4. Comparison of adducts DG1 and DG2 from different sources, by chromatography on a “Jupiter” ODS column (system C): (A) nucleoside adducts made from R,β-dehydro-4-hydroxytamoxifen, (B) same as panel A with added adduct DG1 from diacetoxytamoxifen (elution at 25 min in this system), (C) same as panel A but with added adduct DG2 (26 min), (D) nucleoside adducts made by the method of Marques and Beland (21), (E) same as panel D with added adduct DG1 from diacetoxytamoxifen, and (F) same as panel D but with added adduct DG2.

Reaction of r,β-Dehydro-4-hydroxytamoxifen with DNA. DNA (10 mg) was reacted with about 1.4 mg of R,β-dehydro-4-hydroxytamoxifen (8, mixed isomers) in water/ethanol for 3 days at 37 °C and recovered as described above. A sample was analyzed by 32P-postlabeling and TLC, giving the pattern of spots shown in Figure 3C. This was identical to that obtained from DNA treated with 4,R-dihydroxytamoxifen or R-acetoxy-4hydroxytamoxifen (Figure 3A,B). The level of adducts was 1 per 5000 nucleotides. 8 is therefore much more reactive to DNA than R-hydroxytamoxifen, which gave 1 adduct per 1 million nucleotides under these conditions, but much less reactive than R-acetoxytamoxifen, which gave 1 per 100. Addition of buffers to the 8/DNA reaction medium considerably reduced the yield of adducts; in 0.05 M phosphate or 0.15 M acetate buffer, the yield was only 1 and 3 adducts per 1 million nucleotides, respectively. Another sample of the reacted DNA was digested with enzymes to nucleosides as described above, and separated on ODS as described above (system A). Two small adduct peaks were seen, equal in quantity and coming at 40 and 41 min (Figure 2C). Despite their apparent displacement from DG1 and DG2 of Figure 2B, they were identified as the same products by chromatography of mixtures of the adducts from 8 with DG1 and DG2. The results are shown in Figure 4A-C. Comparison of Adducts with Previously Reported Products (21). Marques and Beland (21) reported the preparation of a reactive intermediate by

Osborne et al.

oxidation of 4-hydroxytamoxifen with manganese dioxide, and tentatively identified this as the tamoxifen quinone methide (9). The substance was not isolated or characterized, but was immediately added to DNA, and its adducts with deoxyguanosine were isolated and identified by mass and proton resonance spectroscopy. We prepared these adducts by the same method, and found them to be identical with our adducts DG1 and DG2 by cochromatography on ODS (Figure 4D-F). We also analyzed DNA containing the “quinone methide” adducts by 32Ppostlabeling, and obtained a pattern of adducts (Figure 3D) which again was identical to that from DNA containing DG1 and DG2 (Figure 3B). It appears likely from the experiments described here that the reactive intermediate prepared by Marques and Beland (21) from 4-hydroxytamoxifen and manganese dioxide is identical with our R,β-dehydro-4-hydroxytamoxifen. It has not been possible to prove this, because neither our ODS column nor any other that we have tried will satisfactorily separate the new compound from an excess of their starting material, 4-hydroxytamoxifen. Yield of Adducts in Reactions of Acetates with DNA. Addition of 1 µmol of R-acetoxytamoxifen to 10 mg of DNA resulted in 1 adduct per 400 bases, and 4,Rdiacetoxytamoxifen gave approximately the same number of adducts. The corresponding figure for R-acetoxy-4hydroxytamoxifen could not be precisely determined, because its rapid hydrolysis in water meant that the quantity added to DNA was only approximately known. According to approximate data, R-acetoxy-4-hydroxytamoxifen reacted with DNA to 3 times the extent that R-acetoxytamoxifen did. The order of reactivity with DNA parallels the order of reactivity with water (Figure 1). Adducts in Rat Hepatocytes. Rat hepatocytes in primary culture were treated with tamoxifen or R-hydroxytamoxifen, and their DNA was analyzed for adducts by the 32P-postlabeling procedure. An identical pattern was obtained from the two substances, as shown in Figure 5B,C (one intense spot and two or three fainter spots). This result was described previously (24) and provided part of the evidence that the tamoxifen adducts are derived via R-hydroxylation. Treatment with 10 µM tamoxifen yielded ca. 0.6 adducts per 1 million nucleotides and, with 1 µM R-hydroxytamoxifen, ca. 3.0 per 1 million. When the hepatocytes were treated with 4-hydroxytamoxifen or 4,R-dihydroxytamoxifen in the same way, no adducts were observed (Figure 5D,E). If these substances had been activated by metabolism to a reactive ester as tamoxifen was, then one would expect to see the adduct spots shown in Figure 3, which are in the same area of the plate as those from tamoxifen. However, no significant activity was found in this area. The minimum level for detection would be about 1 per 109 nucleotides. Likewise, no adducts were found in the DNA of hepatocytes treated with R,β-dehydro-4-hydroxytamoxifen (Figure 5F) even though this reacts directly with DNA, albeit to a small extent. Adducts in Rat Liver. Female rats were given the same substances orally, in tricaprylin solution. After 24 h, their liver DNA was analyzed for adducts by the 32Ppostlabeling procedure. The results were the same as those in hepatocytes in culture; tamoxifen and R-hydroxytamoxifen gave prominent adduct spots (Figure 5H,I), while 4-hydroxytamoxifen, 4,R-dihydroxytamoxifen, and

DNA Adducts of 4-Hydroxytamoxifen

Figure 5. Autoradiographs of adducts as in Figure 3, but obtained from DNA from (A-F) cultured rat hepatocytes or (GL) rat liver. Samples for panels A and G were from untreated cells or animals, and the other samples were obtained after treatment with (B and H) tamoxifen, (C and I) R-hydroxytamoxifen, (D and J) 4-hydroxytamoxifen, (E and K) 4,Rdihydroxytamoxifen, and (F and L) R,β-dehydro-4-hydroxytamoxifen.

R,β-dehydro-4-hydroxytamoxifen gave none (Figure 5JL).

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 157

able that in an aqueous medium, at least, it is rapidly converted to another tautomeric form, R,β-dehydro-4hydroxytamoxifen. We prepared this latter substance by chemical synthesis, and established that it alkylates DNA, though only slowly and in low yield. It has been proposed (5, 18, 20) that 4-hydroxytamoxifen is an important intermediate in the bonding of tamoxifen to DNA in vivo. It produces adducts in the DNA of mouse liver (5, 20), but we were unable to detect any adducts in the liver of rats treated orally with 4-hydroxytamoxifen, or in rat hepatocytes in culture. Pathak et al. (18) and Martin et al. (27) examined the liver of rats treated with tamoxifen, by intraperitoneal injection and in the diet, respectively. Both claimed that one minor adduct (3% of the total) had arisen from 4-hydroxytamoxifen. However, the identification must remain uncertain, being based only on its chromatographic mobility being the same as that of one of the adducts they isolated from DNA treated with 4-hydroxytamoxifen in vitro. There are four possible reasons why 4-hydroxytamoxifen fails to produce significant adducts in rat liver. (a) The rat liver does not oxidize it to produce 4,R-hydroxytamoxifen or the quinone methide. (b) It does not conjugate 4,R-hydroxytamoxifen to give a reactive ester. (c) The esters or quinone methides are formed, but are so reactive that they do not survive long enough to reach the DNA. (d) They are rapidly detoxified, probably by conjugation at the phenolic hydroxyl group. Of these, reason a is insufficient, because the already oxidized derivative 4,R-dihydroxytamoxifen also fails to produce adducts in rat liver. Reason b is possible; conjugation of 4,R-dihydroxytamoxifen may be solely on the phenolic hydroxyl group, giving unreactive esters analogous to the 4-acetoxy-R-hydroxytamoxifen mentioned earlier. Explanation c appears unlikely, given that the same compounds can give adducts in mouse liver. It would appear that the major route by which tamoxifen becomes bonded to rat liver DNA is through R-hydroxylation and not through 4-hydroxylation. These details of mechanism point the way to studies of the possibility of activation of tamoxifen to cytotoxic metabolites in humans. So far, the evidence for DNA adducts in human tissues is equivocal; reports that DNA adducts have been detected in endometrial DNA from women treated with tamoxifen (28, 29) have not been universally accepted (30-32). Whether or not tamoxifen poses a risk of genotoxicity in a given tissue depends on the level of oxidative and conjugative enzymes, and the site of oxidation.

Discussion We have made the R-acetoxy derivative of the tamoxifen metabolite 4-hydroxytamoxifen. This gave a higher level of reaction with DNA than did R-acetoxytamoxifen itself. This was also to be expected, as the phenolic group can assist in the loss of the R-acetoxy group, to give a highly resonance-stabilized carbocation. Thus, as far as the purely chemical experiments are concerned, our results confirm the predictions of Potter et al. (15) and Kuramochi (16). It is unclear whether the quinone methide (9, Scheme 2), formed by loss of a proton from the carbocation, is an intermediate or a byproduct in the reaction of R-acetoxy4-hydroxytamoxifen with DNA. So far, it has not been possible to isolate this quinone methide; it seems prob-

Acknowledgment. We thank B. Nutley for the mass spectra. The work was supported by grants from the Cancer Research Campaign, U.K.

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